Preface - the-eye.euthe-eye.eu/public/Books/BioMed/Heterotrimeric G-Protein Effectors [Methods...

P r e f a c e

Transduction of signals through G-protein pathways is achieved largely by production of intracellular messengers that either directly or through protein kinases regulate cellular biochemical and physiological processes. Several enzymes, including adenylyl cyclase, phospholipase C, and the cGMP phosphodiesterase, are well established as effectors directly regulated by G proteins. These enzymes are covered in this volume. Others such as phospholipase A2 and phospholipase D are often described as G-protein effectors. Though receptors that couple to G proteins undoubtedly regulate the activity of these enzymes, there is no compelling evidence at this time to indicate these enzymes are directly regulated by G-protein subunits. Hence these enzymes are not included.

Over the past two years it has become increasingly obvious that there is considerable molecular and functional diversity of the effector enzymes as well. Eight mammalian Gs-sensitive adenylyl cyclases and four Gq- stimulated phospholipases C-fl have been cloned, and many of these have been characterized as having distinct capabilities for signal input. The varied functional characteristics and the tissue-specific distribution of the effector isoforms allow the various cell types and tissues to develop cus- tomized response systems by altering the mix of the effector isoforms. Several approaches to characterize the molecular and functional identities of the effector isoforms are presented.

Several mitogens use G-protein pathways to communicate prolifera- tive signals. At this time it is not clear if the immediate effectors in G- protein signaling pathways are those that we already know of or are as yet unidentified ones. Hence techniques currently used to study G-protein regulation of cell proliferation measure the activity of downstream elements. These techniques are nevertheless useful in tracing G-protein pathways and are covered in this volume.

G proteins can also modulate cellular functions by presumably direct regulation of channels to alter the flow of ions through the plasma membranes. To date there have been no descriptions of reconstitution experiments in which purified G-protein subunits can alter the function of purified ion channels. The circumstantial evidence for direct regulation is substantial, so it is reasonable to assume that some channels will be G- protein effectors. A number of imaging and electrophysiological techniques relevant to G-protein regulation of channels are included.

In selecting the chapters for this volume an attempt was made to restrict areas covered to those likely to be of direct interest to researchers

xiii

xiv PREFACE

working in cell surface signal transducing systems. Such selections are undoutedly subjective, and I am certain that there are some areas that are not as thoroughly covered as they could be. However, G-protein effector research is a very active area in many laboratories including my own, and undoubtedly techniques emerging from these studies will have to be covered at later dates.

I would like to thank the authors for their contributions. I am especially thankful to those involved in phospholipase C research, a field that moved at a brisk pace during the year these chapters were being com- piled, for providing chapters documenting the very latest advances. I am also grateful to Ms. Lina Mazzella for her unfailingly cheerful help in organizing the chapters.

RAVI IYENGAR

Contributors to V o l u m e 238

Article numbers are in parentheses following the names of contributors. Affiliations listed are current.

ROBERT ALVAREZ (3), Institute of Pharma- cology, Syntex Research, Paid Alto, Cali- fornia 94304

JEFF AMUNDSON (27), Department of Phar- macology, Mayo Foundation, Rochester, Minnesota 55905

RODRIGO ANDRADE (29), Department of Pharmacological and Physiological Sci- ences, St. Louis University School of Medicine, St. Louis, Missouri 63130

NIKOLAI O. ARTEMYEV (2), Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

ANDREW BALL (12), Department of Physiol- ogy, University College London, London WC1E 6J J, United Kingdom

JONATHAN L. BLANK (19), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206

ROBERT D. BLITZER (11, 32), Departments of Psychiatry and Pharmacology, Bronx Veterans Admnistrations Medical Center, and Mount Sinai School of Medicine, New York, New York 10029

ANTHONY A. BOMINAAR (16), Department of Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands

D. A. BROWN (30), Department of Pharma- cology, University College London, Lon- don WC1E 6BT, United Kingdom

KEVlN P. CAMPBELL (28), Howard Hughes Medical Institute, College of Medicine, University of Iowa, Iowa City, Iowa 52242

MONTSERRAT CAMPS (14), Molecular Phar- macology Division, German Cancer Re- search Center, 69120 Heidelberg, Ger- many

M. P. CAULFIELD (30), Department of Phar- macology, University College London, London WCIE 6BT, United Kingdom

JIANQIANG CHEN (8), Department of Phar- macology, Mount Sinai School of Medi- cine, City University of New York, Ne~t York, New York 10029

DAVID E. CLAPHAM (27), Department of Pharmacology, Mayo Foundation, Roch- ester, Minnesota 55905

SHAMSHAD COCKCROFT (12, 13), Depart- ment of Physiology, University College London, London WCIE 6J J, United Kingdom

DERMOT M. F. COOPER (5), Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colo- rado 80262

ADRIENNE D. Cox (23, 24), Departments of Radiation Oncology and Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

EMER CUNNINGHAM 0 2 , 13), Department of Physiology, University College Lon- don, London WCIE 6J J, United Kingdom

MICHAEL DE VIVO (10), Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, New York, New York 10029

NICOLAS DEMAUREX (26), Division of Cell Biology, Hospital for Sick Children, To- ronto, Ontario, Canada M5G 1)(8

CHANNING J. DER (23, 24), Department of Pharmacology, University of North Caro- lina at Chapel Hill, Chapel Hill, North Carolina 27599

MICHEL DE WAARD (28), Howard Hughes Medical Institute, College of Medicine, University of lowa, Iowa City, Iowa 52242

ix

X CONTRIBUTORS TO VOLUME 238

J. H. EXTON (19), Department of Molecular Physiology and Biophysics, Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

ANNE M. GARDNER (22), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206

PETER GIERSCmK (14), Department of Phar- macology and Toxicology, University of Ulm, 89069 Ulm, Germany

ALFRED G. GILMAN (7), Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

HEIDI E. HAMM (2), Department of Physiol- ogy and Biophysics, University of lllinois at Chicago, Chicago, Illinois 60612

T. KENDALL HARDEN (15), Department of Pharmacology, School of Medicine, Uni- versity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

ANNE E. HARWOOD (21), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206, and Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

CRAIG A. HAUSER (23), Cancer Research Center, La Jolla Cancer Research Foun- dation, La Jolla, California 92037

JORGEN HESCHELER (31), lnstitut far Phar- makologie, Freie Universitdt Berlin, 14195 Berlin, Germany

YEE-KIN HO (1), Departments of Biochem- istry and Ophthalmology, University of Il- linois at Chicago, Chicago, Illinois 60612

RAVI IYENGAR (8, 20), Department of Phar- macology, Mount Sinai Medical Center, New York, New York 10029

VEER JACOBOWITZ (8), Department of Phar- macology, Mount Sinai School of Medi- cine, City University of New York, New York, New York 10029

MARISA E. E. JACONI (26), Department of Pharmacology, Mayo Foundation, Roch- ester, Minnesota 55905

DEOK-YOUNG JHON (17), Laboratory of Bio- chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

GARY L. JOHNSON (22), Division of Basic Sciences, National Jewish Center for Im- munology and Respiratory Medicine, Denver, Colorado 80206, and Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

ROGER A. JOHNSON (3, 4), Department of Physiology and Biophysics, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York 11794

STEVEN D. KAHL (28), Howard Hughes Medical Institute, College of Medicine, University of lowa, Iowa City, Iowa 52242

KARL-HEINZ KRAUSE (26), Infectious Dis- eases Division, University Hospital, CH- 1211 Geneva 4, Switzerland

YOSHIHISA KURACHI (34), Division of Car- diovascular Diseases, Departments of ln- ternal Medicine and Pharmacology, Mayo Foundation, Rochester, Minnesota 55905, and Department of Pharmacology H, Faculty of Medicine, Osaka University Medical School, Suita, Osaka 565, Japan

EMMANUEL M. LANDAU (] ], 32), Depart- ments of Psychiatry and Pharmacology, Bronx Veterans Administration Medical Center, and Mount Sinai School of Medi- cine, New York, New York 10029

CAROL A. LANGE-CARTER (22), Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medi- cine, Denver, Colorado 80206

CHANG-WON LEE (18), Laboratory of Bio- chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

KWEON-HAENG LEE (18), Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

P. DANIEL LET (26), Infectious Diseases Division, University Hospital, CH-1211 Geneva 4, Switzerland

CONTRIBUTORS TO VOLUME 238 xi

HAI-WEN MA (20), Department of Pharma- cology, Mount Sinai Medical Center, New York, New York 10029

I. MCFADZEAN (30), Pharmacology Group, King's College London, London SW3 6LX, United Kingdom

JOHN S. MILLS (2), Department of Physiol- ogy and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

ANDREW J. MORRIS (15), Department of Pharmacology, School of Medicine, Uni- versity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

DONGEUN PARK 07), Laboratory of Bio- chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

RICHARD T. PREMONT (9, 20), Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710

HELEN M. RARICK (2), Department of Phys- iology and Biophysics, University of Illi- nois at Chicago, Chicago, Illinois 60612

STEPHEN R. RAWLINGS (25, 26), Founda- tion for Medical Research, Department of Medicine, University of Geneva, CH-1211 Geneva 4, Switzerland

SUE Goo RULE (17, 18), Laboratory of Bio- chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892

YORAM SALOMON (3), Weizmann Institute of Science, Department of Hormone Re- search, Rehovot 76100, Israel

WERNER SCHLEGEL (25, 26), Foundation for Medical Research, Department of Medi- cine, University of Geneva, CH-1211 Ge- neva 4, Switzerland

1LANA SHOSHANI (4), Department of Physi- ology and Biophysics, School of Medi- cine, Health Sciences Center, State Uni- versity of New York at Stony Brook, Stony Brook, New York 11794

N1KOLAI P. SKIBA (2), Department of Physi- ology and Biophysics, University of Illi- nois at Chicago, Chicago, Illinois 60612

LISA STEHNO-BITTEL (27), Department of Pharmacology, Mayo Foundation, Roch- ester, Minnesota 55905

WEI-JEN TANG (7), Department of Pharma- cology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ANDREA TAR (1), Department of Biochemis- try, University of Illinois at Chicago, Chi- cago, Illinois 60612

RONALD TAUSSIG (7), Department of Phar- macology, University of Texas South- western Medical Center, Dallas, Texas 75235

ANDRl~ TERZIC (34), Departments of Inter- nal Medicine and Pharmacology, Mayo Foundation, Rochester, Minnesota 55905

JEAN-MARC THELER (25), Division of Clini- cal Biochemistry, Department of Medi- cine, University of Geneva, CH-1211 Ge- neva 4, Switzerland

GERAINT M. H. THOMAS (12, 13), Depart- ment of Physiology, University College London, London WCIE 6J J, United Kingdom

Tuow D. TING (1), Department of Biochem- istry, University of Illinois at Chicago. Chicago, Illinois 60612

RICHARD R. VA1LLANCOURT (21, 22), Divi- sion of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206

Y. VALLIS (30), Department of Pharmacol- ogy, University College London, London WCIE 6BT, United Kingdom

PETER J. M. VAN HAASTERT (16), Depart- ment of Biochemistry, University of Gro- ningen, 9747 AG Groningen, The Nether- lands

GARY L. WALDO (15), Department of Phar- macology, School of Medicine, Univer- sity of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

SIM WINITZ (21), Division of Basic Sci- ences, National Jewish Center for Immu- nology and Respiratory Medicine, Den- ver, Colorado 80206, and Department of Pharmacology, University of Colorado Medical School, Denver, Colorado 80262

xii CONTRIBUTORS TO VOLUME 238

DERRICK R. WITCHER (28), Howard Hughes Medical Institute, College of Medi- cine, University of lowa, Iowa City, Iowa 52242

YUNG H. WONG (6), Department of Biol- ogy, Hong Kong University of Science and Technology, Kowloon, Hong Kong

MITSUHIKO YAMADA (34), Pharmacology H, Osaka University Medical School, Suita, Osaka 565, Japan

ATSUKO YATANI (33), Departments of Phar- macology and Cell Biophysics, University of Cincinnati, College of Medicine, Cin- cinnati, Ohio 45267

[1 ] P U R I F Y I N G R E T I N A L cGMP P H O S P H O D I E S T E R A S E 3

[1] Pur i f ica t ion of B o v i n e Re t i na l c G M P P h o s p h o d i e s t e r a s e

By ANDREA TAR, TUOW D. TING, and YEE-KIN H o

Introduction

Visual excitation in vertebrate rod photoreceptor cells involves a light-activated cGMP enzyme cascade in the rod outer segment (ROS). Absorption of a photon by the receptor molecule, rhodopsin (R*), leads to the activation of a latent cGMP phosphodiesterase (PDE) which rapidly hydrolyzes cytosolic cGMP. The transient decrease in cGMP concentration causes the closure of cGMP-sensitive cation channels in the plasma membrane and results in hyperpolarization of the cell. 1,2 The PDE can either be bound on ROS disk membranes or exist in a soluble form in the cytosol. In both forms, PDE is a latent enzyme complex composed of three types of polypeptides, P~ (88 kDa), P~ (84 kDa), and P~ (14 kDa), with a ratio of 1 : 1 : 2. Polypeptides P~ and P~ contain separate catalytic sites which are inhibited by the binding of the inhibitory P~ subunits.

Signal coupling between photolyzed rhodopsin (R*) and PDE is mediated by a signal-transducing G protein called transducin (T) via a GTP-binding and hydrolysis cycle. Transducin is a trimeric protein composed of three polypeptides: T~ (39 kDa), T¢ (37 kDa), and T~ (8.5 kDa). In the dark-adapted state, T~ contains a bound GDP (T~-GDP) and interacts with T~. The T~-GDP • T~ complex tightly associates with rhodopsin. On photoexcitation, R* catalyzes a GTP/GDP exchange reaction converting T~-GDP to the active form of T~-GTP. The T~-GTP complex activates PDE by relieving the restraint exerted by the P~ inhibitory subunit on the P~ and Po catalytic sites. After the hydrolysis of the tightly bound GTP, T~-GDP releases P~ and recombines with T~. In turn, P~ shuts off the cGMP hydrolysis, and the cascade is ready for another cycle of activation. 3 This chapter delineates methods for the purification of retinal cGMP PDE from bovine retinas and also describes biochemical asssays for the enzymatic activities.

i M. L. Applebury and P. A. Hargrave, Vision Res. 26, 1881 (1986). -' L. Stryer, J. Biol. Chem. 266, 10711 (1991). 3 Y.-K. Ho, V. N. Hingorani, S. E. Navon, and B. K.-K. Fung, Curr. Top. Cell. Regul.

30, 171 (1989).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved.

4 cGMP PHOSPHODIESTERASE [1]

Purification of Retinal cGMP Phosphodiesterase

The purification procedure was originally developed by Hurley and Stryer and was modified for large-scale preparation from 400 retinas. 4-6 The y subunit of PDE can be isolated from bovine retina according to the procedure of Hurley. 7 Phosphodiesterase can be isolated from Rana catesbiana according to a procedure of Yamazaki et al. 8 This chapter describes the purification of bovine PDE from crude ROS membranes prepared from frozen retinas.

As a peripheral membrane protein, PDE can be extracted from photolyzed ROS membranes by repeatedly washing with low ionic strength buffer. Transducin, the other major peripheral membrane protein, remains tightly associated with R* in the absence of GTP, and transducin subunits are retained on the ROS membrane during low ionic extraction. The extracted PDE is further purified by column chromatography.9 An isolation process using 400 retinas typically yields more than 3 mg of pure PDE. Based on the convenience of the purification scheme, it is recommended that a minimum of 200 retinas be used to obtain optimal yields.

Preparation of Crude Rod Outer Segment Membranes for Phosphodiesterase Extraction

Frozen dark-adapted bovine retinas can be purchased from supply houses (G. A. Hormel Co., Austin, MN, or J. A. Lawson Co., Lincoln, NE). Fresh bovine eyes are collected from local packing companies and kept in the dark for several hours prior to dissection of the retinas under dim red light (Kodak, Rochester, NY, red No. 2 safety light filter). These retinas can be stored in the dark at -70 ° for over a year without loss of activity of the enzymes involved in the cGMP cascade. The isolation procedure is carried out under dim red light to keep rhodopsin in the dark- adapted state. Two liters of ice-cold isolation buffer is required, which consists of 10 mM MOPS [3-(N-morpholino)propanesulfonic acid], 60 mM KCI, 30 mM NaCI, 2 mM MgCI2, 0.1 mM PMSF (phenylmethylsulfonyl fluoride), and 1 mM DTT (dithiothreitol) at pH 7.5. Two 500 ml sucrose solutions are prepared using the isolation buffer, namely, a 50% (w/v) solution and a 38% (w/v) solution.

4 B. K.-K. Fung, J. Biol. Chem. 258, 10495 (1983). 5 B. K.-K. Fung, J. B. Hurley, and L. Stryer, Proc. Natl. Acad. Sci. U,S.A. 78, 152 (1981). 6 Y.-K. Ho and B. K.-K. Fung, J. Biol. Chem. 259, 6694 0984). 7 j. B. Hurley, this series, Vol. 81, p. 542. s A. Yamazald, N. Mild,and M. W. Bitensky, this series, Vol. 81, p. 526. 9 j . B. Hurley and L. Stryer, J. Biol. Chem. 257, 11094 (1982).

[1] PURIFYING RETINAL cGMP PHOSPHODIESTERASE 5

For the preparation of crude ROS membranes, 400 frozen retinas are thawed in 200-350 ml of the 50% sucrose buffer. The suspension is transferred to a beaker and stirred with a magnetic stirrer for approximately 20 min. The retinal suspension is forced twice through a 50-ml syringe without a needle to break up the tissue, then divided equally among sixteen 50-ml centrifuge tubes used in superspeed centrifuges (Du Pont Sorval RC5 centrifuges with SS-34 rotors, Du Pont Company, Wilmington, DE; or Beckman Instruments J2-21 centrifuges with J-21 rotors, Beckman Instruments Inc., Palo Alto, CA). The volume of each tube is adjusted to 40 ml with 50% sucrose buffer. After the contents are mixed by shaking, the suspension is centrifuged at 15,000 rpm (27,000 g) for 15 min at 4 °. Under these conditions, the ROS membranes float to the top of the tubes, while the remainder of the retinal membranes pellet to the bottom.

With a rubber policeman, the floating ROS membranes are scraped off the walls of the tubes and resuspended in the supernatant. The supernatant is gently poured off and distributed equally among 28 clean 50-ml centrifuge tubes. The volume in each tube is increased to 40 ml with isolation buffer without sucrose to dilute the sucrose concentration to approximately 28%. The tubes are shaken and centrifuged at 18,000 rpm (38,000 g) for 15 min at 4 °. The ROS membranes sediment to the bottom of the tubes. After discarding the supernatant, the ROS membrane pellets are resuspended in 150 ml of 38% sucrose in isolation buffer and divided equally among 16 tubes. The volume of each tube is adjusted to 30 ml with the 38% sucrose buffer, and the tubes are centrifuged at 18,000 rpm for 45 min. The ROS membranes float to the top of the tubes and are resuspended in the supernatant.

The 38% sucrose membrane suspension is divided among 16 clean centrifuge tubes. To reduce the sucrose concentration, the volume of each tube is increased to 45 ml with isolation buffer. After mixing, the tubes are centrifuged at 19,000 rpm (43,000 g) for 20 min at 4 ° to pellet the ROS membranes. The supernatants are discarded, and the pellets are resuspended with 40 ml of isolation buffer. The samples are centrifuged again at 19,000 rpm for 20 min at 4 °. Residual sucrose is removed by washing the ROS membranes twice more with isolation buffer. The final ROS membranes are resuspended with 40 ml of isolation buffer and divided equally among four tubes. The crude ROS membranes can be kept in the dark overnight on ice prior to PDE extraction.

Extraction of PDE from crude ROS membranes is carried out under room light. Two liters of low ionic strength extraction buffer containing 5 mM Tris, 0.5 mM MgClz, 0.1 mM PMSF, and 1.0 mM DTT at pH 7.5, a 120-ml Teflon-glass homogenizer, and two 30-ml syringes fitted with 8 cm of Tygon tubing are required. The crude ROS membranes are resus-


pended in 100 ml extraction buffer and homogenized with the Teflon-glass homogenizer by five up and down strokes. The homogenate is divided equally among four 50-ml centrifuge tubes, and the volume of each tube is increased to 45 ml with extraction buffer. The samples are photolyzed on ice under a lamp for 15 min until the red color of the suspension changes to bright orange, indicating the conversion of rhodopsin to the meta-II state (R*). Under these conditions, transducin remains tightly bound to the R*-containing membrane, whereas most of the peripheral proteins, including PDE, can be extracted by the low ionic strength buffer.

After photolyzing the ROS membranes, the suspensions are centrifuged at 19,000 rpm for 30 rain. The supernatants are carefully removed using a 30-ml syringe fitted with Tygon tubing and transferred to four clean 50-ml centrifuge tubes. The supernatants containing the extracted proteins are centrifuged again at 19,000 rpm for 30 min to remove residual ROS membranes. Supernatant from the second centrifugation is removed with a clean 30-ml syringe and stored on ice for subsequent chromatographic separation of PDE. The ROS membrane pellets from the two centrifugation steps are pooled and homogenized again with 100 ml buffer for the second extraction. The repetitive extraction of PDE with double centrifugation steps is carried out at 0°-4 ° for a total of six times.

The protein concentration of each extract is monitored. In general, supernatants from the first four extractions (-480 ml) are pooled for PDE purification by column chromatography steps, and supernatants from the last two extractions are discarded because of the low protein content.The final ROS membrane pellets are pooled and saved for extraction of transducin with GTP. The purification of transducin is described elsewhere in this series ~° and is not elaborated here,

Purification of Phosphodiesterase by Column Chromatographies

DEAE-Sephadex Chromatography. The low ionic strength extract of the ROS membrane is applied to an 18 x 2.5 cm DEAE-Sephadex (Sigma Chemical Co., St. Louis, MO) column equilibrated with buffer A (10 mM MOPS, 2 mM MgC12, 1 mM DTT, pH 7.5) at 4 °. After washing the column with 50 ml buffer A, the bound proteins are eluted with a 500 ml linear gradient of NaC1 from 0.1 to 0.6 M in buffer A. Fractions of 6 ml are collected. The protein content in each fraction is determined by the Bradford assay, ~t and the PDE activity in each fraction is assayed using a pH electrode method after trypsin activation as described in a later section.

~0 j. Bigay and M. Chabre, this series, Vol. 237 [11]. ii M. M. Bradford, Anal. Biochem. 72, 248 (1986).

l l ] PURIFYING RETINAL cGMP PHOSPHODIESTERASE 7

The chromatogram and distribution of PDE activity are shown in Fig. 1A. Two broad peaks are observed, with PDE activity being associated with the second peak at NaCI concentrations between 0.3 and 0.4 M. Fractions containing the PDE activity are pooled and concentrated with an Amicon (Danvers, MA) pressure concentrator equipped with a YM10 membrane. The protein composition as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is shown in Fig. 2: lane 1 shows that in the low ionic strength extract, PDE is the major protein; lane 2 demonstrates that PDE fractions from the DEAE-Sephadex column are approximately 80% pure. Despite about 20% contamination, the crude PDE is suitable for reconstitution assays. Further purification can be accomplished by Sephacryl S-300 and to-aminooctyl-agarose chromatography steps described below.

Sephacryl S-300 Chromatography. The PDE fractions from the DEAE- Sephadex column are concentrated to a final volume of 5 ml. The sample is then applied to a 90 x 2.5 cm Sephacryl S-300 column (Pharmacia LKB Biotechnology, Piscataway, N J) equilibrated with buffer A at 4 °, The proteins are eluted with buffer A, and fractions of 3 ml are collected. An aliquot of the fractions is removed and assayed for protein composition and PDE activity. The chromatogram of the S-300 column separation is shown in Fig. lB. The PDE is eluted as a single peak. The appropriate fractions are pooled, and the protein composition of the pool is shown by SDS-PAGE in Fig. 2 (lane 3). PDE is purified to approximately 95% homogeneity after the S-300 chromatography step and can be purified further by to-aminooctyl-agarose chromatography.

o~-Aminooctyl-agarose Chromatography. The PDE fractions from the Sephacryl S-300 column are applied to an oJ-aminooctyl-agarose column (15 x 1 cm, ICN ImmunoBiologicals, Lisle, IL) equilibrated with buffer A at 4 °. The column is then washed with 50 ml buffer A and subsequently eluted with a 180 ml linear NaC1 gradient from 0 to 0.4 M in buffer A. Fractions of 2 ml are collected, and the protein composition and PDE activity of the fractions are assayed. The chromatogram is shown in Fig. 1C. The PDE activity coelutes with the single protein peak at 0.25 M NaCI. The protein fractions are pooled and concentrated with the Amicon concentrator using YM10 membrane. The protein composition of the purified PDE as analyzed by SDS- PAGE (Fig. 2, lane 4) shows protein bands of P, (88 kDa) and P~ (84 kDa). The P~ polypeptide (14 kDa) stains less well with Coomassie blue and is not readily apparent with the amount of protein present in the gel. The purified PDE is stored in 40% glycerol in buffer A at -20 °. The to-aminooctyl-agarose column can be regenerated by washing the column with 1 M NaCI and is stored in 0.5% toluene.

8 cGMP FHOSPHODIESTERASE [1]

B I

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, , , , , , , , , , ~ 0 , I

10 20 30 40 50 60 70 80 90

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-I 5 4

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100 120 140 160

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20 30 40 50 60

Fraction Number

FIG. 1. Purification of PDE by column chromatographies. The conditions for the chromatographic separation are described in the text. The protein content is determined by the Bradford dye binding assay monitored by the absorbance at 595 nm. The PDE activity is monitored by the decrease of medium pH owing to the hydrolysis of cGMP. (A) Purification of PDE by DEAE-Sephadex. Extract from 400 retinas in 480 ml of extraction buffer is applied to the colunm. The column is eluted using 500 ml of a linear gradient of 0.1-0.6 M NaCI in buffer A. The PDE is eluted during the second peak. (B) Purification of PDE by Sephacryl S-300. The pooled fractions from the DEAE-Sephadex column are concentrated to 5 ml using an Amicon concentrator and applied to a Sephacryl S-300 column. The column

[1] PURIFYING RETINAL c G M P PHOSPHODIESTERASE 9

M W x 10 -3

66

45

36

29 24

20 --~

14,2

1 2 3 4

FIG, 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PDE from each chromatographic step. Each lane contained 15/zg protein. Lane 1, Crude low ionic strength extract from ROS membrane; lane 2, pooled fractions from DEAE-Sephadex step; lane 3, pooled fractions from Sephacryl S-300 step; lane 4, pooled fractions from to-aminooctyl- agarose step. Molecular weight markers include bovine albumin (66 kda), egg albumin (45 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor (20 kDa) and c~-lactalbumin (14.2 kDa). Purified PDE contains three polypeptides of 88 (P~), 84 (P~) and 14 (PT) kDa,

A summary of each step of the isolation procedure is shown in Table I. The purified PDE remains as a latent enzyme, and its activity can be assayed either with trypsin activation or in the presence of transducin- Gpp(NH)p complexes (transducin T~ subunit containing a nonhydrolyze- able GTP analog of guanylyl imidophosphate).

Functional Assays for Phosphodiesterase

Phosphodiesterase Activation Using Trypsin Treatment

The PDE activity assay is based on the procedure described by Yee and Liebman. ~z The activity of purified PDE can be assayed independent

12 R. Yee and P. A. Liebman, J. Biol. Chem. 253, 8902 (1978).

is eluted with buffer A. (C) Purification of PDE by to-aminooctyl-agarose. The fractions containing PDE from the Sephacryl S-300 column are pooled and applied to the to-aminooctyl- agarose column. The column is eluted with a 180 ml linear NaCI gradient from 0 to 0.4 M in buffer A.


<

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[1] PURIFYING RETINAL cGMP PIqOSPHOD1ESTERASE 1 1

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cGMP = 0 A

B

a c t i v a t ~

t FIG. 3. Activation of cGMP phosphodiesterase by transducin. The activity of PDE

was determined by the rate of proton release arising from cGMP hydrolysis using a pH microelectrode. The assays were carried out in 200/zl of PDE assay buffer containing 10 ~g PDE under the following conditions: (A) PDE was activated by 1/xg trypsin for 3 rain, and 5 mM cGMP was added at the time indicated by the arrow; (B) PDE was incubated with 5 t~M R*, 5 tzg transducin, and 5 mM cGMP, and 10/xM GTP was added at the time indicated by the arrow; (C) PDE was incubated with 40/zg TjGpp(NH)p, and 5 mM cGMP was added at the time indicated by the arrow.

of R* and transducin activation by treatment with trypsin. The hydrolysis of cGMP can be monitored as a decrease in the pH of the solution. The PDE assay buffer contains 10 mM MOPS, 200 mM NaC1, and 2 mM MgClz, at pH 7.5. Treatment with trypsin will cleave the P~ subunit of PDE and relieve its inhibition on the catalytic subunits of P~ and PC.

In a typical assay, a 100/zl sample containing 10 ~g PDE is treated with 1 ~g trypsin for 3 min in a well of a microtiter plate. The sample is allowed to equilibrate to achieve a baseline pH. The cGMP hydrolysis reaction is then initiated by the addition of 100/xl of 10 mM cGMP. The change in pH of the reaction medium is monitored by a pH microelectrode (MI-410 Micro combination pH Probe, Microelectrodes Inc., London- derry, NH) connected to a Radiometer PHM 82 pH meter (Radiometer America, Inc., Westlake, OH). The result is recorded on a strip-chart recorder. The change in pH is converted to nanomoles of protons liberated


by titrating the sample with 5-/.d aliquots of 5 mM NaOH. The amount of protons generated is then converted to the amount of cGMP hydrolyzed. A typical progress curve is shown in Fig. 3A. The change of pH in the reaction medium will level off when cGMP is completely hydrolyzed.

Activation of Phosphodiesterase in a Reconstitution System Containing Rhodopsin and Transducin

Latent PDE can be activated in a reconstituted system containing photolyzed rhodopsin (R*) and transducin in the presence of GTP or the nonhydrolyzable analog Gpp(NH)p. Procedures for purifying R* and transducin are described elsewhere ~°'13 and are not repeated here. In a typical assay using R*, transducin, and PDE, the reaction mixture contains 5/zM R*, 5/~g transducin, 10/zg PDE, and 5 mM cGMP in a volume of 100/zl of PDE assay buffer. The PDE remains as a latent enzyme with negligible activity in the absence of GTP. The sample is allowed to equilibrate to achieve a baseline pH. Then GTP (10 /.~M) or Gpp(NH)p (3/zM) is added to activate the PDE. As the hydrolysis ofcGMP proceeds, the pH of the reaction medium decreases, which is monitored by the pH microelectrode. Similar to the above assay, the change in pH of the reaction medium is converted to nanomoles of protons liberated, and the rate of cGMP hydrolysis can be calculated from the initial rate of the pH change. The maximal activity of PDE in this reconstituted assay is approximately 40-60% that of the trypsin-activated PDE (Fig. 3B).

The activation of PDE is due to the interaction of Pv with the activated form of transducin. The T~ subunit of transducin can be purified in its activated form containing tightly bound Gpp(NH)p. The T,~-Gpp(NH)p complex activates PDE directly in the absence of R* and Tar. The efficiency of activation with the reconstitution of soluble PDE and T~-Gpp (NH)p is not as high as that in the presence of rhodopsin-containing membranes. In a typical assay, purified PDE (10/zg) is incubated 150/zl of PDE assay buffer containing 5 mM cGMP to establish the baseline pH. Excess T~-Gpp(NH)p (40/zg) in 50/zl of buffer is added to initiate the PDE activation. The hydrolysis of cGMP is again monitored as the decrease of pH in the reaction medium. The maximum activation of PDE with soluble T~-Gpp(NH)p is approximately 5-10% that of the trypsin-activated level (Fig. 3C).

13 T. D. Ting, S. B. Goldin, and Y.-K. Ho, in "Methods in Neurosciences" (P. A. Hargrave, ed.), Vol. 15, pp. 180-195. Academic Press, San Diego, 1993.

[2 ] P E P T I D E PROBES FOR G PROTEINS A N D EFFECTORS 13

[2] Specific P e p t i d e P r o b e s for G-P ro t e in In t e rac t ion wi th Effec tors

B y HELEN M. RARICK, NIKOLAI O. ARTEMYEV, JOHN S. MILLS, NmOLAI P. SKmA, and HEIDI E. HAMM

In t roduc t ion

In the phototransduction cascade in the rod outer segment (ROS), the activated receptor, rhodopsin (R*), forms a transient complex with the G protein transducin (G O and catalyzes GDP-GTP exchange on the a subunit of G t (Gto~). The binding of GTP to Gto~ enables it to interact with and activate the effector, cGMP phosphodiesterase (PDE). The activated PDE hydrolyzes cGMP to 5'-GMP which then results in closure of cGMP- activated cation channels in the plasma membrane.

The Gta polypeptide, consisting of 350 amino acids, binds both GDP and GTP, contains GTPase activity, undergoes a conformational change on binding to GTP, and interacts with rhodopsin, the /37 subunits of transducin, and the effector, PDE. Bovine PDE consists of two catalytic subunits, Pa (88 kDa) and P/3 (84 kDa), and two identical inhibitory subunits, P3' (11 kDa). 2 The catalytic activity of heterotrimeric PDE is kept low in the dark by the inhibitory subunits. In the light Gta-GTP activates PDE by removing the inhibitory constraint. Bovine Gta-GTPyS has been shown to form a complex with py,3 and thus it has been hypothesized that Gta-GTP activates PDE by interacting with PT and causing its removal or displacement from the Pa/3 catalytic subunits. However, there has also been evidence for Gta interaction with the Pail subunits. 4 The Gta-GDP complex inhibits PDE activated by trypsin, which degrades the inhibitory subunits. 5 This suggests that there may be sites of binding on Gtot for both P7 and Pa/3. Prior to this investigation, however, the location of the site(s) of PDE interaction on the Gta subunit had been completely unknown. Based on the hypothesis that linear regions of amino acid residues on G~a are involved in PDE interaction, we tested synthetic peptides corresponding to specific regions of Gta for functional effects on PDE activity. In

1 L. C. Stryer, Annu. Rev. Neurosci. 9, 87 (1986). 2 p. Deterre, J. Bigay, F. Forquet, M. Robert, and M, Chabre, Proc. Natl. Acad. Sci.

U.S.A. 85, 2424 (1988). 3 p. Deterre, J. Bigay, M. Robert, C. Pfister, H. Kuhn, and M. Chabre, Proteins 1, 188 (1986), 4 A. Sitaramayya, J. Harkness, J. H. Parkes, C. Gonzalez-Ouva, and P. Liebman, Biochem-

istry 25, 651 (1986). s S. Kroll, W, J. Phillips, and R. A. Cerione, J. Biol. Chem. 264, 4490 (1989).



this chapter we discuss general aspects involved in the use of peptides (such as choice, purity, and solubilization), as well as give examples of functional effects of Gtc~ peptides previously reported. 6'7

Choice of Peptides

Because the effector region o n Gtot was unknown, other GTP-binding protein/effector contact studies were examined in order to choose which Gto~ peptides to synthesize. Studies using Gia/G~t~ chimeras have revealed that a 120-residue segment within the carboxyl 40% of Gsa residues is sufficient for adenylate cyclase activation. 8'9 Thus, the putative effector contact region on Gas (which consists of 394 residues) would map between residues 235 and 356. This corresponds to Gtot residues 209-314. Peptides from within this region of the carboxyl terminus of Gtot w e r e synthesized. Based on a model of the three-dimensional structure of Gto~,10 regions that were predicted to be surface exposed, as well as hydrophilic, were chosen. The Gtot peptides that were synthesized from this region (from residues 209 to 314) are Nos. 6-12 in Fig. 1 and Table I. They correspond to G t a residues 224-239 (No. 6), 242-259 (No. 7), 265-280 (No. 8), 288-310 (No. 9), 293-314 (No. 10), 300-314 (No. 11), and 305-329 (No. 12). Sequences for the peptides are given in Table I.

Two Gta peptides, spanning residues 189-210 (Fig. 1, No. 4) and residues 201-215 (Fig. 1, No. 5) were synthesized because they are within a region thought to be involved in the GTP-induced conformational change of the Ga subunit. A mutation at G226 of Gsc~ (which corresponds to G 199 in Gta) results in loss of the ability to activate adenylate cyclase since the "active" GTP-induced conformation is blocked. 11'12 A Gto~ peptide spanning residues 162-181 (Fig. 1, No. 3) was synthesized which corresponds to a region in p21 ras proposed as an effector contact region (Fig. I). Mutational replacements of residues in this region of p21 ras block neoplastic transformation. ~3

6 H. M. Rarick, N. O. Artemyev, and H. E. Hamm, Science 257, 1031 (1992). 7 N. O. Artemyev, H. M. Rarick, J. S. Mills, N. P. Skiba, and H. E. Harem, J. Biol. Chem.

267, 25067 (1992). 8 S. B. Masters, K. A. Sullivan, B. Beiderman, N. G. Lopez, J. Rarnachandron, and

H. R. Bourne, Science 241, 448 (1988). 9 S. Osawa, N. Dhanaseharan, C. W. Woon, and G. L. Johnson, Cell (Cambridge, Mass.)

63, 796 (1990). l0 D. Deretic and H. E. Harnm, J. Biol. Chem. 262, 10839 (1987). II R. T. Miller, S. B. Masters, K. A. Sullivan, B. Beiderman, and H. R. Bourne, Nature

(London) 334, 712 (1988). 12 E. Lee, R. Taussig, and A. G. Gilman, J. Biol. Chem. 267, 1212 (1992). 13 I. Sigal, J. B. Gibbs, J. S. D'Alonzo, and E. M. Scolnick, Proc. Natl. Acad. Sci. U.S.A.

83, 4725 (1986).

[2] PEPTIDE PROBES FOR G PROTEINS AND EFFECTORS 15

1 2 3 4 5 6 7 8 . 9 - 1 3 . 14 peptides ~ [ ] [ J 1 I 1'1 ~'-]~'-'"1 ~ ~ [ ]

100 200 300 . . . . .

Gi/G s CHIMERA

ras p21

i v / / J ~ l . . . . . . . . . . . . . . . . . . . . . . . . . II

EF-Tu

. . . . . . . . . . . . . . . . . . . . . . . . . . ~ t ~ ~/~ I

FIG. 1. Localization of synthesized Gtc~ peptides in relation to a schematic linear map of Gtc~, Gai/Ga s chimera, p21 ras, and EF-Tu. The number of each peptide (Nos. 1-14) is at the top and corresponds to the numbering system given in Table I. The four GTP-binding domains (hatched regions) of each protein are aligned with one another. Putative effector domains of each protein are solid black.

In addition, peptides from the amino-terminal region were examined. The Gtct peptide 53-65 (Fig. 1, No. 2) is from a region on Gta that has the least sequence similarity to other Ga chains and contains an identical sequence with the PDE a subunit (Gtot 59-63 and P~ 120-124 both contain the sequence L-E-E-C-L).14 The region 53-65 on G,a also corresponds to a putative effector binding region in elongation factor-Tu(EF-Tu) ~5 (Fig. 1). The Gta peptide 1-23 (Fig. 1, No. I) as well peptides 311-329 (Fig. 1, No. 13) and 340-350 (Fig. 1, No. 14) from the carboxyl terminus had been previously synthesized for studies involving rhodopsin domain mapping.

14 D. J. Takemoto and J. M, Cunnick, Cell. Signalling 2, 99 (1990). ~5 R. A. Laursen, J. J. L'Italien, S. Nagarkatti, and D. L. Miller, J. Biol. Chem. 256,

8102 (1981).


TABLE I Gta PEPTIDES SYNTHESIZED

Peptide Residues Sequence

1 1-23 2 53-65 3 162-181 4 189-210 5 201-215 6 224-239 7 242-259 a 8 265-280 9 288-310 a

10 293-314 11 300-314 12 305-329 13 311-329 14 340-350

MGAGASAEEKHSRELEKKLKEDA HQDGYSLEECLEF GYVPTEQDVLRSRVKTTGII DLNFRMFDVGGQRSERKKWIHC RSERKKWIHCFEGVT SAYDMVLVEDDEVNRM SLHLFNSICNHRYFATTS NKKDVFSEKIKKAHLS GPNTYEDAGNYIKVQFLELNMRR EDAGNYIKVQFLELNMRRDVKE KVQFLELNMRRDVKE ELNMRRDVKEIYSHMTCATDTQNVK DVKEIYSHMTCATDTQNVK IKENLKDCGLF

a Peptides were insoluble under the PDE assay conditions (pH 8.0) and were not tested further.

Peptide Synthesis and Purification

The Gta peptides are synthesized by the solid-state Merrifield method ~6 on an Applied Biosystems (Foster City, CA) automated synthesizer in the Protein Sequencing/Synthesis Laboratory at the University of Illinois at Chicago (UIC). All peptides (except the amino- and carboxyl-terminal peptides corresponding to residues 1-23 and 340-350) are synthesized with an acetyl group at the amino terminus and an amide group at the carboxyl terminus. Crude peptides are purified by reversed-phase high- performance liquid chromatography (HPLC) on a preparative Aquapore Octyl (C8) column (25 x 1 cm) (Applied Biosysterns) using a 0-60% gradient of acetonitrile in 0.1% trifluoroacetic acid/distilled water (% v/v). After lyophilization, purity is checked by fast atom bombardment (FAB) mass spectrometry at the UIC mass spectrometry facility, analytical HPLC, and at times amino acid analysis. Only those peptides with a single peak corresponding to the predicted molecular weight on the FAB mass spectro- gram are used. All peptides synthesized and used in experiments are listed in Table I. Some peptides are not soluble under the conditions used for the PDE assays (see Table I). Therefore, all peptides are filtered through a 0.22-tzm nylon filter (Micron Separations, Inc., Westboro, MA) and

16 R. B. Merrifield, Fed. Proc., Fed. Am. Soc. Exp. Biol. 21, 412 (1962).


centrifuged at 6000 g prior to use. This is to ensure that only soluble peptides are used in testing functional effects on PDE.

Solubility of Peptides

The solubility of a peptide in aqueous solution is influenced by several factors, such as number of residues, percentage of residues that are charged, number of hydrophobic residues, and pl value of the peptide, t7.t8 When testing functional effects of peptides, it is important to be certain that the peptide is soluble under the assay conditions. For example, because the functional effects of G t a peptides on PDE activity (using the proton evolution assay) are examined within the range ofpH 8.0-8.5, the solubility of each peptide is determined at this pH range.

The G t a peptides were initially dissolved in isotonic buffer A [10 mM HEPES, pH 8,0, 100 mM KCI, 1 mM MgClz, and 1 mM dithiothreitol (DTT)] to give a 1 mM solution. The initial pH of the peptide solutions was less than 8.0; therefore, each solution is then brought to pH 8.0-8.5 with NaOH. In most cases, each peptide is in solution; however, in some cases, the peptides are not soluble. These included peptides 224-239, 242-259, 288-310, 293-314, and 300-314. Specific attempts were made to get them into solution. First, organic solvents were used such as acetonitrile, dimethylformamide, ethanol, dimethyl sulfoxide, 1-methyl-2-pyrroli- dinone, acetic acid, and dichloromethane. However, greater than a 10% solution of these solvents interfered with the PDE activity assay, and this low amount of solvent was not sufficient to dissolve the insoluble peptides. Another approach was to suspend each of these peptides in a high pH buffer (50 mM Tris, pH 9.5). Here, the initial pH of a 1 mM solution of each peptide was about 9. Then, the pH was brought to pH 8.0 with HCI. With this procedure, it appeared that now most of the insoluble peptides were in solution (i.e., the peptide solution is clear and not opaque).

There are only two peptides that were still insoluble: peptides 242-259 and 288-310. It may be that these peptides have pl values (8.1 for peptide 288-310 and 9.2 for peptide 242-259) that are very close to the pH at which the PDE assay is performed, since it is known that a given peptide or protein is least soluble in the neighborhood of its isoelectric point.18 Also, in peptide 242-259, only 5.6% of the residues are charged; in peptide 288-310, 26% of the residues are charged. The reason for their insolubility

t7 G. A. Grant, in "Synthetic Peptides: A Users Guide" (G. A. Grant, ed.), p. 194, Freeman, New York, 1992,

~8 E. J. Cohn, in "Proteins, Amino Acids, and Peptides as Ions and Dipolar Ions" (E. J. Cohn and J. T. Edsall, eds.), p. 573. Reinhold, New York, 1942.


is still unclear. Since peptides 242-259 and 288-310 remain insoluble, they were not tested further.

To determine whether a peptide is completely in solution, first a 1 mM peptide solution (determined by dry weight of the peptide) is made and centrifuged at 6000 g for 10 min at room temperature. A peptide is assumed to be in solution if no pellet is detected by visual inspection. Second, if a peptide has a single tyrosine, such as peptide 293-314, a 1 mM solution of the peptide is made and an absorption spectrum is run using a spectro- photometer before and after filtration through a 0.22-~m filter. The concentration of the peptide is calculated using the molar extinction coefficient (e) of tyrosine at its hma x (e for tyrosine at 278 nm equals 1400 M-1 cml). A peptide is considered completely in solution if the concentration of the sample before and after filtration through a 0.22-tzm filter is the same.

Possible Effects of Gta Peptides

Synthetic peptides can interfere with protein-protein interactions by either blocking the interaction or mimicking the effect of one protein on another. The Gta subunit has GDP bound in its inactive state and then assumes a different "active" conformation when the GDP is exchanged for GTP. Only the active conformation of Gta is able to activate the effector, PDE. On the other hand, the Gtot peptides do not bind nucleotides and could potentially mimic either Gta-GTP (and Gta-GTPyS) or Gta-GDP. The Gto~ peptides could also potentially compete with Gta-GTP and block the interaction with effector. Thus, we have tested whether the syn the t ic Gtot peptides could (1) compete with Gtoz-GTP3JS and block Gta-GTPTS stimulation of PDE activity; (2) mimic Gta-GTPyS and directly activate basal PDE; or (3) mimic Gta-GDP and inhibit activated PDE.

E x a m i n i n g Func t iona l Effects of Gta Pep t ides on Phosphod ies t e ra se Act iv i ty

Purification of Rod Outer Segments, Gta-GTPTS, and Phosphodiesterase

Bovine ROS membranes are isolated using sucrose density gradients according to the procedure of Papermaster and Dreyer 19 with modifications, z° The Gta-GTPyS complex is extracted from ROS membranes using

~9 D. S. Papermaster and W. J. Dreyer, Biochemistry 13, 2438 (1974). 2o M. R. Mazzoni, J. A. Malinski, and H. E. Hamm, J. Biol. Chem. 266, 14072 (1991).

12] PEPTIDE PROBES FOR G PROTEINS AND EFFECTORS 19

GTPTS and purified by chromatography on Blue Sepharose CL-6B 2°'2~ (Pharmacia LKB Biotechnology Inc., Piscataway, N J). The PDE holoen- zyme (Pa/3~/) is extracted by hypotonic washes of bleached ROS. 22 The PDE "extract" is 80% pure, as determined by densitometric scanning of Coomassie blue-stained sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gels. Pure PDE is also obtained as described in Artemyev and Harem. 23 As there is no difference between the functional effects observed using the PDE extract and purified PDE, PDE extract is used in all experiments discussed in this chapter.

Phosphodiesterase Assay

The PDE activity is measured by recording the rate of proton release using a pH microelectrode 24 (Microelectrodes, Inc., Londonderry, NH). Each assay consists of adding various components (purified proteins, peptides, cGMP) to a small vial containing buffer A to a final volume of 200/zl while stirring with a magnetic stirrer. At pH 8.0, the hydrolysis of cGMP yields 1 tool H ÷ per mole cGMP and can be quantified by back- titrating the suspension with known amounts of NaOH.

Inhibition of @~-GTPyS-Stimulated Phosphodiesterase Activity

Each peptide is tested for the ability to inhibit Gta-GTP3,S-activated PDE. Purified Gto~-GTP3,S (1 ~M) is added to isotonic buffer A containing purified PDE (10 nM), and peptide at varying concentrations (Fig. 2A) is then added. The reaction is initiated by the addition of cGMP (4 mM) and the PDE activity measured. We found that one Gtc~ peptide, 53-65, causes an inhibition of Gt~-GTP3,S-stimulated PDE activity (60% inhibition by 1 mM peptide) (Fig. 2A); the IC50 is equal to 60 ktM. 6 The effect is specific since all other peptides had no significant effect.

Inhibition of Trypsinized Phosphodiesterase Activity

It is unclear whether the inhibitory effect of peptide 53-65 on PDE activity is due to blocking activation of PDE by Gtc~-GTPyS or occurs directly on activated PDE. We tested whether peptide 53-65 and the other peptides could inhibit trypsinized PDE (tPDE). Trypsin degrades the inhibitory y subunit of PDE (leaving only the catalytic Pa/3 subunits)

21 C. Kleuss, M. Pallest, S. Brendel, W. Rosenthal, and W. Schultz, J. Chromatogr. 19, 281 (1987).

22 W. Baehr, M. J. Devlin, and M. L. Applebury, J. Biol. Chem. 254, 11669 (1979). z3 N. O. Artemyev and H. E. Hamm, Biochem. J. 283, 273 (1992). 24 p. A. Liebman and A. T. Evanczuk, this series, Vol. 81, p. 532.


A

, ,- , . 1 0 0 ~

@ :.7,

8 0 ~ f~

6 0 g o

~" 4 0 5

2 0 ~

OTHER PEPTIDES

~ 3 - 6 5

0.001 0,01 0.1 1 10

PEPTIDE CONCENTRATION (mM)

FIG. 2. (A) Effects of Gtc~ peptides on Gt~-GTPTS-stimulated PDE activity. A value of 100% PDE activity represents the Gta-GTPTS-stimulated PDE activity in the absence of peptide and equals 7.4/~mol cGMP hydrolyzed/sec/mg PDE. "Other peptides" refers to peptides listed in Table I (except peptide 53-65). (B) Effects of Gtc~ peptides on trypsin- stimulated PDE activity. A value of 100% tPDE represents the maximum PDE activity after trypsin treatment in the absence of peptide and equals 19.8/zmol cGMP hydrolyzed/sec/ mg PDE. "Other peptides" refers to the peptides listed in Table I (except peptides 53-65 and 201-215).

and causes full activation of the enzyme. PDE is trypsinized to give optimal activation by treating PDE extract with trypsin (10: 1, w/w) for 30 min at 4 °. The proteolysis is stopped by the addition of a 5-fold molar excess of soybean trypsin inhibitor. Trypsin pretreated with trypsin inhibitor has no effect on basal PDE activity. Trypsinized PDE (10 nM) and peptide at varying concentrations are added to isotonic buffer A. Addition of cGMP initiates the reaction, and PDE activity is measured.

Figure 2B shows that peptide 53-65 maximally inhibits tPDE activity by 80% at a final concentration of at least 1 mM and has an IC50 equal to 100/.tM. 6 All other peptides have no effect. It is interesting to note that peptide 189-210, which partially overlaps peptide 201-215, has no effect. The data reveal that Gtot peptides 53-65 and 201-215 have a direct effect on activated PDE, which consists of only the Pa/3 subunits (since P7 has


>

M

B

I00~

80%

60~

40~

20%

0% 0.001

OTHER PEPTIDES ~ J - A A

5 3 - 6 5

i . . . . . . i . . . . . . . , I . . . . . . . . i . . . . . . . .

0.01 0. I I 10

PEPTIDE CONCENTRATION (raM)

FIG. 2. (continued)

been proteolytically removed by trypsin). The fact that peptide 53-65 blocks tPDE with a similar potency as Gta-GTPyS-activated PDE suggests that the peptide does not block the ability of Gta-GTPTS to activate PDE (i.e., compete with Gta-GTPyS in activating PDE), but rather inhibits only active PDE itself. A likely interpretation is that Gta peptides 53-65 ane 201-215 are mimicking Gta in the GDP-bound form since Gta-GDP has been shown to inhibit tPDE. 5 Thus, regions 53-65 and 201-215 may interact with the catalytic aft subunits of PDE and play a role in the turning off of PDE activity rather than the activation of PDE.

Direct Activation of Phosphodiesterase

The next type of experiment examines whether any of the Gte¢ peptides can mimic Gta-GTPyS and directly activate PDE. The Gta-GTPyS complex (10/xM) is added to PDE (100 nM) in isotonic buffer A followed by the addition of cGMP (4 mM) and measurement of PDE activity (Fig. 3A). We found that peptide 293-314 and a truncated version of that peptide, peptide 300-314 (not shown), activate basal PDE whereas all other peptides have no effect. 6 Figure 3A shows that at a final concentration of 100/~M, peptide 293-314 activates PDE to the same extent as 10 ~M of purified Gtc~-GTP7S. Thus, the peptide 293-314 is approximately one

o E

L.)

e'.,, ,O e,,.., eL

+

A

N

,.4

B

200

150

100

50

G~

BASAL PDE

I ]

- v t ) i

I 2 3

Time (rain)

t) .¢

t~

50

40

30

20

10

A

/ / /-

293-314

300-314 , - , & , , , A

t OTHER PEPTIDES ~. z • • •

0 I I l t 0 100 2 0 0 300 4 0 0

PEPTIDE CONCENTRATION (/~M)

FIG. 3. (A) Effects of Gtc~-GTPyS and peptide 293-314 on the basal activity ofPDE (G~ stands for Gta-GTP~,S). The trace shows PDE activity (moles H ÷ produced equals moles cGMP hydrolyzed) over time. (B) Effects of increasing concentrations of Gta peptides on basal PDE activity. The PDE activity is expressed as a percentage of maximal trypsinized PDE (10 nM) activity, which equals 19.4 ttmol cGMP hydrolyzed/sec/mg PDE. "Other peptides" refers to the peptides listed in Table I except for peptides 293-314 and 300-314.


order of magnitude less potent than Gto~-GTPyS in stimulating PDE. The maximal activity of PDE stimulated by peptide 293-314 is approximately 40% of trypsinized PDE activity, similar to the effect of Gta-GTP3,S, which is also 40% of trypsinized PDE activity. 6

Dose-response curves of activation of PDE by peptides 293-314 and 300-314 are generated (Fig. 3B). Peptide at varying concentrations is added to PDE (10 nM), cGMP (4 raM) is added, and PDE activity is measured. The activation constants (Ka) for peptides 293-314 and 300-314 are determined to be 8.3 and 40/xM, respectively. 6 In comparison, the K a for Gta-GTPyS stimulation of PDE in isotonic solution is approximately 1-2/xM. 6 There is no difference in the effect of peptide 293-314 on PDE activity in the presence of urea-stripped membranes compared to that in isotonic buffer solution (data not shown). The sequence of peptide 293-314, shown in Table I, contains five negative and four positive charges, with a net charge of - 1. The effect of peptide 293-314 on PDE activation appears to be specific since all other peptides tested, including those with similar charge characteristics, do not activate PDE. The peptide 293-314 has also been shown to mimic transducin in its ability to activate PDE by reversing inhibition by the inhibitory subunit (p~/).6 In addition, the peptide also decreases the affinity of Py for the Pa/3 subunits of PDE. 6

Use of Fluorescence to Detect Peptide Binding to Phosphodiesterase Inhibitory Subunit

A l t h o u g h Gto~ peptide 293-314 activates PDE by mimicking G t a - GTP3,S, it is important to determine to which subunit of PDE the peptide binds. A fusion protein of the PDE inhibitory subunit, PT, is fluorescently labeled. Using a fluorescent assay, we have tested which peptides could bind to the labeled fusion Py subunit.

Fusion Protein of Phosphodiesterase Inhibitory Subunit

A fusion protein of P~/(fP3') consisting of the first 31 residues of hcII protein, the clotting-proteinase (factor Xa) cleavage site (7 residues), and the P7 sequence (87 residues) is expressed in Escherichia coli and purified as described by Brown and Stryer. 25 The fP~/behaves functionally the same as native P~/, that is, it inhibits trypsinized PDE.

25 R. L. Brown and L. Stryer, Proc. Natl. Acad. Sci. U.S.A. 86, 4922 (1989).

24 cGMP PHOSPHODIESTERASE [2l

Preparation of Fluorescently Labeled Phosphodiesterase Inhibitory Subunit

The fPy has a single cysteine residue, Cys-68, which allows introduction of a fluorescent probe in the protein. The fPy is labeled at Cys-68 with the fluorescent reagent Lucifer Yellow vinyl sulfone [4-amino-N-(3- vinylsulfonylphenyl)naphthalmide 3,6-disulfonate] (LY). 26 When Lucifer Yellow vinyl sulfone is excited at a wavelength of 430 nm, it has a characteristic emission spectrum (Fig. 4A, dashed lines), with a maximum fluorescence (hmax) at 520 nm. If another molecule interacts with a site close to the fluorescent LY probe, the fluorescence will increase if the environment around the probe becomes less polar. 27 Lucifer Yellow-labeled fPy (fPyLY) is prepared by the following procedure: fPy (1 mg/ml) in buffer B (50 mM Tris-HC1, pH 8.0, 50 mM NaCI, 1 mM EDTA, and 1 mM DTT) is put through Sephadex G-25 to remove dithiothreitol. A 3-fold molar excess of Lucifer Yellow vinyl sulfone (LY) is added, and the mixture is incubated at 4 ° for 15 rain. The fPyLY is purified by reversed-phase HPLC on a C4 (25 x 0.46 cm) column (Vydac; Rainin Instruments, Woburn, MA) with a 20-100% gradient of acetonitrile in 0.1% trifluoroacetic acid.

Fluorescent Assay

Fluorescent assays are carried out with a Perkin-Elmer (Norwalk, CT) LS5B spectrofluorometer in buffer C (10 mM HEPES, pH 8.0, I00 mM NaCI, and 1 mM MgCI2) with excitation at 430 nm and emission at 520 nm. The concentrations of the Lucifer Yellow-labeled proteins are determined (using 8430 = 12,400 and e2s0 = 24,000 for Lucifer Yellow, 26 and e2s 0 = 6700 for Py). Using these extinction coefficients, the molar ratio of lucifer yellow to Py is about 0.7 mol/mol.

Peptide 293-314 Binding to Phosphodiesterase Inhibitory Subunit

Peptide 293-314 (10/zM) increases the fluorescence of fPyLY (100 nM) 2- to 3-fold (Fig. 4A). 7 All the other peptides have no effect on fPyLY fluorescence. Purified Gta-GTPyS (175 nM) also increases the fluorescence intensity of fPyLY (100 nM) 2- to 3-fold (Fig. 4A). A dose-response curve for the effect of peptide 293-314 on fPyLY fluorescence is shown in Fig. 4B. A Kl/z of approximately 1.2 t~M for the peptide 293-314-fPyLY interaction is calculated from the data using curve-fitting and plotting software (Graph Pad 4). The Kd for the Gta-GTPyS-fPyLY interaction is found to be 36 nM. 7 The Gta-GDP complex causes no

26 W. W. Stewart, J. Am. Chem. Soc. 103, 7615 (1981). 27 D. Freifelder, in "Physical Biochemistry" (D. Freifelder, ed.), p. 415. Freeman, San

Francisco, 1976.

[2l PEPTIDE PROBES FOR G PROTEINS AND EFFECTORS 25

A

i,I f_D Z LO 0 Oq W rl- 0

Lt_

Ld >

-J W

8C

6( 40 ~ 3 - 314

,:',, \ I '~,~\~

/J , , ,"N_, 4B0 ~2o s6o 600 640

80

60

480 520 560 600 640

WAVELENGTH (nm) WAVELENGTH (nm)

B 2.5

0 I, I,

2.0

1.5

1,0 -7 .0 - 6 . 5 - 6 . 0 - 5 . 5 - 5 . 0 - 4 . 5

LOG [ 2 9 3 - 3 1 4 - G a ] M

FIG. 4. (A) Binding of peptide 293-314 and Gt~x-GTPTS to fP7LY. The emission spectra of fPTLY (100 nM) alone ( - - - - ) and in the presence of 10/zM peptide 293-314 or 175 nM Gta-GTP3,S (--) were obtained by exciting the sample at 430 nm. (B) Interaction of peptide 293-314 with fPTLY. Peptide 293-314 binding to fPTLY was estimated by the relative increase in fluorescence (F/F o) after recording the fluorescence of fPTLY (25 nM) (excitation at 430 nm, emission at 520 nm) in the presence of increasing concentrations of peptide 293-314 (F0, fluorescence of fPTLY alone, F, fluorescence of fPyLY plus peptide 293-314). The binding curve fit a Ku2 value of 1.2/zM, and the maximum FIFo ratio was 2.2.

change in fluorescence unless aluminum fluoride is added (AIF4- causes the conformation of Gta to change to an active state that can activate PDE). 7

Peptide 293-314 Binding within Phosphodiesterase Inhibitory Subunit Residues 46-87, but Not within Residues 24-46

A fragment of PyLY that corresponds to residues 46-87 (PT[46-87]LY) is generated by trypsinization of fPTLY as follows: fPyLY (1 mg/ml) is


P ~ [ 4 6 - 8 7 ] L Y 3.5

3,0

2.5

2.0

1.5

1.0 -7 .0

0 I,

b..

I I I I I

-6 .5 -6 .0 -5 .5 -5 ,0 -4 .5 -4 .0

LOG [293-314-Gta ] M

FIG. 5. Binding of peptide 293-314 to Py[46-87]LY. The relative increase of fluorescence (F/Fo) of PT[46-87]LY (25 nM) was determined after addition of increasing concentrations of peptide 293-314. The major characteristics calculated from the curve of peptide 293-314 binding to PT[46-87]LY were as follows: Kv2 = 1.7 IxM and F/Fo = 3.3.

incubated with trypsin (50/~g/ml) for 5 h at 37 ° and then purified twice by reversed-phase HPLC on a C4 (25 × 0.46 cm) column (Vydac). Peptide 293-314 increases the fluorescence of Py[46-87]LY (Fig. 5), as does Gta-GTPTS. 7 The fluorescence increase caused by peptide 293-314 is slightly higher for Py[46-87]LY than for fPyLY; however, the Kv2 value (1.7 #M) is found to be about the same. This result suggests that region 293-314 on Gta and region 46-87 on PT form a distinct interacting pair. Because the K~/2 values for peptide 293-314 interaction with both fPTLY and Py[46-87]LY are about the same, it appears that other regions on Gta-GTPyS do not interact within the region 46-87 on PT. However, this does not rule out the possibility that there are other regions (besides region 293-314) on Gta that interact with other regions (besides region 46-87) on Py.

A peptide corresponding to residues 24-46 of PT is able to block the increase in fluorescence of fPyLY caused by Gtc~-GTPTS, that is, the peptide blocks the binding between fPy and Gta-GTPyS.7 However, the P7 peptide 24-46 is not able to block the binding between Gta peptide 293-314 and fPyLY. 7 These data suggest that there is another site on Gta-GTPyS that binds to PT within the region 24-46 on Py. To determine where the second PT binding site on Gta-GTPyS is, several Gta peptides have been tested for the ability to block the direct interaction between Gtc~-GTPyS and fPyLY using the fluorescence assay. For a list of the


B

FIG. 6. Model of sites of interaction between Gta and PDE. (A) Sites of interaction between Gta-GTP and P3' involved in PDE activation. Pa~, Catalytic subunits of PDE; 3', inhibitory subunit of PDE (only one of the two P3' subunits is shown for clarity); Ga-GTP, GTP-bound form of the a subunit of transducin; ?, unknown site on Ga-GTP that interacts within pv residues 24-46. (B) Putative sites on Ga-GDP that inhibit active PDE. Pail*, Trypsinized PDE; Ga-GDP, GDP-bound form of the a subunit of transducin.

soluble Gta peptides tested, see Table I. It is found that none of the peptides (up to 150 tzM) block Gta-GTPyS-P3'LY interaction. 7 Most likely, these regions o n G t a do not correspond to PDE 3' binding sites.

Thus, it appears that region 293-314 on Gta-GTP3'S binds to a region on P3' within residues 46-87. However, there is another unknown site on Gta-GTP3'S that binds to a region on P3' that is within residues 24-46.

Conclusion

Synthetic peptides have proved to be powerful probes for studying protein-protein interactions involved in visual transduction. The use of synthetic peptides has elucidated the location of PDE binding domains on the a subunit of transducin as well as sites of G t a interaction on the PDE inhibitory subunit. A model of sites of interaction between Gta and PDE obtained from the results presented above is shown in Fig. 6. In

28 cGMP PHOSPI4ODIESTERASE [2]

summary, amino acids residues 293-314 o n Gta are involved in Py binding (within residues 46-87) and activation of PDE (Fig. 6A). Another unidentified region on Gt a appears to be involved in binding to Py (within residues 24-46 on Py) and is also involved in PDE activation (Fig. 6A). In addition, residues 53-65 and 201-215 on Gta interact with the catalytic Pa/3 subunits and most likely are involved in regulating PDE activity (Fig. 6B).

[3] A S S A Y O F A D E N Y L Y L C Y C L A S E C A T A L Y T I C A C T I V I T Y 31

[3] D e t e r m i n a t i o n of A d e n y l y l Cyclase Cata ly t ic Act iv i ty Us ing Single and D o u b l e C o l u m n P r o c e d u r e s

By ROGER A. JOHNSON, ROBERT ALVAREZ, and YORAM SALOMON

Adenylyl cyclase (ATP-pyrophosphate lyase, cyclizing, EC 4.6.1.1, adenylate cyclase) is a family of membrane-bound enzymes that exhibit inactive and active configurations resulting from the actions of a variety of agents, acting indirectly and directly on the enzyme. The nature of some of these agents and the range of resulting activities will influence the assay conditions used for determining the catalytic activity of the enzyme. Enzyme activity may be increased or decreased by stimulatory or inhibitory hormones/neurotransmitters acting via specific hormone receptors coupled to the catalytic unit of the enzyme by the respective guanine nucleotide-dependent regulatory proteins (Gs and G~z, respectively). The G proteins are also activated by aluminum fluoride and are targets for ADP-ribosylation by specific bacterial toxins. The catalytic moiety of adenylyl cyclase from most tissues is stimulated by the diterpene forskolin and is inhibited by adenosine 3'-phosphates, and some forms of the enzyme are also stimulated directly by Ca2+/cal - modulin.

The catalytic activity of adenylyl cyclase is determined by methods that either rely on the measurement of cAMP formed from unlabeled substrate, with cAMP binding proteins or radioimmunoassay procedures, or rely on radioactively labeled substrate followed by isolation and determination of the radioactively labeled product. The two different approaches have different purposes, different sensitivities, and different ease of use. The method of choice will depend in part on the facilities and orientation of a given laboratory. The procedures described here focus on the use of radioactively labeled substrate and isolation of the labeled product with increased emphasis on the single-column isolation procedure. Additional detailed considerations for the assay of adenylyl cyclase by these procedures can be found in the review by Salomon I and that presented earlier in this series by Johnson and Sa- lomon?

I y . Salomon, Adv. Cyclic Nucleotide Res. 10, 35 (1979). z R. A. Johnson and Y. Salomon, this series, Vol. 195, p. 3.


32 ADENYLYL CYCLASES [3]

A. Considerations for Establishing Reaction Conditions

1. Requirements for Metal-ATP and Divalent Cations

Both ATP and divalent cations (Mg 2+ or Mn 2+) are required for adenylyl cyclase-catalyzed formation of cAMP. 3,4 The enzyme conforms to a bireac- tant sequential mechanism in which metal-ATP z- is substrate and free divalent cation is a requisite cofactor. 5 The concentrations of both substrates (metal-ATP 2- and excess cation) may be varied for determining kinetic constants. However, because of the association constants for divalent cation and ATP 2- [ATP • Mg (65,000/M); ATP • Mn (353,000/M)], 6 the concentration of free Mg 2+ or Mn 2+ must be fixed at a concentration above the total ATP concentration. 5'7 This will maintain divalent cation concentrations essentially constant even though ATP concentrations are being varied. Buffers, such as Tris-Cl, that can significantly affect concentrations of free divalent cation (especially Mn 2+) in the reaction mixture should be avoided. Triethanolamine hydrochloride does not have this problem. 5 Examples of Km values for adenylyl cyclases have been reported as follows: detergent-dispersed enzyme from rat brain, Km(MnATP) - 7 - 9 /zM, Km(Mn2+ ) ~2-3/zM, Km(MgATP) ~30-60/zM, Km(Mg2+ ) ~800-900 ~MS; human platelets, Km(Mg2+ ) -1100 /zM and gm(MgATP) ~50 /xMS; liver, Km~UnATP) and Km~r~gATP) similar ( -50 p,M)9; $49 murine lymphoma cyc- cells, K~(UnATP) and Km(MgATPI similar (-- 100-200/xM), with apparent KmCMg) - 2 - 6 mM and Krn(Mn) -0.3-0.7 mM~°; resolved catalyatic unit from cau- date nucleus, Krn(MnATP ) and Kr~(t~gATP) also similar (-110-140/xM). 11 No kinetic constants have been reported for stably expressed forms of recombinant adenylyl cyclases.

2. Contaminating Enzyme Activities

Adenylyl cyclase is a membrane-bound protein of very low abundance and exists in an environment rich in contaminating enzyme activities

3 T. W. Rail and E. W. Sutherland, J. Biol. Chem. 232, 1065 (1958). 4 E. W. Sutherland, T. W. Rail, and T. Menon, J. Biol. Chem. 237, 1220 (1962). 5 D. L. Garbers and R. A. Johnson, J. Biol. Chem. 250, 8449 (1975). 6 D. L. Garbers, E. L. Dyer, and J. G. Hardman, J. Biol. Chem. 250, 382 (1975). 7 W. W. Cleland, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 2, p. 1. Academic

Press, New York, 1970. 8 R. A. Johnson, W. Saur, and K. H. Jakobs, J. Biol. Chem. 254, 1094 (1979). 9 C. Londos and M. S. Preston, J. Biol. Chem. 252, 5957 (1977). ~0 S. G. Somkuti, J. D. Hildebrandt, J. T. Herberg, and R. Iyengar, J. Biol. Chem. 257,

6387 (1982). 11 j. Bender and E. J. Neer, J. Biol. Chem. 258, 2432 (1983).

[3l ASSAY OF ADENYLYL CYCLASE CATALYTIC ACTIVITY 33

(Fig. 1), including a number of nucleotide phosphohydrolases (steps 1, 2, 3, and 6 in Fig. 1), cyclic nucleotide phosphodiesterases (step 9 in Fig. 1), and ATP-utilizing kinases (step I0 in Fig. I). Thus, adenylyl cyclase in membrane preparations competes with other enzymes for ATP, and the cAMP formed is readily hydrolyzed to 5'-AMP. Analogously, GTP, required for hormone-induced activation or inhibition of adenylyl cyclases, is also metabolized. Consequently, the use of regenerating systems to counteract this alternative metabolism of ATP (steps 7 and 8 in Fig. 1) and/or GTP and the use of inhibitors of cAMP phosphodiesterases are nearly unavoidable. These inclusions are particularly important if the single-column chromatography system (see below) is used.

a. Cyclic Nucleotide Phosphodiesterases. cAMP is effectively inactivated through the hydrolysis of its 3'-phosphate bond, yielding 5'-AMP (step 9 in Fig. 1). Because cyclic nucleotide phosphodiesterase activity is substantial in most membrane preparations, these enzymes must be inhibited to measure accurately the rate of formation of cAMP by adenylyl cyclase. This is usually accomplished by the use of unlabeled cAMP in the reaction mixture or use of inhibitors of the enzyme, such as papaverine or alkylxanthines (e.g., 3-isobutyl-l-methylxanthine; IBMX). The inhibitor IBMX also potently blocks adenosine A1 and Az receptors and thereby may be additionally useful. One must be judicious in the selection of a phosphodiesterase inhibitor, however, in that some agents do not block all cAMP phosphodiesterases. For example, the sole use of the imidazoli- dinone derivative Ro-20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazoli- dinone; Hoffmann-LaRoche, Nutley, NJ] is not recommended. The compound has been useful in the study of adenosine receptor-mediated effects on adenylyl cyclases because it is not an adenosine receptor anatagonist as are the alkylxanthines. Although it may substantially suppress the hydrolysis of cAMP in preparations from some tissues (e.g., pig coronary arteries), it does so incompletely in others (e.g., platelets), requiring the

x

P

MgATP ,

N. ~ A M P

Ino

X adenylyl c_yclase MgPPi Tj cAMP

FIG. 1. Relation of adenylyl cyclase to other membrane-bound enzyme activities.


addition of a second phosphodiesterase inhibitor. This is because Ro-20- 1724 is known to inhibit selectively the type IV cyclic nucleotide phosphodiesterase ~2 and tissues typically contain more than this one isoform of the enzyme. The most effective agents for inhibition of hydrolysis of labeled cAMP produced by adenylyl cyclase are the broadly acting 1 mM IBMX (though its use precludes studies of adenosine receptor-mediated responses) and unlabeled cAMP (100/zM), which also can be hydrolyzed to adenosine and can thereby indirectly and directly affect adenylyl cyclase determinations. In our hands other effective combinations of agents have included the following: cAMP (I00/zM) plus papaverine (100 ~M) and 100/xM anagrelide [6,7-dichloro-l,5-dihydroimidazo[2,1-b]quinazolinone monohydrochloride (BL-4162A)] plus Ro-20-1724 (500/zM). 13 The caveat in the use of these agents is that papaverine is selective for the type I phosphodiesterase and anagrelide is selective for the type III enzyme. ~4

b. ATP-Regenerating Systems. The accurate determination of adenylyl cyclase activities is adversely affected by the metabolism of ATP. Cleavage between the/3- and y-phosphates (step 1 in Fig. 1) occurs with various membrane-bound ATPases, nonspecific phosphohydrolases, and flux through membrane-bound kinases (step 10 in Fig. 1) and phosphatases. Cleavage beween the a- and/3-phosphates occurs for ADP (step 2 in Fig. 1) by membrane phosphohydrolases and for ATP by nucleotide pyrophosphatase (step 6 in Fig. 1). Whether by step 2 or by step 6, the result is 5'-AMP, which is rapidly hydrolyzed by 5'-nucleotidase (step 3 in Fig. 1) to adenosine (Ado). Adenosine can stimulate or inhibit (step 5 in Fig. 1) adenylyl cyclase, indirectly via inhibitory (A~) or stimulatory (A2) receptors, or it may inhibit the enzyme directly via the "P" site, through which adenosine 3'-phosphates inhibit. To counteract and minimize the influence of these reactions enzymes are added to the reaction mixture that constitute an ATP-regenerating system and remove adenosine. The most common ATP-regenerating systems have used creatine kinase and creatine phosphate or pyruvate kinase and phosphoenolpyruvate to catalyze the rephosphorylation of ADP to ATP (step 7 in Fig. 1). Rephosphorylation of 5'-AMP to ADP is catalyzed by the additional inclusion of adenylate kinase (myokinase) (step 8 in Fig. 1). Because the action of 5'-nucleotidase can be reversed only through the use of a 5'-nucleoside kinase, the influence of the formed adenosine is best minimized by the use of adenosine deaminase (step 4 in Fig. 1), as the product inosine (Ino) is without effect

J2 C. D. Nicholson, R. A. J. Challiss, and M. Shahid, Trends Pharmacol. Sci. 12, 19 (1991). 13 E. A. Martinson and R. A. Johnson, unpublished observations (1986). 14 R. Alvarez, unpublished observations (1993).

[3] ASSAY OF ADENYLYL CYCLASE CATALYTIC ACTIVITY 35

on adenylyl cyclase. The effects of these additions to enhance apparent adenylyl cyclase activity were reported earlier ~5 and in this series)

The influence of nucleotide pyrophosphatase and to a lesser extent 5'- nucleotidase can be further minimized by pretreatment of membranes with 5 mM EDTA and 3 mM dithiothreitol. ~5'~6 This occurs presumably through the removal of bound zinc cation. The necessity for the additions to the assay or the effectiveness of the membrane pretreatment with chelator and/or dithiothreitol depends on the source and purity of the adenylyl cyclase being studied,

Although both creatine kinase and creatine phosphate or pyruvate kinase and phosphoenolpyruvate have been utilized as the basis of ATP- regenerating systems, neither is without its pitfalls. Both enzymes bind and utilize adenosine phosphates, and both creatine phosphate and phosphoenolpyruvate form weak complexes with divalent cations. These effects could alter concentrations of free nucleotide or cation. Moreover, phosphoenolpyruvate has been shown to cause both stimulatory and inhibitory effects on the enzyme from liver and to inhibit the enzyme from heart,~7 and contaminants in creatine phosphate have been found to cause both stimulatory and inhibitory effects on adenylyl cyclases.15 For these reasons the preferable ATP-regenerating system is creatine kinase and creatine phosphate. Creatine phosphate should be used at concentrations low enough (e.g., 2 mM) to minimize the influence of the contaminants, but high enough to allow ATP concentrations to be maintained for the duration of the incubation. If higher concentrations (e.g., 10 mM) are found to be necessary for the linear formation of cAMP with time, it may be necessary to purify creatine phosphate before use, for example, by anion-exchange chromatography. ~5

Reagent Enzymes

Creatine kinase: Boehringer/Mannheim (Indianapolis, IN) from rabbit muscle (100 ~g/ml final concentration in the assay mixture)

Myokinase: Boehringer/Mannheim, ammonium sulfate suspension from rabbit muscle (100 /zg/ml final concentration in the assay mixture)

Adenosine deaminase: Sigma (St. Louis, MO), ammonium sulfate suspension, type VIII from calf intestinal mucosa (5 U/ml final concentration in the assay mixture)

t5 R. A. Johnson, J. Biol. Chem. 255, 8252 (1980), ~6 R. A. Johnson and J. Welden, Arch. Biochem. Biophys. 183, 2176 (1977). 17 R. A. Johnson and E. L. Garbers, in "Receptors and Hormone Action" (B. W. O'Malley

and L. Birnbaumer, eds.), Vol. 1, p. 549. Academic Press, New York, 1977.


3. Enzyme Concentrations, Reaction Times, and Temperatures

Three additional factors that obviously influence adenylyl cyclase- catalyzed formation of cAMP interdependently are enzyme concentration, time, and incubation temperature.

a. Enzyme Concentration. Adenylyl cyclase is substantially more active in crude membrane preparations from some tissues, especially in stable expression systems of recombinant enzyme, than from others, and the levels of the various contaminating enzymes that utilize adenine nucleotides (Fig. 1) also vary. Consequently, under any given set of reaction conditions it is imperative to establish (i) that sufficient enzyme is used to catalyze the formation of measurable amounts of [3Zp]cAMP or [3H]cAMP; (ii) that concentrations of both ATP and creatine phosphate are adequate for sustaining stable ATP concentrations; and (iii) that formation of cAMP is linear with respect to enzyme concentration. Formation of measurable amounts of [3ZP]cAMP or [3H]cAMP is improved with increasing specific radioactivity of labeled ATP and increasing enzyme concentration. However, increasing concentrations of crude membrane preparations of adenylyl cyclase typically also require increased amounts of creatine phosphate or proportionally decreased incubations times. Hence, enzyme concentation, ATP specific radioactivity, and incubations times must be adjusted to allow linear formation of cAMP with respect to both protein concentration and time. Useful ranges of specific radioactivity of [a-32p]ATP are 10 to 200 cpm/pmol, depending principally on the enzyme source. Obviously, for studies that vary the metal-ATP concentration the range of [a-32p]ATP specific activity will be much greater than this. Examples of, but not necessarily upper limits for, protein concentrations yielding linear product formation at 30 ° with 0.1 mM ATP and 5 mM creatine phosphate, 100/zg creatine kinase/ml, 100/zg myokinase/ml, 5 U adenosine deaminase/ml, and 10 mM MnC12 or MgCI2 would be as follows (in mg/ml): heart, 0.6; liver, 1.2; kidney, 0.7; skeletal muscle, 0.3; adipocytes, 0.2; spleen, 0.5; human platelets, 0.2; bovine sperm particles, 1.0; washed particles from brain, 0.2; detergent-solubilized brain, 0.2.

b. Time. The incubation time for adenylyl cyclase reactions is dictated by a balance between rates of formation of cAMP from ATP, hydrolysis of cAMP to 5'-AMP by contaminating cyclic nucleotide phosphodiesterases, hydrolysis of ATP by a number of membrane-bound phosphohydrolytic enzymes (Fig. 1), inactivation of adenylyl cyclase by regulatory components, and denaturation of the enzyme. It is essential that linearity of product formation with respect to time be established. The reaction is typically linear with respect to time for crude membrane preparations,


with the conditions given above (Section A.3.a), for 2 to 15 rain and for purified enzyme for 60 rain.

c. Incubation Temperature. Temperature can be used to advantage to promote certain characteristic behaviors of adenylyl cyclases as well as to modify rates of alternative substrate utilization and enzyme denaturation. Formation of cAMP is linear with respect to time for a longer period at 30 ° than at a more physiological 37 °. However, in the absence of protective agents the catalytic moiety is readily inactivated by exposure to heat for short periods of time. For example, exposure of adenylyl cyclases from platelets and from $49 lymphoma wild-type and cyc- cells for 8 min at 35 ° causes 70 to 75% inactivation. 18.19 Comparable inactivation of adenylyl cyclases from bovine sperm and detergent-dispersed porcine brain occurs by exposure at 45 ° for 8 and 4 rain, respectively. 18 Partial protection against thermal inactivation is afforded by forskolin (200 ~M), metal-ATP (below millimolar), the "P"-site agonist 2',5'-dideoxyadenosine (250 tzM), guanine nucleotides (/zM), and, for the Ca2+/calmodulin-sensitive form of adenylyl cyclase, by CaZ+/calmodulin (50 I~M/5/.~M) 18-2~ In addition, adenylyl cyclase reactions conducted at different temperatures can be used to enhance selective regulatory properties of the enzyme. For example, inhibition of adenylyl cyclase mediated by guanine nucleotide-dependent regulatory protein (Gi), whether by hormone or stable guanine nucleotide, is more readily shown experimentally at lower temperatures (e.g., 24°), whereas activation, mediated by the stimulatory G protein (Gs), is evident at higher temperatures (e.g., 300). 23

4. Guanine Nucleotides

GTP is required for G s- and Giz-mediated activation and inhibition of adenylyl cyclases. 24-28 The more stable GTP analogs guanosine 5'- (/3,~/-imino)triphosphate [GPP(NH)P] and guanosine 5'-O-(3-thiotriphos-

t8 j. A. Awad, R. A. Johnson, K. H. Jakobs, and G. Schultz, J. Biol. Chem. 258, 2960 (1983). t9 V. A. Florio, and E. M. Ross, Mol. Pharmacol. 24, 195 (1983). z0 M. A, Brostrom, C. O. Brostrom, and D. J. Wolff, Arch. Biochem. Biophys. 191, 341 (1978). 21 R. S. Salter, M. H. Krinks, C. B. Klee, and E. J. Neer, J. Biol. Chem. 256, 9830 (1981). 22 j. p. Harwood, H. L6w, and M. Rodbell, J. Biol. Chem. 248, 6239 (1973). 23 D. M. F. Cooper and C. Londos, J. Cyclic Nucleotide Res. 5(4), 289 (1979). 24 M. Rodbell, L. Birnbaumer, S. L, Pohl, andH. M. J. Krans, J. Biol. Chem. 246, 1877 (1971). :5 K. H. Jakobs, W. Saur, and G. Schultz, FEBS Lett. 85, 167 (1978). 26 C. Londos, D. M. F. Cooper, W. Schlegel, and M. Rodbell, Proc. Natl. Acad. Sci. U.S.A.

75, 5362 (1978). 27 D. M. F. Cooper, W. Schlegel, M. C, Lin, and M. Rodbell, J. Biol. Chem. 254, 8927 (1979). z8 E. Perez-Reyes and D. M. F. Cooper, J. Neurochem. 46, 1508 (1986).


phate) (GTPyS) can substitute for GTP. Effects of these analogs are evident after a distinct lag phase, and preincubation of the enzyme with them will result in persistently activated or inhibited enzyme, depending on incubation conditions. In addition, the effectiveness of Gs and G~2 to regulate adenylyl cyclase is further influenced by divalent cation (type and concentration) and by membrane perturbants (e.g., Mn 2+ and detergents obliterate G~z-mediated inhibition). Half-maximal stimulation of adenylyl cyclase in the presence of hormones is usually observed with 10 to 50 nM GTP, or with 50 to 100 nM GPP(NH)P or GTPyS. Maximum stimulation occurs with at least 1 to 10/zM GTP, GPP(NH)P, or GTPyS. Half-maximal inhibition by hormones occurs with 100 to 500 nM GTP, 10 to 100 nM GPP(NH)P, or 1 to I0 nM GTPyS. Maximum inhibition occurs with GTP~S above 100 nM, GPP(NH)P above 10 nM, and GTP above 1 ~M.

Consequently, even in relatively pure membrane preparations enzyme activity may be increased somewhat by stimulatory hormones owing to endogenous GTP (e.g., present in membranes or as contaminant of ATP; GTP-free ATP can be prepared chromatographically or can be purchased from Sigma). The addition of GTP enhances stimulation further. By comparison, GTP must be added to demonstrate hormonal inhibition of adenylyl cyclase, and GTP-dependent inhibition is often best elicited with enzyme that has been stimulated by forskolin or a stimulatory hormone. In addition, the concentrations of guanine nucleotides necessary for regulation of adenylyl cyclase activity are dependent on the enzyme source and incubation temperature and are influenced by the relative activities and abundances of Gs and Gi2.

B. Radioactive Substrates: [aH]ATP versus [a-32P]ATP

The radiolabels [3H]ATP and [a-32p]ATP are commonly used as substrate for measuring adenylyl cyclase catalytic activity. The use of each has both advantages and disadvantages, some of which are described below.

1. flH]ATP Advantages

The main advantage to the use of [3H]ATP as labeled substrate is its long half-life (-12.3 years). This allows the nearly complete usage of purchased isotope without regard to loss through decay, thereby being cost effective per assay tube. Thus, low usage rates may adequately compensate for its being initially substantially more expensive than [a-3Zp]ATP. A second advantage is due to the low energy of tritium's /3 emission, obviating the use of cumbersome thick Lucite shielding that should be used with 32p-labeled compounds.

[3] ASSAY OF A D E N Y L Y L CYCLASE CATALYTIC ACTIVITY 39

2. [SH]ATP Disadvantages

There are several significant disadvantages to the use of [3H]ATP as substrate in adenylyl cyclase reactions. Tritium-labeled adenine nucleosides and nucleotides are chemically unstable in that the tritium at C-8 exchanges with water, especially under alkaline conditions. This results in a continuous loss of tritium to 3H20 that can occur at the rate of several percent per month. Consequently, for accurate estimations of substrate specific activity the 3H20 must be removed periodically, either chromatographically or by lyophilization. Both procedures necessitate undue handling of and exposure to moderate quantities of isotope and create the potential for major isotope spills in a laboratory environment.

The low energy from the/3 decay of tritium necessitates the use of scintillation cocktails to detect [3H]cAMP, and the long half-life of tritium means that large volumes of liquid radioactive waste, which necessarily also contains large quantities of organic solvents, must be disposed rather than be allowed to dissipate through radioactive decay as would be the case with 32p. Disposal of radioactive waste, especially mixed with a scintillation cocktail, is an expensive and undesirable consequence of the use of tritium-labeled substrate. The low energy of tritium decay also makes it more difficult to detect if there are inadvertant spills or contamination in a laboratory and could thereby lead to undue exposure of laboratory personnel to low-energy radiation.

Breakdown products of [3H]ATP or [3H]cAMP include various nucleotides and nucleosides, as well as xanthine, hypoxanthine, and others, that are also labeled. Chromatographic techniques for the separation of [3H]ATP and [3H]cAMP must take this into consideration. The breakdown products and the continuous formation of 3H20 from tritium-labeled adenine nucleotides contribute to blank values [counts per minute (cpm) for samples in the absence of enzyme] with the Dowex 50/A120 3 column system described below being substantially higher than those obtained with [a-32p]ATP as substrate. With adenylyl cyclases having low enzyme specific activity in crude membrane preparations such high blank values may constitute a substantial percentage of the [3H]cAMP formed enzymatically.

3. [a-32P]A TP Advantages

There are several important advantages to the use of [a-32p]ATP as substrate for adenylyl cyclase reactions. The specificity of labeling of [a-32P]ATP, which is dictated by the enzymatic means typically used for

40 ADENYLYL CYCLASES [3l

its synthesis, 29,3° means that only a-phosphates are labeled and, since the a-phosphate of ATP is not readily transferred to other compounds, that only purine nucleotides immediately derivable from ATP in membrane preparations will be labeled. Additional products that could result from contaminating activities in crude membrane preparations, for example, [a-32p]ADP, [a-3Zp]AMP, [a-32p]IMP, and [32p]Pi, a r e all readily separated from [32p]cAMP because of differences in ionic properties.

Phosphorus-32 is high-energy/3 emitter that allows detection by Ceren- kov radiation and obviates use of scintillation cocktails, that is, it can be detected in aqueous solutions with efficiencies approaching that of tritium in scintillation cocktails, but with little influence of agents that typically quench detection of tritium. The high energy of the/3 emission also allows easy detection of inadvertant spills with a Geiger-MOller detector and thereby actually enhances laboratory safety because of increased aware- ness. Finally, the short half-life of 32p (-14.3 days) allows waste to be decayed off before disposal, effectively eliminating expensive or awkward disposal of radioactive waste, whether solid or liquid.

4. [a-32p]ATP Disadvantages

The short half-life of 3Zp implies that the usefulness of the isotope is often lost to decay before the [a-32P]ATP is fully utilized. Consequently, if usage rates are low the decay of the isotope may result in the cost of [a-32p]ATP approaching that of [3H]ATP. However, the cost of [a-szp]ATP can be substantially reduced if it is enzymatically synthesized in the laboratory. 29

Blank values can depend on the quality of substrate, even with the double-column procedures described below. The quality can vary substantially among different suppliers and in different batches from a given supplier. Blank values for the adenylyl cyclase assay may be supplied on the product data sheet. The quality of [a-3ZP]ATP can be assured by its purification before use or through its enzymatic synthesis in the laboratory from carrier-free [32p]p i ,z9 a process which also includes its purification. The synthesis of 32P-labeled nucleotides has been updated in this series, j°

C. S t o p p i n g t h e R e a c t i o n

There are several good methods for stopping adenylyl cyclase reactions. The choice depends on whether [3H]ATP or [a-32p]ATP is used as substrate, on the method used for estimating loss of labeled cAMP during

29 R. A. Johnson and T. F. Walseth, Adv. Cyclic Nucleotide Res. 10, 135 (1979). 3o T. F. Walseth, P. S. T. Yuen, and M. C. Moos, Jr., this series, Vol. 195, p. 29.


its purification, and on the chromatographic system used for separating labeled product from labeled substrate. Some of these considerations are dealt with below.

I. Stopping with Zinc Acetate~Sodium Carbonate/cAMP or Zinc Acetate~Sodium Carbonate/[3H]cAMP

The use of coprecipitation or adsorption of nucleotides with inorganic salts dates from an early assay for adenylyl cyclase developed by Krishna et al.,3t who used a combination of column chromatography on Dowex 50 and precipitation with ZnSO4 and Ba(OH)z, yielding the insoluble salts BaSO4 and Zn(OH)2, which adsorb phosphomonoesters and polyphos- phates but not cyclic nucleotides. A disadvantage in the use of ZnSO4/ Ba(OH)2 is that cAMP may be formed nonenzymatically from ATP at alkaline pH, especially at elevated temperatures, leading to variable and high blank values. This problem is circumvented by the use of other salt combinations or by the use of acidic inactivation of adenylyl cyclase.

The effectiveness of a variety of combinations of inorganic salts, for example, ZnSO4/NazCO3, CdC1JNazCO3, ZnSO4/BaCI 2, and BaCI2/ NazCO3, to bind labeled ATP, ADP, AMP, cAMP, and adenosine has been cataloged previously.3-' Because comparable separation of ATP and cAMP can be achieved with columns packed with ZnCO332 or AI20 3 ,33 adsorption to the insoluble inorganic salts, rather than coprecipitation with them, is the likely basis of the separation of cAMP from the multivalent nucleotides and hence the basis of their usefulness in assays of adenylyl or guanylyl cyclases. It is important to emphasize that none of the salt combinations alone will separate cAMP from adenosine or inosine. The coelution of [3H]cAMP, formed via adenylyl cyclase, and 3H-labeled nucleoside contaminants (e.g., [3H]adenosine, [3H]inosine, [3H]xanthine, and [3H]hypoxantine) is effectively circumvented by (a) use of a two-step chromatographic procedure, (b) acidification of samples before chromatography on alumina, and/or (c) use of [a-32p]ATP instead of [3H]ATP as substrate. The following procedure, adapted from Jakobs et al . , 34 takes advantage of the nucleotide adsorptive characteristics of the insoluble inorganic salts.

Reagents

Zinc acetate/cAMP: 120 mM Zn(CzH302) 2 • 2H20 (FW 219.49) is prepared in deionized, double distilled, or Millipore (Bedford, MA)

31 G. Krishna, B. Weiss, and B. B. Brodie, J. Pharmacol. Exp. Ther. 163, 379 (1968). 32 p. S. Chan and M. C. Lin, this series, Vol. 38, p. 38. 33 A. A. White and T. V. Zenser, Anal. Biochem. 41, 372 (1971). 34 K. H. Jakobs, W. Saur, and G. Schultz, J. Cyclic Nucleotide Res. 2, 381 (1976).


grade water that has been boiled and then cooled to remove dissolved carbon dioxide, and cAMP is then added (165 mg/liter or to 0.5 mM); the solution is kept refrigerated and tightly capped between uses to minimize precipitation of atmospheric CO2

Zinc acetate/[3H]cAMP: the radiolabeled reagent is prepared as above except that tritiated cAMP, from which 3H20 has been removed, is added to an amount of Zn(C2H302) z needed for a given assay to yield approximately 10,000 to 20,000 cpm/ml, when counted in the same volume of eluate used for samples

Sodium carbonate: 144 mM NazCO 3, anhydrous (FW 106.0) Both Zn(C2H302) 2 and Na2CO3 solutions are stored in and dispensed

from glass repipettors.

Procedure

a. With [a-32p]ATP as substrate adenylyl cyclase reactions, typically 50 to 200/zl in 1.5-ml plastic Eppendorf tubes, are terminated by the addition of 0.6 ml of 120 mM Zn(CzH3Oz)z/cAMP or Zn(CzH302)2/ [3H]cAMP. If [3H]ATP is used as substrate, tritiated cAMP cannot be used for determination of recoveries; unlabeled cAMP, [32p]cAMP, or [14C]cAMP would have to be used. Aliquots of these stopping solutions are taken for determining absorbance (A259 nm) or radioactivity as appropriate, values to be used for quantitating sample recovery.

b. One-half milliliter of 144 mM Na2CO3 is added to precipitate ZnCO3 and adsorb multivalent adenine nucleotides and Pi.

c. Samples are placed on ice or can be kept refrigerated or frozen overnight. The ZnCO 3 precipitate is sedimented by centrifugation in a bench-top centrifuge. Pellets of frozen samples are smaller and heavier than those of unfrozen samples.

d. The supernatant fractions are decanted onto columns for purification of sample cAMP.

e. Assay blanks are prepared by substituting enzyme buffer for enzyme.

A potential disadvantage of this method is that if [3H]cAMP is used, it becomes necessary to use, and hence eventually dispose of, scintillation cocktails for quantitating [32p]cAMP and its recovery. An advantage of this procedure that has led to its use in many laboratories is that over 98% of all multivalent ntlcleotides, namely, substrate [a-3ZP]ATP, [a-32p[ADP, [32p]AMP, as well as any [32p]p i, are retained in the capped Eppendorf assay tubes in the ZnCO3 precipitate. The waste radioactivity is thus highly confined, occupies little volume, and can be allowed to decay off and then be dealt with as normal solid waste.


2. Stopping with ATPISodium Dodecyl Sulfate~cAMP with or without [3H]cAMP

An alternate method, adapted from Salomon et al. 1'2'35 relies on sodium dodecyl sulfate (SDS) to inactivate adenylyl cyclase and depends on unlabeled ATP and unlabeled cAMP to overwhelm adenylyl cyclase and cAMP phosphodiesterases with nonradioactive substrates and thereby effectively prevent the further formation or degradation of [32p]cAMP.

Reagents

Stopping solution: 2% (w/v) SDS, 40 mM ATP, 1.4 mM cAMP, pH 7.5, and approximately 100,000 cpm [3H]cAMP/ml, to monitor recovery of [3Zp]cAMP; alternatively, [3H]cAMP could be omitted from the stopping solution and added separately

Procedure

a. Adenylyl cyclase reactions, typically 50 to 200/xl in 13 x 65 mm glass or plastic tubes or in 1.5-ml plastic Eppendorf tubes, are terminated by the addition of I00 ~1 of the stopping solution.

b. To achieve full membrane solubilization in cases of high membrane content, it is advisable to boil the test tubes for 3 min at this stage. This also accelerates the rate of chromatography. Hence, use heat-stable tubes.

c. The mixtures in the reaction tubes are then diluted and decanted onto chromatography columns for purification of sample cAMP.

d. Assay blanks are prepared by omitting enzyme or by adding enzyme after the stopping solution.

A disadvantage of this procedure is that all radioactive compounds, including unused substrate [a-32p]ATP, [a-32p]ADP, [32p]AMP, [32p]Pi, as well as degradation products of [3H]cAMP, are passed with the labeled cAMP onto the chromatography column and are typically eluted in a fall- through fraction that must be collected and then dealt with as a large volume of liquid radioactive waste. To minimize this waste see Section E below. A disadvantage of either stopping procedure when [3H]cAMP is used to monitor recovery of sample [32p]cAMP is that scintillation cocktails must be used and consequently disposed.

3. Stopping with Hydrochloric Acid with or without cAMP or [3HlcAMP

Because cAMP can be formed nonenzymatically from ATP at alkaline pH, especially in the presence ofMn 2÷ , lower blank values can sometimes

as y . Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974).


be obtained by stopping the adenylyl cyclase reaction with acid. The essence of this procedure was first reported by Nakai and Brooker 36 and has since been verified and modified somewhat by Counis and Mongongu 37 and by Alvarez and Daniels. 3s,39

Reagents

HC1 (2.2 N) To monitor recovery of [32p]cAMP, add either unlabeled cAMP (165

rag/liter or to 0.5 raM) or [3H]cAMP (10,000 to 20,000 cpm per sample) to the hydrochloric acid just before use. When kept cold, cAMP and [3H]cAMP are stable in acid against chemical degradation.

Procedure

a. Adenylyl cyclase reactions, typically 50 to 200/.d in 13 × 65 mm glass or plastic tubes or in 1.5-ml plastic Eppendorf tubes, are terminated by the attention of a sufficient volume of the HCI solution to give 0.2 to 0.5 M HC1 (e.g., 10/A of 2.2 N HCI to a 100-/A reaction volume). Concentrations less than 0.1 M do not result in markedly lower blank values, and concentrations greater than 1 M cause degradation of [32p]cAMP when samples are heated. 36,37 If [3H]ATP is used as substrate, adenine (0.1 raM) may be included in the reaction mixture to reduce the specific activity of nonphosphorylated metabolites of [3H]ATP generated during the reaction. 39

b. The test tubes are then placed in a water bath at 90o-95 ° for 4 to 8 min. Examples are 90 ° for 8-10 rain in 0.95 N HC1, 36 4 min at 95 ° in 0.165 N HCI, 37 or 95 ° for 10 rain in 0.2 N HCI. 3s The heat step hydrolyzes ATP and unknown substances that contribute to assay blanks if [a-32p]ATP is used as substrate. If [3H]ATP is used as substrate, the reaction should not be terminated by heating as this increases the assay blank. 39

c. The mixtures in the reaction tubes are then either diluted and decanted onto chromatography columns for purification of sample cAMP, or they are neutralized and precipitated by the subsequent addition of Zn(C2H3Oz)2/NazCO 3 (see Section C.l,a). Chromatography is either on sequential Dowex 50 and alumina columns or on single alumina columns (see Section D,2).

d. Assay blanks are prepared by omitting enzyme, by adding enzyme after the HCI, or by prior heat denaturation of the enzyme.

36 C. Nakai and G. Brooker, Biochim. Biophys. Acta 391, 222 (1975). 37 R. Counis and S. Mongongu, Anal. Biochem. 84, 179 (1978). 38 R. Alvarez and D. V. Daniels, Anal. Biochem. 187, 98 (1990). 39 R. Alvarez and D. V. Daniels, Anal. Biochem. 203, 76 (1992).


A disadvantage of each stopping procedure when [3H]cAMP is used to monitor recovery of sample [32p]cAMP is that scintillation cocktails must be used and consequently disposed.

D. C h r o m a t o g r a p h i c A l t e r n a t i v e s

The characteristic property of neutral alumina and other insoluble inorganic salts to bind multivalent nucleotides but not cAMP is the central feature of a number of variations of assays for adenylyl and guanylyl cyclases. White and Zenser 33 passed reaction mixtures over columns of neutral alumina that were equilibrated and then developed with neutral buffer. Assay blanks with this procedure were variable and depended highly on the radiochemical purity of the a-32p-labeled substrate and on the quality of the alumina. Salomon et al. 35 and later Wincek and Sweat 4° showed that sequential chromatography on Dowex 50 and alumina produced an assay for adenylyl cyclase that was more consistent than alumina columns alone, a combination that has also been utilized for the assay of guanylyl cyclase. 41 Additional variations on this procedure have been reported by a number of investigators. For example, nearly quantitative separation of cAMP from ATP was achieved by a combination of precipitation with inorganic salts (zinc acetate/NaECO3) followed by chromatography on alumina) 4 This also forms the basis of the assay of guanylyl cyclase as updated in this series. 42 To minimize the influence of variations in the quality of [o~-3ZP]ATP, variations in the behavior of various sources of alumina, and the coelution of potential contaminants, one method of choice has become sequential chromatography on Dowex 50 and then alumina columns.~'2'~5 An effective alternative, though, is an inherently easy single alumina column procedure 38,39 and is also presented (see Section D,2).

I. Sequential Chromatography on Dowex 50 and Alumina

Reagents

Dowex 50: H + form (e.g., Bio-Rad, Richmond, CA, AG50-X8, 100-200 mesh). Before use the Dowex 50 is washed sequentially with approximately 6 volumes each of 0.1 N NaOH, water, 1 N HC1, and water. Dowex 50, in an approximately 2 : 1 slurry, is then poured into columns (-0 .6 × 4 cm). After each use, Dowex 50

40 T. J. Wincek and F. W. Sweat, Anal. Biochem. 64, 631 (1975). 41 j. A. Nesbitt, lII, W. B. Anderson, Z. Miller, I. Pastan, T. R. Russell, and D. Gospodaro-

wicz, J. Biol. Chem. 251, 2344 (1976). 42 S. E. Domino, D. J. Tubb, and D. L. Garbers, this series, Vol. 195, p. 345.


columns are regenerated by washing with 5 ml of 1N HC1 and stored until reused. Before use the columns are then washed once with 10 ml of water. Between uses columns are covered with a dust cover. Columns can be reused dozens of times, though additional resin may need to be added occasionally. If flow rates decrease columns should be regenerated with NaOH, water, and HCI as above.

Alumina: Neutral (e.g., Bio-Rad AG7, 100-200 mesh; Sigma WN-3; ICN, Cleveland, OH, alumina N, Super I). The source of A120 3 is less critical with the two-column procedure than if it is used alone, or if used alone and is washed with acid before elution of cAMP with buffer (see below). The alumina ( -1 g) may be poured dry into columns (e.g., with a plastic scoop or a large disposable plastic syringe from which the alumina is allowed to drain), or an RCBS Uniflow adjustable gun powder measure (Omark Industries, Oro- ville, CA) as suggested by Alvarez and Daniels. 38

Elution buffers: 100 mM imidazole, pH 7.5, as per the original method of Salomon et al. 35 An equally effective and less expensive alternative is 100 mM Tris-C1, pH 7.5, and a more efficient and consistent elution has been reported with 0.1 M ammonium acetate. 38 The purpose of the buffer is to elute cyclic nucleotides. Because eluate from the Dowex 50 columns is acidic, which enhances adsorption of cyclic nucleotides to alumina, elution of cyclic nucleotides is achieved principally through the increase in pH of the buffer as well as through increased ionic strength.

Apparatus . Rapid flow rates for the alumina columns, and consequently short chromatography times, are achieved with glass columns with a large cross-sectional area and a coarse sintered glass plug to retain the alumina (Fig. 2). Satisfactory dimensions are alumina to approximately 1 cm in a column 11 mm inner diameter by 4 to 9 cm attached to a 2 to 4 cm glass funnel (24 mm i.d.). [Smaller columns (e.g., - 0 . 6 × 2 cm, alumina) while allowing satisfactory chromatographic performance are slow.] It is important that the volume above the alumina be sufficient to hold all the buffer necessary for elution of the cAMP. Alternatively, disposable plastic columns can be used as recommended for the single alumina column described below.

The alumina may clog the sintered glass plug in time. This can be minimized by placing a glass-fiber filter disk on the sintered glass plug before adding alumina. The filters (Whatman, Clifton, N J, GF/D) are cut to size with the plastic lip of a Sarstedt polypropylene tube (No. 72-693; 10.8 mm diameter). If necessary a clogged sintered glass plug can be freed


1 E E

1

~-24 mm-~

alumina f

I I * lOmm

intillation vial

/

FIG. 2. Setup for alumina columns.

and restored to initial flow rates by sonication in 6 N nitric acid for 30 min, followed by reverse flushing with water.

Both Dowex 50 and alumina columns are most conveniently used if they are mounted in racks (e.g., Lucite) with spacing the same as that of the racks of scintillation vials to be used. The design of the racks supporting the Dowex 50 columns should be such that the columns can be conviently mounted above the alumina columns so that the eluate of the Dowex 50 columns can drip directly onto the alumina columns. Similarly, the design of the racks supporting the alumina columns should allow the eluate from them to drip directly into scintilation vials.

Procedure. Two alternative procedures are described, the use of which depends on the quality of 32p-labeled substrate. The first procedure should be adequate with all but the poorest quality substrate, and the second procedure should lower blank values further if necessary.

48 ADENYLYL CYCLASES [3l

a. Water Elution of Dowex 50 i. Whether reactions are stopped by the zinc acetate/Na2CO3,

ATP/SDS/cCAMP, or HCI method, the samples are decanted directly onto the Dowex 50 columns.

ii. The Dowex 50 columns are then washed with about 3 ml water. (The actual volume necessary for this step may vary slightly from batch to batch or with the age of the Dowex 50 resin and should be determined. 1) The eluate from the wash contains [32P]P i , [cz-32p]ATP, and [a-32p]ADP and should be disposed of by the procedure described below (Section E).

iii. The Dowex 50 columns are then mounted above a comparable number of alumina columns so that the eluate drips directly onto the alumina. The Dowex 50 columns are washed with 8 ml water. The eluate from the Dowex 50 columns is slightly acidic and causes cAMP to be retarded on the alumina column.

iv. After the eluate from the Dowex 50 columns has dripped onto and through the alumina columns, the alumina columns are placed over scintillation vials.

v. cAMP is eluted from the alumina columns directly into scintillation vials. The volume of elution buffer used depends on whether unlabeled cAMP or [3H]cAMP is used to quantitate sample recovery and on the types of vials used in the scintillation counter. It is important to use sufficient buffer to elute all the cAMP as well as to optimize counter efficiency, which is dictated by the geometry of the phototubes of the counter. Two examples are given here: (i) If recovery is monitored with unlabeled cAMP and [32p]cAMP is determined by Cerenkov radiation in 20-ml counting vials, [32p]cAMP is eluted from alumina columns with 8 ml of 100 mM Tris-Cl. In our counter smaller volumes do not give optimal counting efficiency. Fol- lowing counting, absorbance at 259 nm is determined on an aliquot of the sample to quantitate recovery of unlabeled cAMP. (ii) If recovery is monitored with [3H]cAMP, both [3H]cAMP and [32p]cAMP are eluted with 4 ml of 100 mM imidazole into 10-ml vials to which 5 ml scintillation cocktail is then added. The smaller vials spare expensive cocktail. Sample recovery is determined from dual-channel counting.

vi. Scintillation counting of 32P by Cerenkov radiation is achieved with a single channel with wide open windows.

vii. Counting of samples containing both 3H and 32P can be achieved in a two-channel scintillation counter with windows adjusted such that there is zero cross-over of 3H into the 32p window


and probably small but measurable cross-over of 32p into the 3H window.

b. Acid elution of Dowex 50, adapted from White and Karr 43 i. Before use the Dowex 50 columns are washed with 10 ml of

0.01 N HC1. ii. Whether reactions are stopped by the zinc acetate/Na2CO3,

ATP/SDS/cAMP, or HC1 method, the samples are decanted directly onto the Dowex 50 columns.

iii. The Dowex 50 columns are then washed with 6 ml of 0.01 N HCI. (The actual volume necessary for this step may vary from batch to batch or with the age of the Dowex 50 and should be determined.) The eluate from this wash contains [32p]p~, [~. 32p]ATP, and [a-32p]ADP and should be disposed of by the procedure described below (see Section E).

iv. The Dowex 50 columns are then mounted directly above a comparable number of alumina columns and are washed with 8 ml of 0.01 N HC1, which is allowed to drain through both columns.

v. The alumina columns are then washed with 10 ml water. This eluate is discarded.

vi. The alumina columns are mounted above a rack of scintillation vials and the cAMP is eluted as in the procedure described above.

Note: Dowex 50 is slowly decomposed by HC104 as used originally 4~ and can therefore not be used too many times. This problem is not apparent with HCI, which therefore has been substituted for the perchloric acid.

Column Care. Before initial use alumina columns must be washed once with elution buffer, either 10 ml of 100 mM Tris-Cl or 10 ml of 1 M imidazole, pH 7.5; otherwise, the procedure does not work right away. After each use of the columns are washed with 10 ml of 100 mM Tris-Cl or 10 ml of 100 mM imidazole, pH 7.5. Alumina columns may be reused virtually indefinitely, though additional alumina may need to be added occasionally.

2. Single Alumina Column

Use of a single alumina column for the separation of labeled cAMP from labeled substrate and its various degradation products has been reported by a number of investigators. Typically, the problems with the procedure that have been reported include the following: there was sub-

43 A. A. White and D. B. Karr, Anal. Biochem. 85, 451 (1978).


stantial variation in the behavior of alumina from various sources; the assay blank was considerably higher than that for the double-column procedure above; the assay blank was dependent on the source of [32p]ATP; and when [3H]ATP was used as substrate or [3H]cAMP was used to monitor recovery, enzymatically derived breakdown products (e.g., [3H]xanthine and [3H]hypoxanthine) would coelute with [3H]cAMP, resulting in erroneous values for [3H]cAMP. These concerns have been considerably reduced or eliminated by two modifications to the original methods, namely, the use of acid to stop the adenylyl cyclase reaction and improved elution characteristics when columns are eluted with ammonium acetate instead of Tris-C136-39 The best variations on this method are described below and depend on whether [a-32p]ATP or [3H]ATP is used as substrate. 36-39

Single-Column Procedure with [a-32p]ATP as Substrate

Reagents

Neutral alumina: the source of A120 3 is not critical. Alumina (1.3 g) may be poured dry into columns (see Section D. 1).

Elution buffer: 100 mM ammonium acetate, pH 7 (FW 77.08; 7.7 g/liter)

Apparatus. Alumina columns are the same as those described above (see Section D,1 and Fig. 2).

Procedure

a. Adenylyl cyclase reactions are stopped by the addition of HC1 followed by heating at approximately 95 ° for 4 min, as described above, without or with [3H]cAMP or unlabeled cAMP, depending on whether recovery of sample [32p]cAMP is to be determined. Samples are decanted directly onto dry alumina columns. By use of small sample volumes and by. stopping the reaction with a small volume of concentrated HCI (see above), there will be no fall-through liquid. If the recovery of labeled cAMP is not to be determined, a known aliquot of the sample (e.g., 100 /zl) is applied to the column. The ratio of applied volume to total volume is used in the calculation of reaction velocities (Section F. 1).

b. Place the rack(s) of alumina columns over rack(s) of scintillation vials and elute the labeled cAMP with 4.0 ml of 100 mM ammonium acetate directly into the vials.

c. Counting i. If radioactivity is determined by Cerenkov radiation ([32p]cAMP

only), add 5 ml water to the vials and then place in a liquid scintillation counter.

ii. If [3H]cAMP is used to monitor sample recovery, add scintilla-


tion cocktail and determine [32p]cAMP and [3H]cAMP by dual- channel counting in a liquid scintillation counter.

iii. If unlabeled cAMP is used to monitor sample recovery, determined the absorbance at 259 nm of the stopping solution and, after counting, that of each sample. Use these values to determine sample recovery (see calculations below).

Single-Column Procedure with [3H]ATP as Substrate

Reagents

Acidic alumina: activity grade 1 from ICN Biomedicals GmbH. Alu- mina (1.3 g) may be poured dry into columns (see Section D.1).

Elution buffers: 5 mM HCI; 100 mM ammonium acetate, pH 7 (FW 77.08; 7.7 g/liter)

Apparatus. Alumina columns are the same as those described above (see Section D.1 and Fig. 2).

Procedure

a. Adenylyl cyclase reactions are stopped by the addition of HCI (to 0.2 N final concentration) followed by heating at approximately 95 ° for 4 min, without or with [32p]cAMP, [14C]cAMP, or unlabeled cAMP, depending on whether recovery of sample [3H]cAMP is to be determined, as described above. Samples are decanted directly onto dry alumina columns. If the recovery of labeled cAMP is not to be determined, a known aliquot of the sample (e.g., 100/xl) is applied to the column. The ratio of applied volume to total volume is used in the calculation of reaction velocities (see Section F.1).

b. Wash alumina columns with 8 ml of 5 mM HCI, followed by 1 ml of 100 mM ammonium acetate. Although this step generates liquid radioactive waste, it is important when [3H]ATP is used as substrate in that it removes tritiated nucleoside and base contaminants that otherwise would coelute with cAMP and lead to an overestimation of [3H]cAMP formation.

c. Elute the sample [3H]cAMP with 3.5 ml of 100 mM ammonium acetate directly into scintillation vials.

d. Counting i. If [32p]cAMP or [~4C]cAMP is used to monitor sample recovery,

add scintillation cocktail and determine [3H]cAMP and recovery tracer by dual-channel counting in a liquid scintillation counter.

ii. If unlabeled cAMP is used to monitor sample recovery, remove an aliquot of known volume (e.g., 1.0 ml) from each sample. Determine the absorbance at 259 nm of the stopping solution and that of the aliquots from each sample. Use these values to determine sample recovery. Add scintillation cocktail to the


vials and determine the remaining [3H]cAMP in a liquid scintillation counter.

e. For each elution method see Section F, 1 for use of these values in the analysis of data.

Column Care. For either single alumina column procedure a convenient treatment of columns is to use disposable plastic columns and to repour them for each use as emphasized by Alvarez and Daniels. 38,39 Alumina is inexpensive, and columns can be prepared in a short time with a gun powder dispenser.

If glass columns are used and the alumina is to be reused, columns should be washed after each use with 8 ml of 100 mM ammonium acetate followed by 8 ml of 0.005 N HCI. The isotope and frequency of use may dictate whether this is appropriate for a given laboratory situation. Because reactions are stopped by the addition of acid [rather than a precipitation step with Zn(C2H302)2/Na2CO3], the columns adsorb virtually all the radioactivity in the sample (excepting 3H20 and 3H-labeled nucleosides and bases). Consequently, radioactivity accumulates very rapidly with frequent use, probably to unacceptable levels as far as laboratory safety is concerned. This is more evident with [a-32p]ATP as substrate, but more insidious with [3H]ATP. It will likely also lead to unacceptable increases in blank values. However, if usage is infrequent, especially with 32p as isotope which decays off rapidly, this concern may be unim- portant.

Alternatively, the accumulation of radioactivity and its untoward effects can be lessened by periodically washing columns with 8 ml of 1 N NaOH to elute over 95% of the bound radioactively labeled products, followed by 8 ml of water and then 8 ml of 0.005 N HCI. This radioactive alkaline wash must then be made neutral or acidic before disposal on the alumina-charcoal filters (described below). Aside from the problem of accumulated radioactivity alumina columns may be reused virtually indefinitely, though additional alumina may need to be added occasionally.

E. Disposal of Waste Isotope

To minimize disposal of radioactive waste the investigator should weigh alternative procedures for the individual laboratory and application. For double-column chromatography procedures and for the single alumina column case when [3H]ATP is used as substrate, all radioactive waste is collected and pooled. To avoid contamination of wastewater it is first poured onto a large Biichner funnel, containing perhaps 250 g alumina [e.g., A1203 , anhydrous, Fisher], attached in series to four parallel 100-g carbon filters and a flask attached to a water aspirator. (The purpose of the flask is to allow an aliquot of the filtered waste to be monitored for


radioactivity before the waste is discarded down the drain.) By use of alumina and carbon filters, virtually no 32p radioactivity is discarded in wastewater, though some 3H20 will be lost if tritiated nucleotides are used ([3H]ATP or [3H]cAMP). An important additional advantage of this is that adsorbed radioactive waste can then be treated as compact solid waste. This is especially useful for tritiated nucleosides and nucleotides. The 32p_ labeled solid waste can be allowed to decay off. The alumina can be used almost indefinitely, whereas the carbon filters tend to clog with prolonged use and need to be replaced periodically (e.g., annually).

Stopping the adenylyl cyclase reaction by use of the zinc acetate/ Na2CO 3 step offers an advantage with regard to waste disposal. Whether used to stop the reaction (Section C. 1) or to precipitate nucleotides after stopping the reaction by acid and neutralization (Section C.2), precipitation with zinc acetate/Na2CO3 traps most of the radioactivity in the ZnCO3 pellet, including unused and unhydrolyzed substrate, whether [t~-32p]ATP or [3H]ATP is used as substrate, and all nucleotide degradation products and [32P]P i . This step prevents most of the radioactivity from being applied to the chromatography columns and allows it to be treated immediately as solid waste. Otherwise, with the sequential Dowex 50/alumina procedure radioactive substances are eluted in a large wash fraction.

If disposable plastic columns are used with the single alumina column, virtually all of the 32p_ and most of the 3H-labeled compounds are adsorbed. The spent columns can then be treated as solid radioactive waste with [3H]ATP as substrate or be allowed to decay offand then treated as normal waste with [a-32p]ATP as substrate.

Apparatus. Carbon filters for the removal of adsorbable radioactive materials from column eluates are available from Gelman (Ann Arbor, MI, No. 12011) as carbon capsules, each containing 100 g activated charcoal.

F. Data Analysis

Calculation of adenylyl cyclase activities determined with radioactive substrates is straightforward. It is typically easier for the single alumina column chromatography system than for the double-column system, as the single alumina column is often run without determination of sample recovery since near quantitative (>98%) recovery of cAMP is usually observed. For double-column chromatography systems calculations must take into consideration the loss of sample cAMP that occurs during chromatography. Consequently, volumes applied to columns must be known or sample recovery must be determined, either with unlabeled cAMP or with cAMP that is labeled with a second isotope. In addition, because many adenylyl cyclases exhibit low activities, especially under basal assay


conditions, the radioactivity measured in the sample in the absence of enzyme (no enzyme blank) can represent a sizable percentage of that measured with enzyme. Consequently, it becomes important to consider how this value is to be treated in the calculation of activity.

If there is measurable nonenzymatic formation of cAMP from ATP, as may be the case under alkaline assay conditions, especially in the presence of manganese, the labeled cAMP must be corrected for sample loss during purification. However, if it can be established that the sample radioactivity in the absence of enzyme is due to labeled contaminants in the sample, that is, for example, 3Zp-labeled compounds not adsorbed by alumina 43 or as determined through alternative chromatographic techniques, the blank value should not be corrected for sample recovery. Such a correction would give rise to an erroneously high blank value, and the apparent enzyme activity would be lower than it should be. Both sample recovery and assay blank adjustments to the determination are readily made with programmable calculators, although they are more conveniently done with a computer. Programs can easily be written to accommodate variable amounts of protein, substrate concentrations, assay times, and volumes; they can be extended to the computation of enzyme kinetic constants, and output can be interfaced with graphic plotters. This is readily done with commercially available spreadsheet programs. Exam- ples of calculations are given below.

1. Calculation without Sample Recovery

The calculation of adenylyl cyclase activities without determination of sample recovery is simplest and is the same whether [3H]ATP or [a-32p]ATP is used as substrate. The example below assumes [a-3Zp]ATP is substrate.

Velocity = (sample 32p cpm - no enzyme 32p cpm) (ATP concentration) (reaction volume)/([a-3:p]ATP cpm - no enzyme 32p cpm)/fraction of sample applied to column/fraction of sample counted/time/protein

The fraction of sample applied to the column is determined from the volume of the reaction divided by the volume of the reaction plus the stopping solution (e.g., HCI in the single-column procedure).

2. Calculation with [3H]cAMP Used for Sample Recovery

For the calculation of adenylyl cyclase activities with [3H]cAMP used for sample recovery, the assumption is made that the windows for the 32p


and 3H channels of the scintillation spectrometer have been set so there is zero crossover of 3H counts into the 32p channel. The calculation com- pensates for crossover of 32p counts into the 3H channel.

Velocity = (sample 32p cpm - no enzyme 32p cpm) (ATP concentration) (reaction volume)/fraction of sample counted/([o~-32P]ATP cpm - no enzyme 32p cpm) ([3H]cAMP-std cpm)/{sample 3H cpm - [(sample 32p cpm - no enzyme 32p cpm) (32p cpm in 3H channel)/ ([a-32p]ATP cpm)]}/time/protein

[3H]cAMP-std cpm is the value that would represent 100% recovery of the added [3H]cAMP, for example, the total 3H counts in the 0.6 ml of zinc acetate containing [3H]cAMP used to stop the reaction, counted under comparable quench conditions used to count the samples.

3. Calculation with Unlabeled cAMP Used for Sample Recovery

An analogous though simpler calculation is used for activities when unlabeled cAMP is used for sample recovery and is the same whether [a-32p]ATP or [3H]ATP is used as substrate tracer.

Velocity = (sample cpm - no enzyme cpm) (ATP concentration) (reaction volume)/fraction of sample counted/ (substrate cpm - no enzyme cpm) (cAMP standard A259)/ sample A~59/time/protein

cAMP standard A259 is the optical density at 259 nm that would represent 100% recovery of the added unlabeled cAMP. This value usually also includes a factor to compensate for the volume of the final sample. In the example given here for samples chromatographed first on Dowex 50 then on A1203 columns, samples are 8 ml. For example, the optical density (A259) of the 0.6 ml of zinc acetate containing unlabeled cAMP is typically determined on an aliquot diluted 40-fold in 100 mM Tris-Cl, pH 7.5, and gives a value of approximately 0.2. In this example, 0.2 × 40 × 0.6 ml/ 8 ml yields 0.6 for the cAMP standard A259.

4. Reporting of Values

Velocities are in nanomoles cAMP formed per minute per milligram protein when the substrate concentration is entered as micromolar, time is minutes, protein is micrograms per tube, and reaction volume is microliters. The value for the term fraction of sample counted is usually 1. It would be less than 1 only if an aliqout of the sample were used for some

56 A D E N Y L Y L CYCLASES ~ ]

other purpose, for example, use of [3H]ATP as substrate, use of unlabeled cAMP to monitor recoveries, and the necessary removal of an aliquot of sample to determine recovery before scintillation cocktail is added. If protein is not known or if it is not desirable to normalize to protein, a value of 1 is used and velocities are picomoles cAMP formed per minute per tube. For the calculations of Sections F,2 and F,3, the determinations of velocity assume that 32p counts observed in the absence of enzyme is not cAMP, and no correction is made for loss during purification of those samples. This is an important assumption only in instances when enzyme activity is low and the radioactivity observed in the absence of enzyme represents a sizable percentage of sample counts.

Acknowledgments

Yoram Salomon is the Charles and Tillie Lubin Professor of Hormone Research. Research in the laboratory ofR. A. J. was supported by National Institutes of Health Grant DK38828.

[4] P r e p a r a t i o n a n d U s e o f " P " - S i t e - T a r g e t e d Aff in i ty L igands for A d e n y l y l Cyc la ses

By ROGER A. JOHNSON and ILANA SHOSHANI

Introduction

All mammalian adenylyl cyclases (EC 4.6.1.1; adenylate cyclase), with the exceptions of the enzymes from sperm and testes, are potently and directly inhibited by analogs of adenosine via a domain that is referred to as the "P" site because of an apparent requirement for an intact purine moiety. ~-4 Although this is actually a misnomer, "P"-site-mediated inhibition may be characterized pharmacologically by (1) a strict requirement for an intact adenine moiety; (2) a requirement for a fl-glycosidic linkage at the ribosyl moiety; (3) substantially increased inhibitory potency of 2'- deoxy- and especially 2',5'-dideoxyribosyl moieties; (4) a strong preference for phosphate at the 3' position; and (5) a tolerance for large substitutions at the 3' position. 4 The most potent inhibitors are 2',5'-dideoxyade-

C. Londos and J. Wolff, Proc. Natl. Acad. Sci. U.S.A. 74~ 5482 (1977). 2 j . Wolff, C. Londos, and D. M. F. Cooper, Adv. Cyclic Nucleotide Res. 14, 199 (1981). 3 R. A. Johnson, W. Saur, and K. H. Jakobs, J. Biol. Chem. 254, 1094 (1979). 4 R. A. Johnson, S.-M, H. Yeung, D. Sttibner, M. Bushfield, and I. Shoshani, Mol. Pharma-

col. 35, 681 (1989).


[4] "P"-SITE AFFINITY LIGANDS 57

nosine (2',5'-ddAdo; IC50 -2 /xM), 2'-d,3'-AMP (IC50 -1 ~M), Y-AMP (IC50 - 9 ~M), and 2',5'-dideoxy-, 3'-AMP (2',5'-dd,3'-AMP; IC50 <0.1 tzM). Of these, 2'-d,3'-AMP and Y-AMP are naturally occurring and are nucleic acid metabolites. Inhibition is noncompetitive with respect to substrate metal-ATP and is dependent on divalent cations, and greater potency of inhibitors is exhibited with ATP and 2'-dATP as substrates than with y-thio,/3~/-imido, or/3y-methylene analogs of ATP. 3-5

Available evidence suggests that the "P" site is distinct from, yet homologous with, the catalytic domain, 5-8 though these two domains share apparent requirements for substrate structure. Neither domain tolerates modification of the adenine moiety, and 2'-deoxy analogs and phosphorylated analogs are preferred at both. Catalysis requires 5'-phosphorylated substrate, whereas inhibition via the "P" site is preferential for 3'-phosphorylated analogs.

As an approach toward elucidating the structure-function relationships of adenylyl cyclases, covalent affinity ligands should be particularly useful. Obvious targets are the catalytic active site, the "P" site, and calmodulin- and G-protein-binding domains. The approaches our laboratory has taken target both nucleotide binding sites, but this chapter focuses on affinity ligands targeted to the "P" site and emphasizes the synthesis and use of reagents that take advantage of "P" site tolerance for large substitutions at the Y-ribose position. The purpose is to develop ligands that can be radioactively tagged and have the potential for covalent attachment to adenylyl cyclase. The preparation and use of two classes of affinity ligands are presented. One relies on sulfonyl fluoride as reactive functional group, and the other relies on light-induced reactivity of an arylazido moiety.

Synthesis of 2 ', 5'-Dideoxyadenosine

Given the increased potency of deoxyribose analogs of adenosine and of 2',5'-ddAdo in particular, 4 we have used the dideoxy analog as starting compound for several of the syntheses described here. The 2',5'-ddAdo is prepared essentially by the methods described by Beacham 9 and Wang et al.~° Anhydrous 2',5'-ddAdo is stable desiccated at -20 ° indefinitely.

R. A. Johnson and I. Shoshani, J. Biol. Chem. 265, 11595 (1990), 6 S.-M. H. Yeung and R. A. Johnson, J. Biol. Chem. 265, 16745 (1990). 7 j. Krupinski, F. Coussen, H. A. Bakalyar, W.-J. Tang, P. G. Feinstein, K. Orth, C.

Slaughter, R. R. Reed, and A. G. Gilman, Science 244, 1558 (1989). 8 W.-J. Tang, J. Krupinski, and A. G. Gilman, J. Biol. Chem. 266, 8595 (1991). 9 L. M. Beacham III, J. Org. Chem. 44, 3100 (1979). J0 y. Wang, H. P. C. Hogenkamp, R. A. Long, G. R. Revankar, andR. K. Robins, Carbohydr.

Res. 59, 449 (1977).


In aqueous solutions it is important that the pH be neutral and that the solution be kept on ice before dilution into incubations. At room temperature and especially at acidic pH hydrolysis of the adenine-ribosyl bond occurs.

Synthesis of Unlabeled and Tritiated 2 ',5'-Dideoxy-3 '-p-fluorosulfonylbenzoyladenosine

The sulfonyl fluoride moiety of 2',5'-dideoxy-3'-p-fluorosulfonylbenzoyladenosine (2',5'-dd-3'-FSBAdo) is a reactive functional group that can act as an electrophilic agent in covalent reactions with several classes of amino acids, including tyrosine, lysine, histidine, serine, and cysteine, and it has been used to bind covalently with various nucleotide utilizing enzymes (see Ref. 11).

Reagents

2',5'-ddAdo, prepared as described, 9,1° is dried over P205 under vacuum at least overnight; it is important that the reagent be anhydrous

Fluorosulfonylbenzoyl chloride (FSO2BzCI; Sigma, St. Louis, MO) must be stored desiccated; it is dried over P205 under vacuum at least overnight

Dimethyl sulfoxide [(CH3)2SO4] is good if freshly purchased, best if redistilled under vacuum

Hexamethylphosphoric triamide (HMPTA; Aldrich Chemical Co., Milwaukee, WI) is stored under dry nitrogen

Silica gel thin-layer plates (DC-Plastikfolien, Kieselgel 60 F~54, No. 5735 from EM Science, Cherry Hill, NJ) are developed with methyl ethyl ketone/acetone/water (60 : 20 : 15, v/v)

General solvents: methyl ethyl ketone, acetone, petroleum ether, ethyl acetate, diethyl ether

Procedure. The derivative 2',5'-dd-3'-FSBAdo is prepared by reaction of 2',5'-ddAdo with FSO2BzC1 by slight modifications 6 of the procedures described by Pal et al.~2'13 The procedure described below is used if 2',5'- ddAdo is limiting, but it is readily increased in scale (e.g., 10-fold) simply by proportionally increasing the amounts of the reagents.

1. Dissolve 2',5'-ddAdo (I0.6 rag, 42 /zmol) in 100 /A hexamethylphosphoric triamide. (This is instead of dimethylformamide as called for by Pal et al. lz13 and additional base is not added.)

~I R. F. Colman, Annu. Rev. Biochem. 53, 67 (1983). 12 p. K. Pal, W. J. Wechter, and R. F. Colman, J. Biol. Chem. 250, 8140 (1975). 13 p. K. Pal, W. J. Wechter, and R. F. Colman, Biochemistry 14, 707 (1975).


2. Add FSOzBzCI (26.7 mg, 120/xmol). After approximtely 45 min at room temperature a further 9.4 mg (42/zmol) FSO2BzCI is added to drive the reaction and to compensate for hydrolyzed FSOzBzCI. The reaction is then allowed to continue for 30 min more.

3. Monitor the progress of the reaction by silica gel thin-layer chromatography (TLC). Small aliquots ( -1 /zl) of reaction mixture are spotted on thin-layer strips (1 × 5 cm). These are developed with a solvent system consisting of methyl ethyl ketone/acetone/water (60:20: 15, v/v), the strips develop in approximately 5 min and spots are observed under UV light.

4. Extract the resulting mixture twice with 300 t~l petroleum ether. 5. Centrifuge the petroleum ether suspension in a bench-top centrifuge

for 5 min at about 1000 g. The top layer is discarded, and 2',5'-dd-3'- FSBAdo is precipitated from the lower layer by the slow addition of 400 ~1 acetate/diethyl ether (1 : 1, v/v).

6. Collect the precipitate by filtration on Whatman (Clifton, N J) No. 50 paper and wash it three times with ethyl acetate/diethyl ether (1:1, v/v).

7. Dry the washed material in a gentle N2 stream and then store it in a desiccator at 4 ° until 2' ,5'-dd-3'-FSBAdo is purified by high-performance liquid chromatography (HPLC).

Preparation of Tritiated 2',5'-Dideoxy-3'-p-fluorosulfonylbenzoyladenosine. Tritiated 2',5'-ddAdo is obtained commercially (Amersham Corp., Arlington Heights, IL) through a tritium-labeling service from unlabeled 2',5'-ddAdo sent to the company.

1. In a reaction vial (Reacti-Vial; Pierce, Rockford, IL) dry 5-10 mC~ 2',5'-dd[3H]Ado together with a small amount (< 1 mg) of unlabeled 2',5'-ddAdo over P205 until all visible fluid is removed, then place the vial over P205 under vacuum overnight. This removes HzO and 3H20. The carrier 2',5'-ddAdo protects 2',5'-dd[3H]Ado and allows product 2',5'- dd-3'-FSBAdo to be visualized during extraction and precipitation steps. The amount of carrier 2' ,5'-ddAdo added correspondingly dilutes the specific radioactivity of product 2',5'-dd-3'-FSB[3H]Ado and consequently should be held to a minimum.

2. Add 50 ~1 HMPTA and FSOzBzC1 (25 mg, -112 /zmol); further additions of FSO2BzCI are not made.

3. All subsequent steps are similar to those for a small preparation above.

Purification of Compounds. The 2',5'-dd-3'-FSBAdo and 2',5'-dd-3'- FSB[3H]Ado are purified by HPLC on CI8 reversed-phase or silica gel


columns. The size of the column used depends on the size of the preparation. An analytical or semianalytical reversed-phase column (4.6 × 150 mm or 4.6 × 250 ram) is used for the purification of2',5'-dd-3'-FSB[3H]Ado. A semipreparative column (10 × 250 ram) is used for smaller unlabeled preparations, and a preparative column (22 x 250 mm) is used for larger ones. For reversed-phase chromatography samples are injected onto a column previously equilibrated with 30% acetonitrile (or methanol), and elution is performed with an acetonitrile (or methanol 6) gradient. Recover- ies have been higher with acetonitrile. The 2',5'-dd-Y-FSBAdo can be identified by its characteristic UV spectrum with absorbance peaks at 258 nm (e 16300 cm -l M -1) and 232 nm (e 20100 cm -t M-l), 13 as well as by other analytical techniques. We verify the structure (Fig. 1) by comparison of retention times, UV spectra, ~H nuclear magnetic resonance (NMR) spectra, and fluorine NMR spectra in deuterated (CH3)2504 with those obtained with FSO2BzC1, 5'-FSBA, 2',5'-ddAdo, and adenosine. The peak containing 2',5'-dd-3'-FSBAdo is collected, evaporated to dryness, and stored in a desiccator at -20 °.

NH 2

5' CH3 0 /

0 /

0~° ~C~O

F FIG. 1. Structure of 2',5'-dd-3'-FSBAdo (2',5'-dideoxy-3'-p-fluorosulfonylbenzoylade-

nosine).

[4] "P"-SlTE AFFINITY LIGANDS 61

Synthesis of 3'-arylazidoiodo-2',5'-dideoxyadenosine Photoaffinity Probes

Arylazido analogs have been used as covalent affinity probes for many nucleoside binding domains. 14'15 Exposure of the ligand to UV light causes the formation of several intermediates that subsequently react with proteins. Although the synthesis of 3'-substituted arylazido analogs of 2',5'- ddAdo should be straightforward based on procedures used for other nucleotides,~5.~6 in practice we have encountered poor coupling efficiencies with these methods and substantial difficulties owing to the insolubility of the 2',5'-ddAdo in the solvents that are typically used for these reactions. To circumvent these problems an alternative synthetic approach has been developed that involves the intermediate formation of symmetric aryl anhydrides (see Scheme l; boldface Roman numerals in text refer to products and boldface arabic numbers refer to intermediates). In this chapter we describe the synthesis of two 3'-substituted 2',5'-ddAdo affinity ligands: I, 3'-(p-azido-m-iodophenylacetyl)-2',5'-ddAdo; and II, 3'-(p- azido-m-iodophenylbutyryl)-2' ,5'-ddAdo. 17 The compounds differ only in the length of the carbon chain linking the arylazido moiety with 2',5'- ddAdo.

Reagents 2',5'-ddAdo, prepared as described above, is dried over P205 un-

der vacuum Dimethyl sulfoxide [ ( CH 3) 2804 ] is best if redistilled under partial

vacuum Deuterated (d6) dimethyl sulfoxide (Aldrich; 99.9 atom %; No.

29614-7) Silica gel thin-layer plates (DC-Plastikfolien, Kieselgel 60 F254 , EM

Science No. 5735) Silica gel for flash chromatography (40-63 tzm, 230-400 mesh ASTM:

EM Science #9385-3) 1,3-Dicyclohexylcarbodiimide (DCC; Aldrich No. D8,000-2) 4-Dimethylaminopyridine (DMAP; Aldrich No. 33,245-3) p-Aminophenylacetic acid (Aldrich No. A7, 135-2) 4-(p-Aminophenyl)butyric acid (Aldrich No. 33,533-9) Sodium iodide

14 H. Bayley and J. R. Knowles, this series, Vol. 46, p. 69. ~5 R. J. Guillory and S. J. Jeng, this series, Vol. 46, p. 259. 16 S. Chen and R. J. Guillory, J. Biol. Chem. 252, 8990 (1977). 17 I. Shoshani, H. Qiu, F. Johnson, and R. A. Johnson, Nucleosides Nucleotides, in

press (1994).


OH ~=o

(+H2). NaI / ThC13

P

NH2

1 n = l 2 n = 3

O

o=c ~ ~ c = o (+H2)n

N3 N3 7 n = l 8 n = 3

OH OH &o d=o

NH2 N 3

3 n = l 5 n = l 4 n = 3 6 n = 3

CH3CN, TEA, DM.~P <..~..N.......u--.~N/.,.

I ~ . C ~ ,

,::, II:n = 3

3'-(p-azido, m-iodophenylacetyl)- 2'5'-ddAdenosine

SCHEME 1. Synthesis of photoaffinity probes. (From Shoshani et al. 17)

Thallium trichloride (Aldrich No. 33,322-0): extreme care must be exercised in the use of this highly toxic compound

Sodium metabisulfite Solvents used in syntheses: dichloromethane (DCM), dimethylform-

amide (DMF; Fisher), triethylamine (TEA; Aldrich No. 23,962-3), acetonitrile; DCM, TEA, and acetonitrile are redistilled over calcium hydride and are stored under dry nitrogen, and DMF is stored over previously dried molecular sieves

General solvents: ethyl acetate, methanol, chloroform, toluene, acetic acid, sulfuric acid

Preparation of Iodinated Precursors

p-Amino-m-iodophenylacetic Acid (3)

1. Suspend p-aminophenylacetic acid (1 in Scheme 1 ; 0.907 g, 6 retool) and sodium iodide (0.9 g, 6 retool) in 180 ml of 0.1 M sodium acetate

[4] "P"-SlTE AFFINITY LIGANDS 63

buffer, pH 4.2, in a 500-ml three-port round-bottomed flask as described by Lowndes et al. TM This is done under nitrogen, which is introduced through one of the ports. A reflux condenser is attached to a second port.

2. The mixture is refluxed on a steam bath and is agitated both with a gentle stream of nitrogen gas and with magnetic stirring.

3. Sodium acetate buffer (50 ml) containing 2.2l g of thallium trichloride is added dropwise over a 1-hr period.

4. After an additional 1 hr the reaction is stopped by the addition of 0.69 g of sodium metabisulfite in 15 ml of water, and the mixture is then cooled to room temperature and transferred to a separatory funnel.18

5. Rinse the reaction flask with 100 ml ethyl acetate and add this wash to the separatory funnel. The aqueous phase is then extracted twice more with 100 ml of ethyl acetate.

6. The combined ethyl acetate extracts are concentrated by rotary evaporation, and the oily residue is redissolved in approximately 2 ml of chloroform/acetic acid (20: 1, v/v).

7. Purify the product by flash chromatography 19,2° on silica gel with the same solvent mixture. Pool fractions containing product and remove solvent by rotary evaporation. Wash the material twice with methanol.

8. Add just sufficient methanol to dissolve the oily residue on the bottom of the flask and add water ( -100 ml) to precipitate the product. Water and methanol are then removed by rotary evaporation. Preparations have yielded 0.76 g of pure 3 (46%), Rf 0.45 on silica gel TLC (chloroform/ acetic acid, 20: 1, v/v)

4-(p-Amino-m-iodophenyl)butyric acid (4). The same experimental procedure is used as in the case of 3, starting from p-aminophenylbutyric acid (2). The desired iodinated derivative (4) is again separated from starting material by flash chromatography but with chloroform/acetic acid (40 : 1, v/v) as the eluant. In our preparations product 4 (1.09 g; yield 60%) was shown to be homogeneous by TLC17: R f 0.34 on silica gel TLC (with chloroform/acetic acid (20: 1, v/v) and Rf 0.44 on silica gel TLC with toluene/acetonitrile/acetic acid (20 : 1 : 2, v/v)

Preparation of lodoazido Precursors

p-Azido-m-iodophenylacetic Acid (5). The synthesis is done under subdued light.

1. Suspend compound 3 (0.7 g, 2.53 mmol) in 3% sulfuric acid (50 ml) cooled to 4°. ~8

58 j. M. Lowndes, M. Hokin-Neaverson, and A. Ruoho, Anal. Biochem. 168, 39 (1988). 19 W. C. Still, M. Kahn, and A. Mitra, J. Org. Chem. 43, 2925 (1978). z0 D. F. Taber, J. Org. Chem. 47, 1351 (1982).


2. Dropwise add sodium nitrite (0.35 g, 5.06 mmol) in ice/water (25 ml) and stir the reaction at 4 ° for 30 min.

3. Dropwise add sodium azide (0.33 g, 5.06 mmol) in ice-cold water (4.2 ml) and stir at 4 ° for an additional 45 rain.

4. The desired product (5) is recovered by extracting the aqueous reaction mixture three times with approximately 50 ml of chloroform. The extractions are combined, and the chloroform is removed by rotary evaporation. The residue is washed twice by being resuspended in 50 ml of methanol and removing the methanol by rotary evaporation. Just sufficient methanol is added to dissolve the oily residue on the bottom of the flask and then water (I00 ml) is added. As the solution is evaporated to remove the methanol compound 5 precipitates. The water is then removed by rotary evaporation under reduced pressure. In routine preparations the solid product 5 (0.613 g; 80% yield) was shown to be homogeneous by TLC and is used without further purification. The product can be verified by the characteristic IR spectral band (in chloroform) for arylazido at 2123 cm -~. Reference values for TLC are as follows: Rf 0.37 on silica gel TLC with toluene/acetonitrile/acetic acid (20 : 1 : 2, v/v) and R e 0.88 on silica gel TLC with butanol/acetic acid/water (8.5 : 0.5 : 1, v/v).

p-Azido-m-iodophenylbutyric acid (6). The procedure used is the same as in the case of 5 and is also done under subdued light. In our preparations yield was 0.734 g (86.7%). Reference values are as follows: Rf 0.56 on silica gel TLC with toluene/acetonitrile/acetic acid (20 : 1 : 2, v/v) and Rf 0.90 on silica gel TLC with butanol/acetic acid/water (8.5 : 0.5 : 1, v/v).

Preparation of Symmetric Anhydride Precursors

p-Azido-m-iodophenylacetic Anhydride (7)

1. Dissolve p-azido-m-iodophenylacetic acid 5; 1.00 g, 3.3 mmol in 20 ml of DCM.

2. Cool the solution in an ice/water bath, then add DCC (0.34 g, 1.65 mmol) and stir the mixture at 0 ° under dry N2 for 1 hr.

3. Remove the insoluble N,N'-dicyclohexylurea formed during the reaction by filtration under N2 and evaporate the solvent under reduced pressure. The conversion of the acid to anhydride can be verified by IR spectroscopy. The C ~ O band of the COOH group (1706 cm -l) disappears, and the carbonyl bands characteristic for anhydrides (1752 and 1819 cm -1) appear. The crude anhydride is then used as such.

p-Azido-m-iodophenylbutyric Anhydride (8)

1. Use the same procedure as in the case of 7. Dissolve p-azido-m- iodophenylbutyric acid 6; 1.06 g, 3.21 mmol) in 15 ml of DCM.

[4] "P"-S1TE AFFINITY LIGANDS 65

2. Cool the solution in an ice/water bath, add DCC (0.34 g, 1.65 mmol), and stir the mixture at 0 ° under dry N2 for 2 hr.

3. Dry the oily residue over P205 under vacuum overnight. The dried anhydride is then used as such for further reactions.

Preparation of Iodoazidodideoxyadenosine Affinity Probes

3'-( p-Azido-m-iodophenylacetyl)-2',5'-dideoxyadenosine (!)

1. Mix 2' ,5'-ddAdo (300 rag, 1.275 mmol), triethylamine (0.195 ml, 1.4 retool), and DMAP (100 rag) together in 10 ml of acetonitrile. DMF (6 ml) is added to dissolve completely the 2',5'-ddAdo.

2. Dissolve compound 7 (1.059 rag, 1.8 retool) in I0 ml of acetonitrile. 3. Stir the reaction mixture at room temperature for 48 hr. 4. Remove the solvent under reduced pressure. Purification of the

reaction mixture by flash chromatography on silica gel, with ethyl acetate ethanol (9 : 2, v/v), as solvent, results in I (330 mg; yield 49%).

5. Purified product I must be kept in the dark and is stable indefinitely desiccated at -65 ° .

3'-(p-Azido-m-iodophenylbutyryl)-2',5'-dideoxyadenosine (IlL Use the same procedure as for the preparation of I. In our experience, the reaction of 2',5'-ddAdo with 4-azido-3-iodophenylbutyric anhydride (8) goes to completion much faster than with the acetic anhydride (6). We have observed that after 2 hr TLC analysis indicated approximately 10% residual 2',5'-ddAdo. Even so, the reaction is allowed to continue overnight. By this procedure we have detected only one major product by TLC. The product is purified by flash chromatography as for I, and yields have been high (89.5%). Purified product II must be kept in the dark and is stable indefinitely dessicated at -65 °.

Use of 2',5'-Dideoxy-3'-p-fluorosulfonylbenzoyladenosine

The sulfonyl fluoride moiety of T,5'-dd-3'-FSBAdo reacts with a number of amino acids, but of particular importance in experiments with adenylyl cyclase is its reactivity with thiol (SH) groups. Dithiothreitol is typically used to protect adenylyl cyclase during isolation. It is therefore necessary that the protective thiol be removed before exposure of the enzyme to 2',5'-dd-3'-FSBAdo. We previously reported the irreversible inactivation of a partially purified adenylyl cyclase from bovine brain by 2',5'-dd-3'-FSBAdo. 6 We have extended this to include the purified bovine enzyme and, in collaboration with Dr. A. G. Gilman (UTSW Medical School, Dallas, TX), recombinant type I adenylyl cyclase expressed in Sf9 (Spodoptera frugiperda, fall armyworm ovary) cells. Inhibition of


adenylyl cyclase by 2',5'-dd-3'-FSBAdo characteristically exhibits two phases. Whereas inhibition of the enzyme by 2',5'-ddAdo is immediate in onset and the rate of cAMP formation is linear with respect to time, inhibition by 2',5'-dd-3'-FSBAdo increases with longer incubation times. 6 This slow phase for inhibition by 2',5'-dd-Y-FSBAdo is due to a slowly developing irreversible inactivation of the enzyme that is concentration- dependent and is not reversed on extensive dialysis, whereas inhibition by 2',5'-ddAdo is freely reversible (Fig. 2). Thus, since reaction of the sulfonyl fluoride is not induced by light but occurs as a function time, temperature, and reactant concentrations, these must be used to advantage to effect covalent attachment.

Procedure

1. Dialyze crude detergent-dispersed preparations of adenylyl cyclase overnight against 10 mM MOPS [3-(4-morpholino)propanesulfonic acid], pH 7.5, 1 mM MgCI 2 , I% (v/v) Lubrol PX, and 5% (v/v) glycerol, principally to remove dithiothreitol. The purified catalytic moiety of adenylyl cyclase is dialyzed against the same buffer with 0.1% (v/v) Tween 60 substituting for 1% (w/v) Lubrol PX.

1.0

0.8

z $

> 0.6

0.4

z 0.2

• ~ ddAdo After Dialysis

"~'D ~ . - - ~ ddFSBAdo Before Dialysis

o

ddAdo Bofore Dialysis ddFSBAdo After Dia~ysi~

r , i ~o 3 o i . . . . . I 3 I 100 300

NUCLEOSIDE CONCENTRATION ( ~ M )

FIG. 2. Concentration curve for irreversible inactivation of adenyiyl cyclase by 2',5'-dd- 3'-FSBAdo. Partially purified adenylyl cyclase was incubated for 45 rain at 30 ° with the indicated concentrations of 2',5'-dd-3'-FSBA (O, 0) or 2',5'-ddAdo (I-1, II). Adenylyl cyclase activities were measured in the presence of 50 gM forskolin with 50-/~1 aliquots of samples after dialysis (solid lines) or 30/zl of samples before dialysis (dashed lines). (From Yeung and Johnson. 6)


2. Incubate the adenylyl cyclase preparation with 100 txM 2',5'-dd- 3'-FSBAdo at 30 ° for 45 rain in buffer containing 10 mM MOPS, pH 7.5, 1 mM MnCI 2 , 10 mM MgCI2,0.1% Tween 60, 5% glycerol, 100/xM ATP, and 0.3 mg/ml bovine serum album in a final volume of 220 txl.

3. At the end of the incubation add 20/zl bovine serum albumin to give a final concentration of I mg/ml. Bovine serum albumin does not interfere with the action of 2',5'-dd-3'-FSBAdo on adenylyl cyclase. It serves to protect the small amount of adenylyl cyclase protein during dialysis and also has allowed us to monitor recovery of protein on the microdialyzer. Protein recovery has always been greater than 95% of that added.

4. Several techniques have been used to separate bound and free 2',5'- dd-3'-FSBAdo, but the most effective has been dialysis. Load all or part the resulting solution (240 /zl) into each well of a Microdialyzer S-500 (from Pierce), which has been modified by adding five more dialysis wells so that ten samples and/or controls can be treated at once in the same dialysis system. Dialyze overnight at 4 ° against 10 mM MOPS, pH 7.5, 10 mM MgC12, 0.1% Tween 60, 5% glycerol, with a flow rate of 0.7 ml/min. When crude detergent-dispersed adenylyl cyclase is used, we increase Tween 60 to 0.2%. In control reactions adenylyl cyclase is incubated under similar conditions, except that 2',5'-dd-3 '-FSBAdo is replaced with either solvent [ ( CH 3) 2504 ] o r |00 ~M 2',5'-ddAdo, a reversible "P"-site inhibitor. If the concentration of (CH3)2SO4 carried into the adenylyl cyclase incubation is less than 3% it will not affect enzyme activity.

5. lftritiated 2',5'-dd-3'-FSBAdo is used, reaction conditions and dialysis procedures are the same as above. Treated enzyme is then resolved by denaturing slab gel electrophoresis or is precipitated with antibody.

6. To verify that the reaction occurs at the " P" site it should be prevented by another "P' '-site ligand such as 2' ,5'-ddAdo and not affected by ATP. 6 To establish whether coupling is via cysteine, inactivation should be at least partially reversible by treatment of the enzyme with dithiothreitol.6,~L2t-~3

Use of 3' -( p-Azido-m-iodophenylacetyl)-2', 5' -dideoxyadenosine (I} and 3'-(p-Azido-m-iodophenylbutyryl)-2',5'dideoxyadenosine (II}

The use of the arylazido compounds I and II is complicated by several factors. First are the characteristics of arylazido compounds in general,

21 A. E. Annamalai and R. F. Colman, J. Biol. Chem. 256, 10276 (1981). 22 C. T. Togashi and E. Reisler, J, Biol. Chem. 257, 10112 (1982). 23 M. R, E1-Maghrabi, T. M. Pate, G. D'Angelo, J. J. Correia, M. O. Lively, and S. J. Pilkis,

J. Biol. Chem. 262, 11714 (1987).


namely, that reactions are induced by UV light, that a number of reactive intermediates are involved, and that delayed reactions can and do occur. 24 Second is the poor solubility of these compounds in aqueous buffers compatible with adenylyl cyclase activity determinations. Third is the sequence of reactions used in the synthesis which implies that for the preparation of ~25I-labeled ligand the isotope must be introduced early in the synthesis. This necessitates considerable handling of radioactive reagents. Also, the need to obtain the highest specific radioactivity possible precludes increasing reactant concentrations to drive reactions. Fourth, adenylyl cyclase is extremely sensitive to inactivation by oxidation and is inactivated by irradiation with UV light (Fig. 3). 25 Noteworthy is that UV inactivation of adenylyl cyclase is attenuated both by substrate and by a nitrogen atmosphere (Fig. 3).

Nonetheless, the potential value of such compounds in elucidating structure-function relationships for adenylyl cyclase reactive domains is high, and information can be obtained with them that will complement data obtained by other techniques. The procedures delineated below have been found to be useful for several forms of adenylyl cyclase; crude enzyme dispersed by detergent from rat or bovine brain and from Sf9 cells expressing the type I enzyme, in collaboration with Dr. A. G. Gilman: membrane-bound expressed type I enzyme from Sf9 cells; and partially purified forms of the enzyme from bovine brain. As with many affinity ligands, the cleaner the enzyme preparation the better the specificity.

Procedure

I. Remove dithiothreitol or 2-mercaptoethanol from the adenylyl cyclase preparation. Aryl azides are rapidly reduced to the corresponding amines by dithiols, although the rate of reaction of 2-mercaptoethanol with aryl azides is three orders of magnitude slower than that of dithiothreitol. 24,26

a. For membrane preparations, whether expressed in Sf9 cells or obtained from bovine or rat brain, this is accomplished by three repeated centrifugations (29,000 gav, 10 min, 4 °) and resuspensions in 20 mM HEPES [N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)], pH 7.4, 5% glycerol.

24 H. Bayley and J. V. Staros, in "Azides and Nitrenes, Reactivity and Utility" (E. F. V. Scriven, ed.), Academic Press, New York, 1984.

25 I. Shoshani, H. Qui, F. Johnson, R. Taussiq, A. G. Gilman, and R. A. Johnson, in review (1994).

26 j . V. Staros, H. Bayley, D. N. Standring, and J. R. Knowles, Biochem. Biophys. Res. Commun. 80, 568 (1978).

14] "P"-SITE AFFINITY L1GANDS 69

• , , A I ~ E N2 +MnATP

O < _ . . I

D- 1 0 -

I , - - Z i.u 0 r'.r" ILl 0 . .

T . . . . . .

2 ~ .- O ~ C O N T B O L - N2OONTROL i

L . .

- L ; - -

u v - I R R A D I A T I O N (min)

FIG. 3. Protection against UV irradiation by MnATP and by N2. A detergent-dispersed rat brain adenylyl cyclase, in a buffer containing 50 mM HEPES, pH 7.5, 0.1% Lubrol PX, without or with 100 tzM ATP and 10 mM MnCI2, was irradiated with UV light for the indicated times in an atmosphere of air (02) or nitrogen (N2). The photochemical reactor was housed in a plexiglass incubator box, and the temperature within the reactor was maintained at 10 ° by passage of precooled air or nitrogen through it. Adenylyl cyclase activity was measured with MnCIa and ATP as substrates. (From Shoshani et al. 25)

b. Detergent-dispersed enzyme from rat brain is dialyzed overnight against 10 mM HEPES, pH 7.5, 1 mM MgCI2, 1% Lubrol PX,

5 % glycerol. c. Detergent extract of Sf9 cells expressing hexahistidine-tagged type

I cyclase is dialyzed against 10 mM HEPES, pH 8.0,400 mM NaCI, 1 mM MgCI 2 , 20% glycerol, 0.1% Lubrol PX.

2. Prepare 10 mM stock solutions of the arylazido compounds in 100% (CH3)2SO 4 (5.2 mg I/ml, 5.48 mg ll/ml). It is important that the (CH3)2804 be of very high quality for this purpose (e.g., redistilled over Call2). We have found that typical commercial (CH3)2504 has contaminants which, when exposed to UV irradiation, even for the brief time used in these experiments, can cause unpredictable effects (stimulation or inhibition) on adenylyl cyclase.

3. In quartz tubes incubate adenylyl cyclase for I0 min at 30 ° with compound I or U in medium containing 50 mM HEPES, pH 7.5, 10 mM

70 AOENVLYL CYCLASES [4]

All i 20~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . After uv irradiation

=" 18:_ ~ [ W i~fter dialy sis .... "~ 1 6 - . . . . . . . . . ' . . . . . . . L . . . . . . . . . . . . . . . L i } 14: ......... i ........ ~ ! ...................... ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

l a #

aj

~ 12; ........ i~

loi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8-- . . . . .

d ......

" 41

21 . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

NONE 2'5'ddAdo I-Az-Ph-Act- I-Az-Ph-But- ddAdo ddAdo

ADDITIONS

FIG. 4. Irreversible inactivation of adenylyl cyclase by iodoazidoaryl 2',5'-ddADO analogs (I) and (II). A detergent-dispersed adenylyl cyclase from rat brain, in a buffer containing 50 mM HEPES, pH 7~5, 0.1% Lubrol PX, 100/zM ATP, and 10 mM MnCI2, was exposed to UV light for l min in the photochemical reactor maintained at 8 ° in a nitrogen atmosphere, with either carrier [10% (CH3)2SO4], 1 mM 2',5'-ddAdo, l-azidoiodophenylacetyl-ddAdo (I), or azidoiodophenylbutyryl-ddAdo (I1). After stopping inactivation by addition of dithiothreitol, aliquots were taken and adenylyl cyclase activity determined. The remainder of the samples were assayed for activity after dialysis overnight in the microdialyzer. (From Shoshani e t al. 25)

MnC12, 100/zM forskolin. ATP (100/zM) can be included in the medium to help protect the enzyme (cf. Fig. 3). The presence of forskolin enhances the sensitivity of adenylyl cyclase to inhibition by the 2 ' ,5 ' -ddAdo analogs. For detergent-dispersed forms of the enzyme include 0.1% Lubrol PX in the medium. Total volume is 100 to 200/~1.

4. Transfer the quartz tubes to the UV reactor (the Rayonet Photo- chemical Reactor from the Southern New England Ultraviolet Company is equipped with 254 nm UV lights) and expose to UV light for 1.0 rain at room temperature. It is important that the exposure time be precise for reproducible results and to circumvent UV-induced inactivation of the enzyme with longer exposures (cf. Fig. 3).

5. Stop the photolysis by addition of a small volume of dithiothreitol to a final concentrat ion of 50 mM.

[ 5 ] C a 2 + - S E N S I T I V E ADENYLYL CYCLASES 71

6. If it is desirable to remove excess unreacted compound and other reaction constituents, we have found dialysis to be effective. Dialyze overnight against 10 mM HEPES, pH 7.5, 5 mM dithiothreitol, 5% glycerol, 0.1% Lubrol PX. The dithiothreitol is reintroduced to the adenylyl cyclase at this point to stabilize remaining enzyme activity.

7. For arylazido photoaffinity probes there are several important con- trois that must be included in experiments. These are needed to establish that coupling is specific and is due to the probe itself. First, an experiment should be run without the UV irradiation step to establish that light is required for inactivation. A corollary of this experiment is to remove the ligand following incubation, but before exposure to UV irradiation, to establish that both light and the arylazido ligand are required for inactivation. Second, the compound (e.g., I or II) should be irradiated and then treated with dithiothreitol as above before exposure to enzyme. This establishes that inactivation is not due to some downstream reaction product. Third, the experiment including irradiation should be conducted with carrier solvent [(CH3)2804 in these experiments]. Fourth, to establish site specificity the effects of substrate and/or other "P"-site ligands should be studied together with the affinity ligand. We have used from 0 to 1 mM ATP and/or 1 or 2 mM 2',5'-ddAdo for this purpose. Both iodoazido- phenylacetyl and iodoazidophenylbutyryl analogs of 2',5'-ddAdo inhibit the brain adenylyl cyclase irreversibly, whereas inactivation by 2',5'- ddAdo is freely reversible (Fig. 4). This inactivation is both ligand-dependent and light-dependent. Interestingly, it is partially prevented by high concentrations of either ATP of 2',5'-ddAdo, 25 suggesting that these ligands may interact with both the catalytic active site and the "P" site and may be useful in analysis of both domains.

[5] R e g u l a t i o n o f Ca 2 + -Sens i t ive A d e n y l y l Cyc la ses by C a l c i u m Ion in Vitro a n d in Vivo

By DERMOT M. F. COOPER

Introduction

The fact that adenylyl cyclase (EC 4.6.1,1, adenylate cyclase) in brain could be stimulated by Ca2+/calmodulin was first described in



1975. l'z The early emphasis was on the fact that this was a calmodulin- regulated activity, but the realization that cytosolic Ca 2+ levels ([CaZ+] 0 changed dynamically in response to neurotransmitters and to the state of membrane polarization, while calmodulin is present in most tissues at very high concentrations, shifted the emphasis to the fact that this was a Ca2+-sensitive enzyme. 3 Of paramount importance to the physiological relevance of these observations was the concentration of Ca z + that elicited an effect on the enzyme. For instance, based on one set of assumptions of free [Ca 2+] in adenylyl cyclase assays, sometime ago it was claimed that basal [Ca2+]i levels were such that the enzyme would always be stimulated. 4 Consequently, a primary issue is to replicate the intracelullar range of Ca 2+ concentrations in vitro and determine whether these concentrations regulate Ca: +-sensitive cyclases. We now know that [Ca :+ ]i varies dynamically in neurons and--should adenylyl cyclase be sensitive to these changes--it could provide an important modulation of cAMP pathways.

Thus, there are two issues in considering Ca2+-sensitive cyclases: (1) whether physiologically achieved concentrations of Ca 2+ modulate the cyclases in vitro and (2) whether a physiological elevation in [Ca z + ]i elicits the anticipated effect on cAMP synthesis in vivo. More recently, adenylyl cyclase activity from a number of peripheral sources, including pituitary, 5 cardiac tissue, 6 somatotrophs, 7 and various cell lines (including pituitary- derived GH3,8 C6 glioma, 9 and NCB-20 neuroblastoma cells~°), has been found to be inhibited by the same range of concentrations of Ca 2+ that stimulate the brain enzyme in in vitro assays. The same issues of whether physiologically achieved concentrations elicit this response in vitro and whether the effect actually occurs in vivo following [Ca:+]i elevation sur- round CaZ+-inhibitable adenylyl cyclases. The question is compounded by the fact that all adenylyl cyclases are inhibited by Ca 2+ in the millimolar concentration range, which is clearly nonphysiological and is believed to

I W. Y. Cheung, L. S. Bradham, T. J. Lynch, Y. Lin, and E. A. Tallant, Biochem. Biophys. Res. Commun. 66, 1055 (1975).

a C. O. Brostrom, Y.-C. Huang, B. M. Breckenridge, and D. J. Wolff, Proc. Natl. Acad. Sci. U.S.A. 72, 64 (1975).

3 D. M. F. Cooper, M. K. Ahlijanian, and E. Perez-Reyes, J. Cell. Biochem. 36, 417 (1988). 4 S. Tomlinson, S, Mac Nell, and B. L. Brown, Clin. Endocrinol. 23, 595 (1985). 5 G. Giannatasio, R. Bianchi, A. Spada, and L. Vallar, Endocrinology (Baltimore) 120,

2611 (1987). 6 R. A. Colvin, J. A. Oibo, and R. A. Allen, Cell Calcium 12, 19 (1991). 7 N. Narayanan, B. Lussier, M. French, B. Moor, and J. Kraicer, J. Endocrinology (Balti-

more) 124, 484 (1989). 8 C. L. Boyajian and D. M. F. Cooper, Cell Calcium 11, 299 (1990). 9 M. A. DeBernardi, T. Seki, and G. Brooker, Proc. Natl. Acad. Sci. U.S.A. 88, 9257 (1991).

t0 C. L. Boyajian, A. Garritsen, and D. M. F. Cooper, J. Biol. Chem. 266, 4995 (1991).

[5] Ca2+-SENSITIVE ADENYLYL CYCLASES 73

represent competition between Mg 2+ and Ca 2+ for the catalytic site of the enzyme. 11,12 The latter phenomenon gives rise to a bell-shaped response of brain adenylyl cyclase to an extended range of [Ca 2 +] and to two inhibitory phases, separated by a plateau, in a CaZ+-inhibitable adenylyl cyclase. 3"J° Hence, the purpose of this chapter is to outline some practical considerations in the detection of the regulation by physiologically relevant concentrations of Ca 2 ÷ ofadenylyl cyclases in vitro and to indicate some strategies for the detection of effects of [CaZ+]i on such activities in intact cells.

Detection of Regulation of Adenylyl Cyclase by Calcium Ion Concentrations in Physiologically Significant Ranges

The concentration range over which Ca 2 ÷ exerts its effects on adenylyl cyclase is critical in determining the potential physiological relevance of the effect. Physiologically significant concentrations would be expected to be those that are achieved inside cells on stimulation of Ca 2 ÷-mobilizing processes, such as phospholipase C or modulation of ion channel activity. The cytosolic range is generally approximately 0.1/zM at rest and around 0.8-1 ~M on stimulation. These concentrations of Ca 2÷ are much lower than those found extracellularly (millimolar range) and are also lower than the ambient concentrations found, for instance, in glass-distilled water ( - 10 t~M).

The most readily available method for obtaining graded concentrations in these very low ranges is to use an EGTA-buffering system and a computer program that allows the estimation of free [Ca2+], based on the association constants of the various complexes that can form among the components of an adenylyl cyclase assay which impact on Ca 2+ , such as Na + , M g 2+ , ATP 4-, Ca 2 +, and EGTA 4-. Concentrations that are predicted to be in the supramicromolar range can be confirmed by the use of Ca 2÷- sensitive electrodes, although the presence of Mg 2+ (> ! mM) may con- found these measurements. Calibration of the concentrations with a Ca z +- specific indicator such as Fura-2 (Molecular Probes, Eugene, OR) can also be envisaged, although such measurements also depend on assumed values for the association between Ca z+ and Fura-2.

The computational approach is widely used, and all of the computer programs use identical association constants from standard sources 13 and yield similar results. An example of the effects of a range of C a 2+ c o n c e n -

it L. Birnbaumer, Biochim. Biophys. Acta 300, 129 (1973). 12 M. L. Steer and A. Levitzki, J. Biol. Chem. 250, 2080 (1975). ~3 A. E. Martell and R. M. Smith, "Critical Stability Constants," Vol. 2, p. 269. Plenum,

New York, 1975.


trations calculated by such means on the adenylyl cyclase activity of both cerebellar and NCB-20 cell plasma membranes is shown in Fig. 1. The data illustrate the striking difference between the response of adenylyl cyclase to Ca 2÷ concentrations in the physiological range in most brain areas, compared to certain peripheral tissues and cultured cells.

Assay of Adenylyl Cyclase Activity

The adenylyl cyclase activity of purified plasma membranes (prepared by sucrose density gradient methods, t° generally 6-10 /zg protein per assay) is measured in the presence of the following components (final concentrations): 4 m M phosphocreatine (disodium salt), 20 units/ml creatine phosphokinase, 0.1 mM cAMP, 1 unit/ml adenosine deaminase, 1 mM MgCI2, 80/~M ATP (disodium salt), 3-5 × 10 6 cpm/assay [a-32p]ATP [tetra(triethylammonium) salt] (Amersham, Arlington Heights, IL), 70 m M Tris-HC1, pH 7.4, 10/xM GTP, and 5/zM prostaglandin E1 (PGE0, as required, 200/zM EGTA, and a range of Ca 2 + concentrations. The reaction mixture (final volume 100/zl) is incubated at 30 ° for 20 min. Reactions are stopped, and the [32P]cAMP formed is quantified by the standard method of Salomon et al.t4

Determination of Free Calcium Ion Concentrations

Free concentrations of Ca 2÷ are calculated as described previously.I° This involves an iterative computing program that solves the equations describing the complexes formed in a mixture comprising the assay ingredients that affect free divalent cation concentration, namely, ATP, GTP, EGTA, H +, Mg 2 +, Na ÷, and Ca 2 ÷. 13 Final concentrations (in micromolar) of added CaC12 in the assay mixture (along with 200/~M EGTA) give rise to the free Ca 2+ concentrations (in micromolar), indicated in parentheses, as follows: 132 (0.080), 152 (0.10), 155 (0.14), 168 (0.22), 178 (0.33), 185 (0.49), 191 (0.81), 197 (1.7), 202 (4.0), 210 (10), 223 (23), 241 (40), and 260 (58). A background contribution of 10/zM Ca 2+ is assumed.

Comments on Assay

Chelator. Although theoretically it is possible to calculate an appropriate range of free Ca 2 ÷ concentrations using any concentration of EGTA, in practice it seems more useful to use a relatively low final concentration. One reason for this choice is that the binding of Ca 2+ by EGTA results

i4 y. Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974).

A p-

E 0

E

<~ U l

:>.,

" 0

300

2 0 0

100

" - C a M

• + l ) u

0 . . . . . " " l . . . . . . . . i ' ' • ' ' ' ° ° ~

10-8 10-7 10-6 10-5

C .m

E 6~ 95

0

E

~ 8 5

U

~ 75

~ 6 5

C

" 0

55

b

0 10 "7 10 -6 10 "5

free [Ca2+] (M)

FIG. 1. Differential effects of Ca2+/calmodulin on brain and NCB-20 adenylyl cyclase. Effects of Ca-' + and calmodulin on adenylyl cyclase activity were studied in EGTA-washed membranes prepared from (a) rat cerebellum and (b) NCB-20 cells. Adenylyl cyclase activity was measured (a) in the absence of stimulating hormone and (b) in the presence of 5 txM PGE~ and 10 txM GTP. Concentrations of free Ca 2 ÷ were determined by an iterative computer program, as described in the text. Data are triplicate determinations from a representative experiment that was replicated at least four times. (Reproduced from Boyajian e t a l . , ~°

with permission.)


in the release of protons and the lowering of pH; thus, lower EGTA concentrations require less buffering. Second, for reasons that are not quite clear, the concentration of EGTA that is used affects the apparent breadth of the range over which effects of Ca 2+ are detected. There also appears to be some variability in the purity of EGTA from different suppliers. Thus it is important to recalibrate EGTA solutions from time to time, either chemically by titration or fluorescence measurements or biochemically by Ca 2+ responsiveness. In our laboratory, Ca 2+ dose- response curves of brain adenylyl cyclase preparations are regularly monitored to control for unanticipated changes in background [Ca z÷] or [EGTA].

Magnesium Ion. The concentration of Mg 2+ exerts only a minor influence on responses to submicromolar Ca z+. The fact that responses to Ca z+ can be seen against a 1000- to 10,000-fold excess underlines the specificity of the Ca 2+ effects. Concentrations of Mg 2 + ( -1 mM) are used that simulate intracellular conditions and which, incidentally, enhance the sensitivity of adenylyl cyclase to hormonal regulation.

Creatine Phosphokinase. Creatine phosphokinase (along with bovine serum albumin, any other proteins used in the assay that are commercially purified, or unwashed plasma membrane preparations) should be considered as possible sources of calmodulin, as first pointed out by Goldhammer and Wolff. 15 Such contamination could mask an apparent requirement for calmodulin by a Ca 2+/calmodulin-sensitive enzyme. The most efficient test for contamination of assay components (other than membranes) for calmodulin is to perform an adenylyl cyclase assay of washed brain membranes and determine the lowest concentration of exogenous calmodulin that will elicit a significant stimulation of activity. Under standard conditions used in our laboratory, a small stimulation can be elicited by 5 nM added calmodulin; this indicates a low background and can be used as a bioassay for contributions from changing assay componentsl6; in other words, if higher concentrations of calmodulin are required to yield the same small stimulation, an increased background would be indicated.

Manganous Ion. Manganous ions, which can be used to enhance basal adenylyl cyclase activity, antagonize the effects of Ca 2+ at binding to most of its biological targets, including calmodulin. Hence, use of Mn 2+ is not recommended in studying effects of Ca 2+ .

Inhibition of Adenylyl Cyclase by Calcium Ion. Detection of inhibition of adenylyl cyclases by submicromolar [Ca 2÷ ] can be made quite difficult

15 A. R. Goldhammer and J. Wolff, Anal. Biochem. 124, 45 (1982). 16 K. K. Caldwell, C. L. Boyajian, and D. M. F. Cooper, Cell Calcium 13, 107 (1992).

[5] Ca2÷-SENSI'[IVE ADENYLYL CYCLASES 77

by a combination of factors: (1) no source yet described displays inhibition by submicromolar [Ca 2÷ ] of more than 50%; (2) in many tissues the inhibition can be considerably less than 50%; and (3) if Ca 2÷ concentrations are not accurately known, the profound contribution from submillimolar Ca 2 + can overwhelm the effect, giving the impression that submicromolar concentrations elicit either no inhibition or total inhibition. For these reasons, it is worthwhile to perform a Ca2+/calmodulin stimulation of brain adenylyl cyclase in a parallel assay in order to calibrate the Ca ,-+ concentrations and to ensure that the inhibitory effect observed is at least as potentially relevant as CaZ+/calmodulin stimulation of brain adenylyl cyclase. Ultimately, however, the only means of ensuring that an effect of Ca 2+ on cAMP synthesis is physiologically significant is to replicate the effect in intact cells, as a function of a physiological elevation of [Ca2+]i (see later).

Detection of Regulation of Adenylyl Cyclase by Calmodulin

Regulation of adenylyl cyclase by increasing concentrations of Ca 2t indicates a role for some type of Ca2+-mediated process, including but not restricted to calmodulin; Ca 2÷-dependent proteases or kinases, which may also modulate adenylyl cyclase, could mediate such effects. (Leupep- tin, which inhibits CaZ+-dependent proteases, should always be included in either adenylyl cyclase assays or membrane preparations; see Cooper and CaldwelF 7 for further discussion of artifactual inferences of calmodulin regulation of adenylyl cyclase.) Because most cells, including nervous tissue, contain high concentrations of endogenous calmodulin, the demonstration of a calmodulin-dependent adenylyl cyclase can only be attempted in two ways: either by antagonizing the effects of the endogenous calmodulin or by removing the endogenous calmodulin and demonstrating a requirement for exogenous calmodulin for the mediation of a Ca2+-depen - dent regulation of adenylyl cyclase.

Removal ofCalmodulin. Calmodulin is removed by washes in EGTA- containing buffers, since the binding of calmodulin to most of its targets is promoted by Ca2+; the chelation of Ca 2÷ promotes the dissociation of such complexes. For brain preparations, this may require two or three washes with I mM EGTA, including a 0.5 M NaCI step, in order to minimize the calmodulin content. The effectiveness with which calmodulin has been removed can be evaluated by comparing the ratio of Ca 2÷- stimulated activity in the presence and absence of added calmodulin. An

17 D, M. F. Cooper and K. K. Caldwell, Biochem. J. 254, 935 (1988).


example of the stimulation of rat cerebellar adenylyl cyclase by increasing concentrations of Ca 2+, acting via calmodulin in calmodulin-depleted membranes, is shown in Fig. la.

Some investigators also solubilize calmodulin from washed membranes and perform bioassays with Ca2+/calmodulin-stimulated phosphodiesterase to assess depletion. This strategy is informative with regard to the overall calmodulin content of a membrane preparation, but it does not address whether a particular adenylyl cyclase that might be associated with calmodulin has been depleted of calmodulin, since adenylyl cyclase is in such low concentrations relative to other membrane proteins.18 Fur- thermore, the lack of a need for exogenous calmodulin addition does not exclude the possibility that calmodulin is bound tightly to its target in a Ca2+-independent manner and is therefore not dissociable by EGTA washes.

Antagonism of Effects of Endogenous Calmodulin. Antagonism of endogenous calmodulin is an equivocal approach, owing in large part to the low potency of the currently available "antagonists." It turns out that the blockade of the association of calmodulin with its targets is a more readily antagonizable process than antagonism of the actions of calmodulin that is already bound. Thus, if calmodulin is depleted from membranes, such that adenylyl cyclase is dependent on exogenous calmodulin, then a simultaneous addition of calmodulin with graded doses of calmodulin antagonists, such as W-7, trifluoperazine, and calmidazolium (Sigma, St. Louis, MO) will reveal a graded antagonism of the calmodulin stimulation by modest concentrations ( -10 /zM) of these compounds. However, if the compounds are added to calmodulin-replete membranes, then very high concentrations (-500 /zM) will be required, calling into question whether they are acting as calmodulin antagonists or as nonspecific membrane perturbants (see, e.g., Ahlijanian and Cooper19).

Intact Cell Studies of Calcium Ion Effects

As mentioned above, the central issue concerning Ca 2 +-regulated adenylyl cyclases is whether they are regulated as a result of the elevation in the intact cell of [Ca2+]~. Regardless of the veracity of the assumptions concerning the applicability of calculated in vitro concentrations to concentrations that are achieved on [Ca 2 + ]i elevation in cells, a crucial requirement is to prove that a physiological elevation in [Ca2+]i actually causes

J8 j. M. Girardot, J. Kernpf, and D. M. F. Cooper, J. Neurochem. 40, 848 (1983). 19 M. K. Ahlijanian and D. M. F. Cooper, J. Pharmacol. Exp. Ther. 241, 407 (1987).

[51 Ca2+-SENSITIVE ADENYLYL CYCLASES 79

the changes in activity that would be anticipated from in vitro studies. Following are comments on a number of strategies that might be attempted.

Hormones. Intuitively, the most obvious strategy for determining whether a Ca'- +-sensitive adenylyl cyclase is regulated by increased [Ca 2+ ]~ is to take a hormone that elevates [Ca2+]i and determine whether it elicits the anticipated effect on cAMP accumulation. Ideally, a comparison of the kinetics of the [Ca2+] i rise and the change in the rate of cAMP synthesis should be made (in the presence of phosphodiesterase inhibitors; see Boyajian et al.l°). However, there are problems and ambiguities associated with the use of hormones. (I) Hormones that elevate [Ca2+]i should also be expected to activate protein kinase C, which affects various adenylyl cyclases differently. A strategy to address this problem is to desensitize protein kinase C by overnight treatment with a phorbol ester and determine whether an effect of hormone is preserved. 2° (2) Hormones that act via G proteins may liberate fly subunits, which exert type-specific effects on different adenylyl cyclases. 2~ This problem can be difficult to circumvent except perhaps by precluding the [Ca2+]i rise by the use of inhibitors or intracellular Ca 2+ chelators and determining the consequence of the action of the hormone. A loss of the hormone effect on cAMP synthesis by such treatment would strongly implicate the [Ca2+]~ rise. (3) Hormones may affect undetermined signal transduction pathways that may be responsible for the observed effects. Apart from eliminating known and amenable candidates, such as phospholipase A 2 (PLA2) or cGMP, the possibility of additional mechanisms should not be dis- counted.

lonomycin. Although a Ca 2+ ionophore, such as ionomycin, would appear to circumvent some of the problems associated with the use of hormones, there are complications in the use of ionophores. High concentrations of ionomycin (>1 txM) increase [Ca2+]~ to levels that are generally considered unphysiological. In addition, low concentrations of ionomycin can cause a [Ca2+] i rise which resembles that elicited by hormones in populations of cells, but the source of the [Ca2+]~ is uncertain; the mechanism of the [Ca 2 + ]i elevation is certainly unphysiological, and therefore any effects on cAMP synthesis are of uncertain physiological significance.

Thapsigargin. The sesquiterpene thapsigargin has the potential to provide a more physiological elevation of [Ca2+]i than ionophore, since it

20 A. Garritsen, Y. Zhang, J. A. Firestone, M, D. Browning, and D. M, F. Cooper, J. Neurochem. 59, 1630 (1992).

,.1 W. J. Tang and A. G. Gilman, Science 254, 1500 (1991).

80 ADENYLYL CYCLASES lSl

selectively causes the net release of intracellular Ca 2+ stores. 2z In a cell type where there is rapid flux between uptake and release from stores, thapsigargin could be anticipated to yield effects on CaZ+-sensitive adenylyl cyclases. However, if the flux was not particularly rapid, the effects of thapsigargin might be so slow in elevating [CaZ+]i that insufficient concentrations would be achieved to regulate the cyclases. Even in such situations, however, thapsigargin can be useful in depleting intracellular stores and, by such means, providing a tool for evaluating the contribution of Ca 2+ to the action of a [Ca2+]i-mobilizing hormone. 2°

Depolarization. Alteration of the membrane potential by increasing the extracellular [K ÷] may be a useful strategy in excitable cells. This would tend to elevate [Ca2+]~ to levels that are normally achieved on the firing of action potentials. In nonexcitable cells, of course, this would not be a feasible approach.

Concluding Comments

This chapter has outlined a number of strategies for detecting effects of perceived physiologically significant concentrations of Ca 2÷ and calmodulin on Ca2+-sensitive adenylyl cyclases in in vitro assays. The procedures outlined should allow the detection of a signal where such cyclases represent a significant fraction of the cyclases expressed in a particular tissue. A second theme was a discussion of the means whereby the physiological relevance of the in vitro effects of Ca 2 ÷ on adenylyl cyclase could be evaluated. None of the strategies presented is without reservations in their application. However, informed use of a number of these approaches leads to the conclusion that indeed the elevation in [CaZ+]~ can regulate Ca~+-sensitive adenylyl cyclases. 7,9A°'2°

It is curious, in confronting this apparently simple issue of whether [Ca2+]i rises may regulate Ca2+-sensitive adenylyl cyclases, that gaps in our knowledge about cellular [Ca 2 + ]i homeostasis are highlighted, in terms of spatial organization of pools within cells, the routes and mechanisms whereby Ca 2÷ enters cells, etc. Consequently, the question will soon need to be rephrased more explicitly, that is, which aspect of the elevation of [Ca2+]i is of most importance in regulating Ca2÷-sensitive adenylyl cyclases: the routes of Ca 2÷ entry, the spatial organization of [Ca2+] i, the rate of local elevation of [Ca2+]i rather than overall concentrations, etc. ? Thus, the study of Ca2+-sensitive adenylyl cyclases may provide unexpected insight into how [Ca2+]i may be organized to perform its intracellular functions or, conversely, how systems may have evolved around Ca 2÷

22 O. Thastrup, Agents Actions 29, 8 (1990).

[6] Gi ASSAYS IN TRANSFECTED CELLS 81

entry or mobilization mechanisms so that they respond most efficiently to this messenger.

Acknowledgments

The au thor ' s studies are supported by Nat ional Inst i tutes of Heal th Grants GM 32483

and NS 28389.

[6] G i Assays in T r a n s f e c t e d Cel ls

By YUN6 H. WON6

Introduction

The Gi proteins were initially purified from human erythrocytes and rabbit liver as substrates for ADP-ribosylation by pertussis toxin (or islet- activating protein, lAP) from the bacterium Bordetella pertussis. These proteins have been implicated in the hormonal inhibition of adenylyl cyclase (ATP pyrophosphate-lyase, cyclizing, EC 4.6.1.1; adenylate cyclase) and were thus named G i, by analogy to G~ which mediates the hormonal stimulation. Subsequent cloning and immunological studies have revealed that there are three distinct Gi proteins termed G~, Gi2, and Gi3. The o~ subunits of the G~ proteins are structurally similar to one another and exhibit greater than 85% similarity with respect to their amino acid sequences. A large number of hormone and neurotransmitter receptors are coupled to G~ proteins for mediating their physiological effects. It is widely accepted that the G i proteins play a pivotal role in signal transduction since effectors such as phospholipases C and A2, calcium, and potassium channels are known to be regulated by one or more pertussis toxin substrates. To delineate the specificity of various signaling pathways, it is of vital importance to decipher the functional role(s) played by each distinct G~ protein.

With the advent of genetic cloning and transfection techniques, it has become possible to study the function of G i proteins by expressing them in appropriate cell lines; ideally the cell lines should lack endogenous G~ proteins. However, most if not all cells endogenously express more than one subtype of G i proteins, making it extremely difficult to analyze the results. One way to overcome this problem is to overexpress a particular subtype of G~ in cultured cells with the hope that interference by other endogenous Gi proteins will be minimal. A better alternative is to express



constitutively activated mutant a subunits in cultured cells. Mutations at either of two conserved amino acids involved in the binding and hydrolysis of GTP have been shown to inhibit drastically the intrinsic GTPase, thereby allowing the a subunits to remain in the GTP-bound and active state, and consequently their corresponding effectors are constitutively regulated. Cultured cells expressing the mutant a subunits have been successfully used to identify some of the effectors regulated by G~, ~ Gz, 2 as well as Gq 3 proteins. The procedures described here focus on the use of transfected cells to study G~ function. It should be noted that the basic approach could be easily adopted for studying the function of other G proteins.

Choice of Transfection Procedures

The choice between transient and stable mammalian cell expression systems is primarily determined by the nature of the experiment. In general, transient expression assays allow one to evaluate rapidly the functional significance of the tranfected gene product. The ability to cotransfect a wide variety of cDNAs that encode different receptors, G-protein subunits, and effector molecules makes the transient system extremely powerful and attractive. In contrast to transient expression, if one wishes to study the effects of chronic G-protein activation, then establishing a stable expression system cannot be avoided. Stable cell expression is more labor- intensive and time-consuming, but it has the merit that, once established, it could be used repeatedly in many different types of experiments. Proce- dures that involve long-term measurements such as cellular proliferatio# should utilize the stable rather than the transient cell expression system.

Establishing Cell Lines That Stably Express G-Protein a Subunits

There are two general methods for establishing stable cell expression systems: the DNA of interest can be introduced into the cells via viral infection or by direct transfer. Viral infection is highly efficient (sometimes achieving close to 100% targeting of cells) but suffers from the fact that multiple steps are involved. Direct transfer of DNA into mammalian cells by calcium phosphate coprecipitation is simple and has become a popular

1 y. H. Wong, A. D. Federman, A. M. Pace, I. Zachary, T. Evans, J. Pouyss$gur, and H. R. Bourne, Nature (London) 351, 63 (1991).

2 y. H. Wong, B. R. Conklin, and H. R. Bourne, Science 255, 339 (1992). 3 B. R. Conklin, O. Chabre, Y. H. Wong, A. D. Federman, and H. R. Bourne, J. Biol.

Chem. 267, 31 (1992). 4 A. M. Pace, Y. H. Wong, and H. R. Bourne, Proc. Natl. Acad. Sci. U.S.A. 88, 7031 (1991).

[6] G~ ASSAYS IN TRANSFECTED CELLS 83

technique. However, in the case of establishing cell lines that stably express G-protein a subunits, introduction of the DNA by retrovial infection seems to have a better chance of success than the calcium phosphate coprecipitation method. Numerous G-protein o~ subunits have been successfully expressed in a variety of mammalian cell lines including the $49 cyc- cells] Swiss 3T3,6,7 NIH 3T3 ,I,2,6 and rat-1 fibroblasts 4'6 via retroviral infection. Attempts to express the same constructs in identical cell lines by calcium phosphate coprecipitation have had difficulties (Y. H. Wong, unpublished results). Nonetheless, other researchers were able to express the ~ subunits of Go and Gij in adrenal Y1 s and BALB/c 3739 cells, respectively, by calcium phosphate coprecipitation, albeit at a lower transfection efficiency. If setting up a retroviral infection procedure does not present a major problem, it is desirable to use this method in order to enhance the possibility of a successful outcome.

RetrotJiral Infection of Mammalian Cells

The criteria for selecting a suitable retroviral vector l°,~j and the protocols for retroviral infection are detailed elsewhere. 11 The procedures for retroviral infection of eukaryotic cells using the retroviral vector pMV-75 are briefly described below. The pMV-7 vector is flanked by the long terminal repeats (LTRs) of the Moloney routine sarcoma virus (MSV) and contains the selectable drug resistance gene neo. ~2 Two packaging cell lines, PAl2 and q~2, are transfected with the pMV-7 vector containing the gene of interest and the recombinant virus subsequently produced are used to infect a variety of mammalian cells.

Preparing DNA Samples. The procedures for constructing mutant and chimeric ~ subunits are described elsewhere in this series. 12~ For the purpose of transfection, the plasmid DNA could be purified on commer-

K. A. Sullivan, R. T. Miller, S. B. Masters, B. Beiderman, W. Heideman, and H. R. Bourne, Nature (London) 330, 758 (1987).

6 S. K. Gupta, C. Gallego, J. M. Lowndes, C. M. Pleiman, C. Sable, B. J. Eisfelder, and G. L. Johnson, MoL Cell. Biol. 12, 190 (1992).

7 I. Zachary, S. B. Masters, and H. R. Bourne, Biochem. Biophys. Res. Commun. 168, 1184 (1990).

8 D. B. Bloch, J. V. Bonventre, E. J. Neer, andJ. G. Seiman, Mol. Cell. Biol. 9, 5434 (1989). 9 Z. Cui, M. Zubiaur, D. B. Bloch, T. Michel, J. G. Seidman, and E. J. Neer, J. Biol.

Chem. 266, 20276 (1991). ~0 R. J. Kaufman, this series, Vol. 185, p. 487. ~ A. M. C. Brown and M. R. D. Scott, in "DNA Cloning Volume III" (D. M. Glover, ed.),

p. 189. IRL Press, Oxford and Washington, D.C., 1987. ~2 p. T. Kirschmeier, G. M. Housey, M. D. Johnson, A. S. Perkins, and 1. B. Weinstein,

DNA 7, 219 (1988). 12~ S. Winitz, M. Russell, and G. L. Johnson, this series, Vol. 237 [25].


cially available columns (e.g., columns from Qiagen, Chatsworth, CA) according to the manufacturer's recommendations. However, it is not necessary to purify the plasmid DNA by the CsC1 method.

Cell Culture. The PAl2 cells are maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). The ~2 cells are cultured in DMEM containing 10% calf serum (CS). Host cells such as Swiss 3T3, Rat-l, and Ltk- fibroblasts are maintained in DMEM supplemented with 10% FCS, whereas NIH 3T3 cells are normally grown in the same medium with 10% CS. All cell lines are maintained in humidified incubators aerated with 5% CO2.

Method

1. Seed PAl2 cells into 100-mm tissue culture plates at a density of 2.5 x 105 cells per plate. The next day, refeed the cells with fresh medium 2-4 hr prior to transfection.

2. Transfect PAl2 cells with the pMV-7 vector containing the DNA of interest (20 /zg of plasmid DNA per plate) by the standard calcium phosphate coprecipitation method. ~3 Briefly, equal volumes (250 ~1) of 2 × HEPES-buffered saline (280 mM NaCI, 1.5 mM Na2HPO4 • 2H2 O, 50 mM HEPES, pH 7.05) and a solution containing 250 mM CaCI 2 and DNA are gently mixed together. The mixture is incubated at room temperature for 20-30 min to allow for the formation of precipitates. Distribute the precipitate evenly over the cell monolayer and leave the precipitates on the cells for 16 hr. Include at least one plate for mock transfection to act as a control.

3. Wash the cells several times with growth medium to ensure the complete removal of precipitates and then add fresh medium. Allow the cells to grow for 2 days without changing the medium. On the following day seed ~2 ceils into 100-mm tissue culture plates at a density of 2.5 × l0 s cells per plate.

4. Remove the conditioned medium from the PAl2 cells, filter sterilize, and add to an equal volume of fresh medium containing 16 ~g/ml Poly- brene. Subculture the PAl2 cells and induce selection by adding 0.5 mg/ ml G418 (Geneticin, GIBCO, Grand Island, NY) to the growth medium. The selection of transfected PAl2 cells will take approximately 2 weeks and can be monitored by cell deaths in the mock-transfected control plate. On completion (no live cells in control plate), the cells can be frozen for future use as a source of recombinant virus.

13 C. Gorman, in "DNA Cloning Volume II" (D. M. Glover, ed.), p. 143. IRL Press, Oxford and Washington, D.C., 1985.

[6] Gi ASSAYS IN TRANSFECTED CELLS 85

5. Add the sterile PA12-conditioned medium obtained in the previous step to washed qh cells that have been seeded the day before. Incubate tO2 cells in the PA12-conditioned medium for 16 hr.

6. Remove the supernatant from tO2 cells and wash the cells three times with DMEM containing 10% CS. Allow the qh cells to grow in fresh medium for 8 hr and then replace the supernatant with fresh medium containing 0.5 mg/ml G418.

7. Allow the +2 cells to propagate under G418 for about 2 weeks, subculturing the cells if necessary. After the selection, grow the selected qh cells in G418-free complete medium for 48 hr. Seed host cells (e.g., NIH 3T3) into 100-mm plates at 1-2 x 105 cells per plate.

8. Collect the conditioned medium from the tO2 cells and infect the host cells by repeating Steps 4 to 6. Freeze the ~b 2 cells for future use as a stock of recombinant virus. Select infected host cells with emperically determined concentrations of G418.

9. Clonal lines of infected host cells can be generated during the selection procedure by picking G418-resistant colonies using sterile cloning rings or by dilution cloning from the G418-selected population.

10. After verifying that the G-protein subunit is indeed being expressed (e.g., by immunological means or by pertussis toxin-catalyzed [32p]ADP- ribosylations), the infected cells can be seeded into 24-weU plates at 1 × 105 cells per plate and subjected to [3H]adenine assay, or they may be used in other types of experiments.

Comments. The use of two packaging cell lines greatly increases the titer of recombinant virus and results in less risk of rearrangement of the vector, thus allowing the gene to be properly expressed. The two packaging cell lines can be replaced by a single cell line, PA317, ~4 which produces broad host range amphotropic stocks free of detectable helper virus. The titer of the virus stock can be estimated by a number of methods that rely on physical, biochemical, or biological assays. ~ The whole procedure takes approximately 2 months to complete but usually leads to high transformation efficiency.

Transient Expression of G-Protein a Subunits in Mammalian Cells

The cell line most often used for transient expression is the monkey kidney cell line COS. The COS cells express high levels of the simian virus 40 (SV40) large tumor antigen, which results in the amplification of the plasmid copy number to greater than 10,000 per cell. Unfortunately, the COS cells are not suitable for studying Gi-mediated inhibition of adenylyl

~4 A. D. Miller and C. Buttimore, Mol. Cell. Biol. 6, 2895 (1986).


TABLE I EXPRESSION OF G-PROTEIN-COUPLED RECEPTORS IN 293 CELLS

G protein Receptor Ref.

G~ Rat luteinizing hormone a Human/32-adrenoceptor b Human dopamine Dj c Rat dopamine Dm d Human dopamine D5 e Human thyroid-stimulating hormone f Rat follicle-stimulating hormone g

Gi/Go Porcine c~2-adrenoceptor 1-3, 16 Human dopamine D2 2, 16 Rat adenosine Ai 2, 16 Rat neuropeptide Y~ h Rat 5-HT~B i Human 5-HT~E j Human muscarinic m2 k Human muscarinic m4 k Human interleukin 8 l Human formylpeptide chemoattractant m, n Human C5a n

Gq/G~I Porcine calcitonin o Human muscarinic ml k Human muscarinic m3 k Murine bombesin p Rat 5-HT2 q

K. C. McFarland, R. Sprengel, H. S. Phillips, M. Kohler, N. Rosemblit, K. Nikolics, D. L. Segaloff, and P. H. Seeburg, Science 245, 494 (1989).

b M. yon Zastrow and B. K. Kobilka, J. Biol. Chem. 267, 3530 (1992). c Q. y. Zhou, D. K. Grandy, L. Thambi, J. A. Kushner, H. H. Van Tol,

and R. Cone, Nature (Lonidon) 347, 76 (1990). a M. Tiberi, K. R. Jarvie, C. Silvia, P. Falardeau, J. A. Gingrich, N.

Godinot, L. Bertrand, T. L. Yang-Feng, R. T. Fremeau, and M. G. Caron, Proc. Natl. Acad. Sci. U.S.A. 88, 7491 (1991).

e D. K. Grandy, Y. Zhang, C. Bouvier, Q. Y. Zhou, R. A. Johnson, L. Allen, K. Buck, J. R. Bunzow, J. Salon, and O. Civelli, Proc. Natl. Acad. Sci. U.S.A. 88, 9175 (1991).

f A. L. Frazier, L. S. Robbins, P. J. Stork, R. Sprengel,. D. L. Segaloff, and R. D. Cone, Mol. Endocrinol. 4, 1264 (1990).

g A. L. Schneyer, P. M. Sluss, R. W. Whitcomb, K. A. Martin, R. Sprengel, and W. F. Crowley, Jr., Endocrinology (Baltimore) 129, 1987 (1991).

h j. Krause, C. Eva, P. H. Seeburg, and R. Sprengel, Mol. Pharmacol. 41, 817 (1992).

i M. M. Voigt, D. J. Laurie, P. H. Seeburg, and A. Bach, EMBO J. 10, 4017 (1991).

J G. McAllister, A. Charlesworth, C. Snodin, M. S. Beer, A. J. Noble, D. N. Middlemiss, L. L. Iversen, and P. Whiting, Proc. Natl. Acad. Sci. U.S.A. 89, 5517 (1992).


cyclase owing to the presence of a type II-like adenylyl cyclase.15 Activa- tion of an exogenous inhibitory receptor such as the ~2-adrenoceptor results in an increase rather than a decrease of intracellular cAMP in COS cells; this is presumably due to the stimulation of type II-like adenylyl cyclase by/3y subunits released from the activated G i proteins, J6 An alternative transient expression system utilizes an adenovirus-transformed primary human embryonic kidney cell line, the 293 cells (ATCC, Rockville, MD, CRL 1573). Unlike the COS cells, activation of the o~2-adrenoceptor in transfected 293 cells results in an inhibition of cAMP accumulation. ~,2 Thus the 293 cells provide a suitable cellular environment for the study of Gi functions.

Because only a fraction of the transfected cells (usually less than 50%) will transiently express the protein of interest, it is necessary to somehow target this subset of cells for functional assays, In the case of evaluating inhibition of cAMP accumulation, one could cotransfect the cells with a hormone receptor that stimulates adenylyl cyclase through Gs. By stimulating adenylyl cyclase through the exogenous receptor, one effectively targets the subset of cells that can support transient expression. For example, in 293 cells cotransfected with cDNAs encoding the rat luteinizing hormone receptor (LHR) and the porcine o~2-adrenoceptor, the cAMP accumulation stimulated by an LHR agonist is reduced in the presence of an c~2-adrenoceptor agonist. ~ Thus, both receptors are expressed in the same subpopulation of 293 cells. The cotransfection technique has been extensively used in the study of receptor, G protein, and effector functions. 1-3,16 Many different types of G-protein-coupled receptors have been expressed in the 293 cells (Table I). The ability of endogenous or exoge-

15 W.-J. Tang and A. G. Gilman, Science 254, 1500 (1992). ~6 A. D. Federman, B. R. Conklin, K. A. Schrader, R. R. Reed, and H. R. Bourne, Nature

(London) 356, 159 (1992).

k j. Sandmann, E. G. Peralta, and R. J. Wurtman, J. Biol. Chem. 266, 6031 (1991). J W. E. Holmes, J. Lee, W. J. Kuang, G. C. Rice, and W. I. Wood, Science 253,

1278 (1991). " R. J. Uhing, T. W. Gettys, E. Tomhave, R. Snyderman, and J. R. Didsbury, Biochem.

Biophys. Res. Commun. 183, 1033 (1992). n j. R. Didsbury, R. J. Uhing, E. Tomhave, C. Gerard, and N, Gerard, FEBS Lett. 297,

275 (1992). " O. Chabre, B. R. Conklin, H. Y. Lin, H. F. Lodish, E. Wilson, H. E. Ives, L.

Catanzariti, B. A. Hemmings, and H. R. Bourne, Mol. Endocrinol. 6, 551 (1992). P Y. H. Wong, unpublished results (1992). q J. A. Apud, D. R. Grayson, E. De Erausquin, and E. Costa, Neuropharmacology 31,

1 (1992).


nous G i proteins to interact with various receptors and effectors can therefore be studied.

A number oftransfection procedures are readily available for establishing eukaryotic transient expression systems. ~7,~8 These include calcium phosphate coprecipitation, DEAE-dextran-mediated transfection, electroporation,and lipofection. The DEAE-dextran-mediated DNA transfer technique is described briefly, and the merits of other transfection procedures are also discussed.

DEAE-Dextran-Mediated Transfection of 293 Cells

The DEAE-dextran technique has been widely used for transient expression of cloned genes. Many variants of the basic protocol have been published, 17'18 and it is desirable to adopt well-tested procedures for any given cell type whenever possible. The following method has been extensively applied to the 293 cells.

Cell Culture and Preparation of Plasmid DNA. The 293 cells are routinely cultured in minimum essential medium (MEM) with Earle's salts and 10% FCS. A large frozen stock of low-passage 293 cells should be maintained since the cells have a propensity to senesce with long-term culturing. Alternatively, the cells can be cultured in defined serum (2% Ultroser G, GIBCO). Vectors that support high-level transient expression in 293 cells include CDM8,19 pcDNAI (Invitrogen, San Diego, CA), and pCMV, each of which contains the SV40 replication origin together with the cytomegalovirus (CMV) promoter. The presence of the CMV promoter seems particularly important for high-level expression in 293 cells. The G-protein cDNAs should be subcloned into vectors such as pcDNAI and purified over Qiagen columns prior to use.

Solutions. Plasmid DNAs purified form Qiagen columns are dissolved in 1 mM EDTA, 10 mM Tris-HCl (pH 7.4) and stored at a concentration of 0.1 mg/ml. A stock solution (40 mg/ml) of DEAE-dextran (Pharmacia, Piscataway, NJ, MW ~500,000) is made up in water, sterile-filtered, and stored in 1-ml aliquots at -20 °. Chloroquine (Sigma, St. Louis, MO) is stored as a sterile stock solution (I0 mM) in 1-ml aliquots at -20 °.

Method

1. On day 1, approximately 1 × 106 293 cells are seeded onto 60- mm tissue culture plates. The monolayer should be just subconfluent the next day.

17 B. R. Cullen, this series, Vol. 152, p. 684. 18 W. A. Keown, C. R. Campbell, and R. S. Kucherlapati, this series, Vol. 185, p. 527. 19 B. Seed and A. Aruffo, Proc. Natl. Acad. Sci. U.S.A. 84, 3365 (1987).

[6] G i ASSAYS IN TRANSFECTED CELLS 89

2. On day 2, for each 60-ram plate prepare 4 ml of transfection cocktail in sterile tubes as follows. Add plasmid DNA (0.1 to 3.0 ~g per construct) to MEM containing 10% FCS, mix, and then add DEAE-dextran and chloroquine to final concentrations of 0.4 mg/ml and 0.1 mM, respectively. Mix and keep at 37 ° .

3. Wash each plate once with 2 ml of serum-free MEM and add the transfection cocktail to the ceils. Place cells at 37 ° for 2 h. At the end of the 2 hr, approximately 50% of the cells will appear sick with a "rounded- up" morphology.

4. Aspirate supernatant medium and add 4 ml of phosphate-buffered saline (PBS) containing 10% dimethyl sulfoxide (DMSO). Leave the cells at room temperature for approximately 2 min.

5. Aspirate and wash each plate once with 6 ml of PBS. Replace the PBS with 4 ml of MEM containing 10% FCS. Return plates to the incubator.

6. The transfected cells should look normal by the following morning. Reseed the cells onto I2-well or 24-well plates. Each 60-mm plate should have enough transfected cells to seed onto 6-8 wells of a 12-weU plate. About 2-3 hr later, the transfected cells can be labeled with [3H]adenine as described in the next section.

Comments. The DEAE-dextran method cannot be used to obtain stable transfectants. 2° It has been noted that the DNA introduced into cells by either the DEAE-dextran method or by calcium phosphate coprecipitation has a high rate of mutation. 2~,22 As high as 40-50% of the cell population could be effectively transfected by this method. The efficiency of gene transfer could be further increased by the sequential treatment of mammalian cells with DEAE-dextran and the DNA of interest. 23

Other Methods of DNA Transfection

Another widely used method of introducing DNA into eukaryotic cells for transient expression is the calcium phosphate coprecipitation method. 24 Indeed, a modified verison of the calcium phosphate coprecipitation method with enhanced efficiency z5 works just as well as the DEAE-dextran procedure in 293 cells. However, variability between experiments is much

20 G. Milman and M. Herzberg, Somatic Cell Genet. 7, 161 (1981). 21 M. P. Calos, J. S. Lebkowski, and M. R. Botchan, Proc. Natl. Acad. Sci. U.S.A. 80,

3015 (1983). 22 C. R. Ashman and R. L. Davidson, Somatic Cell Mol. Genet. 11, 499 (1985). 23 W. Holter, C. M. Fordis, and B. H. Howard, Exp. Cell Res. 184, 546 (1989). 2~ M. Wigler, S. Silverstein, L. S. Lee, A. Pelticer, V. C. Cheng, and R. Axel, Celt (Cam-

bridge, Mass.) 11, 223 (1977). 25 C. Chen and H. Okayama, Mol. Cell. Biol. 7, 2745 (1987).


higher with cells transfected by the calcium phosphate coprecipitation when compared to those transfected by DEAE-dextran.

For cells refractory to traditional methods of transfection, it may be possible to introduce the DNA into the cells using high-voltage electric shocks.26 Electroporation is a straightforward procedure which has proved to be effective in many cell types. The parameters of electroporation, especially the peak voltage and the fall time of the discharge waveform, should be optimized for each cell type. 27

A more recently developed technique that relies on the use of DNA- containing liposomes to introduce genetic material into mammalian cells is rapidly gaining popularity. Lipofection utilizes a synthetic cationic lipid, N-[ l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and has been reported to be 6-10 times better than DEAE- dextran? 8 The a subunits of Gq and G~l have been transiently expressed in COS-7 cells by lipofection, z9 Nevertheless, lipofection does not seem to be any better than DEAE-dextran in the 293 system. Some cell lines including Chinese hamster ovary (CHO) and Rat-1 fibroblasts do not support transient expression by DEAE-dextran transfections. An alternative method using a synthetic lipopolyamine (Transfectam, Promega, Madison, WI) appears to produce transient expressions in these cells. Thus, the choice of transfection procedures primarily depends on which cell line is being used.

Methods for Assaying Inhibition of Adenylyl Cyclase

There are several ways in which one could monitor the activity of adenylyl cyclase, usually by estimating the enzymatic activity or by determining the amount of cAMP produced. In many respects the adenylyl cyclase assay 3° is a more accurate measure of the enzyme activity. How- ever, the method requires the preparation of membranes and preservation of enzyme activity. For indirect determinations of adenylyl cyclase activity, it is more convenient to use intact cells and measure the amount of intracellular cAMP. Methods for measuring cAMP levels include protein binding assay 31 and radioimmunoassay (RIA). 32 Alternatively, one could

26 T. K. Wong and E. Neumann, Biochem. Biophys. Res. Commun. 107, 584 (1982). 27 j . C. Kuntson and D. Yee, Anal. Biochem. 164, 44 (1987). 28 D. L. Feigner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop,

G. M. Ringold, and M. Danielsen, Proc. Natl. Acad. Sci. U.S.A. 84, 7413 (1987). 29 D. Wu, C. H. Lee, S. G. Rhee, and M. I. Simon, J. Biol. Chem. 267, 1811 (1992). 3o y . Salomon, C. Londos, and M. Rodbell, Anal. Biochem. 58, 541 (1974). 31 A. G. Gilman and F. Murad, this series, Vol. 38, p. 49. 32 A. L. Steiner, this series, Vol. 38, p. 96.


label the intracellular adenine nucleotide pool with [3H]adenine and measure the subsequent conversion to [3H]cAMP on cyclase stimulation. 3~ The [3H]adenine assay has been used to examine receptor- and G-protein- mediated inhibition of cAMP accumulation in both stably and transiently transfected cells. L2 The following section describes a simplified version of the basic protocol 33 for the [3H]adenine assay.

[3H]Adenine Assay

The [3H]adenine assay procedure determines the cAMP levels in intact stimulated cells relative to unstimulated controls. To label the intracellular adenine nucleotide pool, the cells are incubated with [3H]adenine. Washed cells are then treated with or without various test substances in assay medium. The reaction is terminated by the addition of trichloroacetic acid (TCA), to extract the [3H]adenine nucleotides. Isolation of [3H]cAMP is achieved by double chromatography over Dowex and alumina columns.

Solutions. The assay medium is made up of the appropriate serum- free culture medium which has been buffered with HEPES (20 raM, pH 7.4) and contains 1 mM 3-isobutyl-l-methylxanthine (IBMX). IBMX is a phosphodiesterase inhibitor and is dissolved in DMSO as a stock of 100 raM. IBMX can be replaced by 1 mM Ro 20-1724 in situations where IBMX acts as an antagonist at the adenosine receptors. The ice-cold stop solution contains 5% TCA, 1 mM ATP, and 1 mM cAMP. Hormones and stimulants are freshly dissolved in assay medium.

Method

1. Label the transfected cells in 12-well plates with 2 p.Ci/ml of [3H]ade - nine and incubate the cells for 16-20 hr. For most cell types the [3H]adenine can be directly added to the growth medium. With certain cell types such as Swiss 3T3 fibroblasts, it is important to use quiescent cells, and thus the labeling should be performed in serum-free medium.

2. Where necessary, add pertussis toxin (100 ng/ml; List Biologicals, Campbell, CA) to the cells at least 3-4 hr before challenging the cells with agents that regulate adenylyl cyclase activity. An overnight treatment with pertussis toxin works well for many cell types except Swiss 3T3, which strictly requires addition of the toxin 3-4 hr prior to the assay.

3. Wash off the extracellular [3H]adenine with 2 ml of assay medium. Discard the radioactive waste in accordance with institutional disposal regulations.

4. To each well, add 1 ml of assay medium containing the various test reagents. Incubate at 37 ° for 30 rain.

33 y. Salomon, this series, Vol. 195, p. 22.


5. Terminate the reaction by aspiration immediately followed by the addition of 1 ml of ice-cold stop solution to each well. Incubate at 4 ° for 30 rain to facilitate the extraction of soluble nucleotides. Alternatively, the cells under TCA extraction can be stored at - 2 0 ° until one is ready to separate the nucleotides on columns.

6. Remove the cell free, clear TCA extracts from the plates and directly load onto Dowex columns that have been regenerated (see below).

Comments. For assays performed on 24-well plates, the volumes required to wash off the extracellular [3H]adenine and for drug treatment are reduced by one-half. Certain cell types do not take up the [3H]adenine efficiently and thus cannot be assayed in this manner. A prime example is the Ltk- fibroblast. Therefore, it is imperative to determine whether a particular cell type can indeed be prelabeled with [3H]adenine. The activity ofadenylyl cyclase has to be stimulated before one could induce inhibition. For stable cell lines, 50-100/zM forskolin usually stimulates the cAMP levels by 20- to 50-fold over the basal level. If a stimulatory drug is required, prostaglandin El and isoproterenol (10/~M) have been reported to be effective in a wide range of systems, and they are good starting points for finding a positive stimulus appropriate to the system being used. In general, the forskolin response is more robust and provides a better basis for examining inhibitory responses. However, the use of forskolin should be completely avoided in transient systems because it acts on the entire population of cells. Instead, adenylyl cyclase should be stimulated only via Gs-coupled receptors that are being coexpressed with the protein of interest. Receptor-mediated stimulations of adenylyl cyclase can increase the cAMP levels by I0- to 20-fold over basal values in transient expression systems.

Isolation of [3H]cAMP

The method of choice to isolate [3H]cAMP is sequential chromatography on Dowex and alumina columns. 3° This simple procedure has been widely used in the separation of cAMP from other nucleotides and constitutes a major part of the adenylyl cyclase assay as well as the [3H]adenine assay. Detailed description of the separation procedure can be found elsewhere. 3°'34 A simplified version is briefly described below.

Setting up Dowex Columns. Dowex (AG50-X8, 100-200 mesh from Bio-Rad, Richmond, CA) is washed sequentially with 2 volumes each of 0.1 N NaOH, water, 1 N HCI, and water, a 2 : 1 (v/v) slurry of Dowex is prepared in water, and 2-ml aliquots are distributed into Poly-Prep

34 R. A. Johnson and Y. Salomon, this series, Vol. 195, p. 3; see also R. A. Johnson, R. Alvarez, and Y. Salomon, this volume [3].


chromatography columns (Bio-Rad). The Dowex slurry is mixed continuously to prevent sedimentation during division into aliquots. Before and after each use, the Dowex columns are regenerated by washing with 2 ml of 1 N HC1 followed by 10 ml of water. The columns can be reused dozens of times provided that they are stored immersed in water and not allowed to dry out.

Setting up Alumina Columns. Neutral alumina (Bio-Rad AG7 or Sigma WN-3) can be poured dry into Poly-Prep columns. This is easily achieved by weighing out 0.5 g of alumina in an Eppendorf tube, marking the tube at the level of the weighted alumina, cutting off the upper part of the tube at the mark, and using the lower, cut end as a scoop to transfer 0.5 g of alumina to each column. Wash new columns once with 10 ml of 1 M imidazole, pH 7.5. Before and after each use, regenerate alumina columns with 10 ml of 0.1 M imidazole, pH 7.5. Columns are stored immersed in water.

Method

1. Place the regenerated Dowex columns on top of 20-ml scintillation vials and load the entire 1 ml cell-free TCA extract onto each column. Allow the TCA extracts to drain through the columns to be collected in the scintillation vials.

2. Add 3 ml of water to each column and collect the eluate in the same scintillation vials. The eluate contains [3H]ATP and [3H]ADP. Add 5 ml of scintillation liquid to each vial and transfer the vials to the scintillation counter for determination of radioactivity.

3. Place the Dowex columns over a comparable number of alumina columns so that the Dowex eluate can drip directly onto the alumina. Add 10 ml of water to the Dowex columns.

4. Remove the Dowex columns and mount the alumina columns over a new set of scintillation vials. Elute the [3H]cAMP from the alumina by adding 6 ml of 0.1 M imidazole (pH 7.5) to each column. Add 7.5 ml of scintillation fluid to each vial and transfer to the counter. Regenerate both Dowex and alumina columns accordingly.

Data Analysis. Results are most conveniently expressed as the ratio of [3H]cAMP to total [3H]adenine nucleotides. [3H]cAmP can be determined by counting the vials obtained in Step 4. Total [3H]adenine nucleotides is calculated as the sum of [3H]cAMP plus that of [3H]ATP and [3H]ADP, which is obtained in Step 2. In the unstimulated basal state, this ratio is usually in the region of 1-2 × 10 -3. On stimulation by agents such as forskolin, the ratio can reach as high as 1-2 x 10 -~. Blanks obtained by running 1 ml of TCA without radioisotope should be subtracted from all the counts before analyzing the results. This serves as an


indicator of column contamination and is particularly important when the stimulatory response is not robust. The [3H]ATP and [3H]ADP count obtained in Step 2 is a fair reflection of the cell density, and thus for relative determinations it is not necessary to normalize the results by measuring the protein content of the samples.

Miscellaneous Assays

After establishing cell lines that express G-protein subunits in either a transient of a stable manner, it is necessary to confirm expression at the protein level. A number of G-protein antisera that can be utilized in immunoblots are now commercially available (Du Pont -NEN, Boston, MA). Specificity of various antisera against G-protein" and fly subunits are described elsewhere in this series. 3s'36 Additional detection methods are available in the case of G i proteins. For example, both the wild type and mutationally activated a subunits of G~2 can be detected by pertussis toxin-catalyzed [32p]ADP-ribosylation. 1 The methods for pertussis toxin- catalyzed ADP-ribosylation are described elsewhere in this series. 37 How- ever, it should be noted that both immunoblotting and ADP-ribosylation can only detect a relative increase in protein levels; they cannot distinguish between endogenous and exogenous G protein subunits. To quantitate and monitor the expression of exogenous G protein subunits, it may be necessary to tag the proteins with a specific epitope.

Acknowledgments

I thank Dr. Henry R. Bourne for unfailing interest in our work and for valuable advice and encouragement during my stay at the University of California, San Francisco. I am also indebted to Dr. Paul Wilson and Ann Pace for generously sharing technical knowledge with me. This chapter is based on experiments supported in part by grants from the National Institutes of Health and the March of Dimes allocated to Dr. Henry R. Bourne at UCSF.

35 G. Milligan, this series, Vol. 237 [21]. 36 A. N. Pronin and N. Gautam, this series, Vol. 237 [38]; J. D. Robishaw and E. A. Balcueva,

this series, Vol. 237 [39]. 37 D. J. Carty, this series, Vol. 237 [6].

[7] E X P R E S S I O N O F A D E N Y L Y L C Y C L A S E S I N S f 9 C E L L S 95

[7] Expression and Purification of Recombinant Adenylyl Cyclases in Sf9 Cells

B y RONALD TAUSSIG, WEI- JEN TANG, and ALFRED G. GILMAN

Introduction

The activities of adenylyl cyclases (EC 4.6.1.1, adenylate cyclase), which catalyze the synthesis of cyclic AMP from ATP, are modulated by several cellular regulators, including Ca2+/calmodulin and both the a and /37 subunits of signal transducing, guanine nucleotide-binding regulatory proteins (G proteins). Different forms of adenylyl cyclase were purified during the 1980s, 1-4 and in 1989 Krupinski et al. ~ described the isolation of a cDNA encoding a calmodulin-sensitive form of adenylyl cyclase from bovine brain (now termed the type I isoform). Subsequently, cDNAs encoding five additional isoforms of mammalian adenylyl cyclase have been isolated using low-stringency hybridization and the polymerase chain reaction. 6-12 Some of the salient properties of these adenylyl cyclases are summarized in Table I.

The availability of an abundant cell type expressing predominantly a single isoform of adenylyl cyclase is of obvious utility in elucidating the individual regulatory properties of the enzymes. To this end, our laboratory has employed the recombinant baculovirus-driven Sf9 (Spodoptera

1 E. Pfeuffer, R.-M. Dreher, H. Metzger, and T. Pfeuffer, Proc. Natl. Acad. Sci. U.S.A. 82, 3086 (1985).

2 M. D. Smigel, J. Biol. Chem. 261, 1976 (1986). 3R, E. Yeager, W. Heideman, G. B. Rosenberg, and D. R. Storm, Biochemisto, 24,

3776 (1985). 4 F. Coussen, J. Haiech, J. D'Alayer, and A. Monneron, Proc. Natl. Acad. Sci. U.S.A.

82, 6736 (1985). 5j. Krupinski, F. Coussen, H. A. Bakalyar, W.-J. Tang, P. G. Feinstein, K. Orth, C.

Slaughter, R. R. Reed, and A. G. Gilman, Science 244, 1558 (1989). 6 p. G. Feinstein, K. A. Schrader, H. A. Bakalyar, W.-J. Tang, J. Krupinski, A. G. Gilman,

and R. R. Reed, Proc. Natl. Acad. Sci. U.S.A, 88, 10173 (1991). 7 H. A. Bakalyar and R. R. Reed, Science 250, 1403 (1990). s B. Gao and A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 88, 10178 (1991). 9 y . Ishikawa, S. Katsushika, L. Chert, N. J. Halnon, J. I. Kawabe, and C. J. Homcy, J.

Biol. Chem. 267, 13553 (1992). 10 S. Katsushika, L. Chen, J. I. Kawabe, R. Nilakantan, N. J. Halnon, C. J. Homcy, and

Y. Ishikawa, Proc. Natl. Acad. Sci. U.S.A. 89, 8774 (1992). H M. Yoshimnra and D. M. F. Cooper, Proc. Natl. Acad. Sci. U.S.A. 89, 6716 (1992). 12 R. T. Premont, J. Q. Chen, H. W. Ma, M. Ponnapalli, and R. Iyengar, Proc. Natl. Acad,

Sci. U.S.A, 89, 9809 (1992).



TABLE I PROPERTIES OF MAMMALIAN ADENYLYL CYCLASES

Effect of G

proteins Amino acid Ca2+/

Type residues Expression Gsa BY Calmodulin Forskolin a Ref.

I 1134 Brain +b _ + +7 5 II 1090 Brain, lung + + 0 +5.8 6 III 1144 Olfactory + 0 + + 3.3 7 IV 1064 Brain, others + + 0 + 1.2 8 V 1184 Heart, brain, others + 0 0 +7 9, 12 VI 1165 Heart, brain, others + 0 0 +4 10-12

a Numbers shown are adenylyl cyclase activities (nmol/min/mg) of Sf9 cell membranes after expression of the indicated isoform. All but type I l i were assayed in the presence of 5 mM MnC12 and 100/zM forskolin; type III was assayed with 100/zM forskolin plus 50 nM recombinant Gsa.

b +, Activation; - , inhibition; 0, no effect.

frugiperda, fall armyworm ovary) cell system to express the six isoforms of adenylyl cyclase known to date. We have also constructed mutant adenylyl cyclases containing adjacent histidine residues, which facilitates purification by virtue of the high-affinity interaction of the histidines with chelated metals. Type I and type II adenylyl cyclases have been purified after expression in Sf9 cells by a combination of forskolin affinity and chelated Ni 2+ affinity or lectin chromatography. We confine our comments in this review of techniques to the expression and purification of type I and type II adenylyl cyclases.

Plasmid Construction

Methods for construction of baculoviruses encoding type I, type II, and histidine-tagged type I adenylyl cyclases have been described previously. ~3-15 Restriction fragments of cDNAs containing the entire coding regions of bovine type I (NcoI-EcoRI) and rat type II (EcoRI-EcoRI) adenylyl cyclases are subcloned into shuttle vectors pAcC4 or pVL1392. The choice of vector is dependent on the availability of restriction sites in the cDNAs.

13 W.-J. Tang, J. Krupinski, and A. G. Gilman, J. Biol. Chem. 266, 8595 (1991). t4 W.-J. Tang and A. G. Gilman, Science 254, 1500 (1991). ~5 R. Taussig, L. M. Quarmby, and A. G. Gilman, J. Biol. Chem. 268, 9 (1993).

[7] EXPRESSION OF ADENYLYL CYCLASES IN Sf9 CELLS 97

For the construction of cDNAs encoding histidine-tagged type I adenylyl cyclase, plasmid pAcC4 encoding the type I enzyme is first partially digested with the appropriate restriction enzyme (Table II) to yield a population of molecules with a single cut. The partially digested plasmids are ligated to double-stranded adaptors that (1) encode the amino acid sequence Ser-(Gly)3-(His)6-(Gly)3-Ser in the correct reading frame, (2) contain the appropriate overhanging sequences complementary to the overhangs generated by the restriction enzymes, and (3) abolish the restriction sites at both junctions when ligated into the plasmids. Following transformation of the ligated plasmids into bacteria, insertion mutants are identified by hybridization of radiolabeled mutagenic oligonucleotides to colonies. The site of insertion is determined by digestion ofplasmid DNA isolated from positive clones with appropriate enzymes; the position of the adaptor insertion is marked by loss of the restriction site at that position. Proper orientation of the adaptor is verified by sequencing the double-stranded plasmid DNA at the insertion site.

Expression of type I adenylyl cyclases containing hexahistidine inserts at the amino terminus (position I), the site of a putative extracellular loop in the M2 domain (position 778), or near the carboxyl terminus (position 1114) yielded Sf9 cell membranes with adenylyl cyclase activities comparable to that seen with the wild-type enzyme (Table II). The regulatory properties of the enzymes were also unaltered (not shown). However, introduction of hexahistidine-containing sequences at positions 279 or 702 largely destroyed enzymatic activity.

Cell Culture

Fall armyworm ovarian (Sf9) cells are grown in suspension in IPL-41 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum

TABLE II CONSTRUCTION OF HISTIDINE-TAGGED TYPE 1 ADENYLYL CYCLASES

Restriction Site of Specific activity' site insertion" Region b (nmol/min/mg)

Aval 1114 COOH 6 Ncol 1 NH2 4.5 NcoI 778 Extracellular loop of M2 4.7 Ncol 279 C1 0.7 Sacl 702 Extracellular loop of M z - -

Amino acid numbering is from Krupinski et al? b Nomenclature is from Tang et al. 13

c Assays of membranes expressing wild-type or histidine-tagged type I adenylyl cyclases were performed in the presence of 100 tzM forskolin and 5 mM MnCI2.


and 0.1% (w/v) pluronic surfactant F68 (GIBCO, Grand Island, NY). Cells are maintained at 5 x l0 s to 4 x 10 6 cells/ml. For cultures larger than 1 liter, cells are adapted to grow in IPL-41 medium containing lipid mix (GIBCO, Grand Island, NY) and 0-2% (v/v) serum. To adapt cells, cultures are alternately grown to a density of 2 x 10 6 cells/ml and diluted 4- fold into low-serum medium until the desired volume of cells is obtained. Viability is reduced at higher densities when cells are grown in medium containing low concentrations of serum.

Production of Recombinant Baculovirus

Procedures for production, cloning, and amplification of baculovirus are described by Summers and Smith. ~6 Sf9 cells (1-2 x 106) are seeded onto a 35-ram culture dish, allowed to adhere for 1 hr, washed twice with serum-flee IPL-41 medium, and overlayed with 1 ml of the same medium. A mixture of transfer vector DNA encoding adenylyl cyclase (2 /xg), linearized AcRP23-LacZ viral DNA (0.2/zg digested with Bsu361)J 7 and lipofectin (15 tzg; GIBCO) in a total volume of 45/zl is incubated for 15 rain and applied dropwise to the dish of cells. The medium is replaced 6-15 hr later with IPL-41 medium containing 10% fetal bovine serum. The medium containing baculovirus is then harvested 3 days later, at which time the virus is plaque purified. The titer of virus in the harvested medium should be greater than 105 plaque-forming units/ml, and appropriate dilutions should be utilized to ensure well-isolated plaques. The use of AcRP23-LacZ viral DNA facilitates the identification of recombinant baculovirus. Nonrecombinant viral plaques appear blue when X-GAL (5-bromo-4-chloro-3-indolyl-fl-D-galactoside) is added to the agarose that overlays the infected Sf9 cells; approximately 50% of white plaques are recombinant and encode adenylyl cyclase. The agarose above each plaque is transferred to a 35-ram culture dish containing 1 × 106 Sf9 cells, and the culture is grown for 4 days. The medium from this culture is stored as viral stock, while the cells are assayed for expression of adenylyl cyclase by immunoblotting and enzymatic assay (see below).

I so l a t ion o f S f9 M e m b r a n e s

Crude membranes can be prepared from cells grown in dishes by a rapid procedure that yields material suitable for the characterization of

16 M. D. Summers and G. E. Smith, "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures." Texas Agricultural Experiment Station, Bulletin No. 1555, College Station, Texas, 1987.

i7 p. A. Kitts, M. D. Ayres, and R. D. Possee, Nucleic Acids Res. 18, 5667 (1990).

[7l EXPRESSION OF ADENYLYL CYCLASES IN Sf9 CELLS 99

the initial viral isolates. Cells grown in dishes are scraped from the surface in 1 ml of HME buffer [20 mM Na-HEPES (pH 8.0), 2 mM MgCI2, 1 mM EDTA] plus protease inhibitors [ 16 p.g/ml each of L-1-tosylamido-2-pheny[ ethyl ketone, 1-chloro-3-tosylamido-7-amino-2-heptone, and phenylmethylsulfonyl fluoride (PMSF); 3.2 ~g/ml each of leupeptin and lima bean trypsin inhibitor; and 2 tzg/ml of aprotinin], transferred to a microcentrifuge tube, and frozen in liquid nitrogen. The cell suspension is then thawed and centrifuged for 15 min at 4 ° and the supernatant discarded. The pellet is suspended in 100/zl of HME plus protease inhibitors for assay.

For large-scale production of membranes, 4 liters of Sf9 cells (1 × 106 cells/ml) are infected with recombinant baculovirus encoding type I, type If, or histidine-tagged type I adenylyl cyclase (1 plaque-forming unit/cell). Cells are harvested 46-55 hr after infection by centrifugation at 2000 g for 5 rain at 4°; suspended in 300 ml of 20 mM Na-HEPES (pH 8.0), 150 mM NaCI, 5 mM EDTA, 1 mM EGTA, 2 mM dithiothreitol, and protease inhibitors; and lysed by nitrogen cavitation at 600 psi for 30 rain on ice. Nuclei are removed by centrifugation at 500 g for 10 rain at 4 °, and the pellet is discarded. Membranes are harvested by centrifugation at 70,000 g for 30 rain at 4°; suspended in 350 ml of 20 mM Na-HEPES (pH 8.0), 200 mM sucrose, I mM dithiothreitol, and protease inhibitors; and centrifuged at 70,000 g for 30 min at 4 °. The final membrane pellet is suspended in 50 ml of the same buffer and stored at -80 °.

The time course of expression of type I adenylyl cyclase in Sf9 cells is shown in Fig. 1. Whereas adenylyl cyclase activity measured in Sf9 cells decreases after infection with wild-type baculovirus, that measured in cells infected with baculovirus encoding type I adenylyl cyclase increases dramatically after 30 hr of infection and reaches a maximum at 2-3 days. Increases in immunoreactivity correlate well with enzymatic activity during the first 2 days after infection. However, levels of immunoreactivity then decrease, although enzymatic activity remains high. It is possible that proteolytic fragments of adenylyl cyclase retain catalytic activity.

Immunoblot Analysis

Baculovirus-directed expression of recombinant adenylyl cyclase in Sf9 cells can be assessed by immunoblotting. Samples are subjected to polyacrylamide gel electrophoresis [in the presence of sodium dodecyl sulfate (SDS-PAGE)] and transferred to a nitrocellulose membrane at 10 ° for 1 hr at 25 V and then at 80 V overnight. The resolution of adenylyl cyclase in such gels is markedly improved by alkylating membrane proteins with N-ethylmaleimide prior to electrophoresis. Samples are prepared by heating membranes (10/zl) with 5/zl of 10% sodium dodecyl sulfate and

I00 ADENYLYL CYCLASES [7]

A

~ 1 2

E

E

0 E

4 0.

/mm

/ /

/ do~ ° < •

O.oN~ m , ~ _~ 0 24 48 72 96

Time of Infection (hours)

FIG. 1. Expression of type I adenylyl cyclase in Sf9 cells. (A) Adenylyl cyclase activity of St9 cell membranes prepared at different times following infection with baculovirus: (circles) no virus, (triangle) wild-type virus, (squares) type 1-encoding virus. Membranes were assayed for 10 rain in either (open symbols) I0 mM MgCI2 and 5 mM MnCI2 or (filled symbols) the same plus I00/zM forskolin. (B) Western blot analysis of membranes prepared from Sf9 cells harvested at different times after infection with baculovirus encoding type I adenylyl cyclase. Membrane proteins (35 p.g) were separated by SDS-PAGE, transferred to nitrocellulose, and stained using affinity-purified rabbit anti-type I adenylyl cyclase-specific antisera. The corresponding Mn2+-forskolin-stimulated activities of the membranes are shown at the top. (Reprinted from Tang et al.13)

1 mM dithiothreitol for 5 min at 80 °. Sample buffer is added after the addition of 5/zl of 300 mM N-ethylmaleimide and a second incubation for 10 min at room temperature.

Antipeptide antibodies directed against the carboxyl termini of type I and type II adenylyl cyclase are used to detect the appropriate isoform in immunoblotting experiments. Antibody C 1-1115 was generated against a peptide equivalent to residues 1115-1134 of type I adenylyl cyclase, whereas antibody C2-1077 was generated against a peptide equivalent to residues 1077-1090 of type II adenylyl cyclase. In addition, an internal antibody, C1-251, was generated against residues 251-263 of the type I enzyme. The antibody (1 : 1000 dilution) is reacted with the nitrocellulose membrane and is localized by enhanced chemiluminescence (ECL from Amersham, Arlington Heights, IL) according to the manufacturer's protocol. An example of such analysis is shown in Fig. 2, which demonstrates the specificities of the antibodies for the respective adenylyl cyclases.

[7] EXPRESSION OF ADENYLYL CYCLASES IN Sf9 CELLS 10 l

B Cyclic AMP.

Hours 72 7 90 101241 01 13,1 1, "IS' =1 181

kDa

~---110

Ab C1-1115

~¢-- 43 36

Ab CI-251 110

FIG. 1. (continued)

Adenylyl Cyclase Assays

Adenylyl cyclase activity is assayed as described by Smigel2; all assays contain 10 mM MgCI 2 and are performed at 30 ° in a volume of 100 ~1. To monitor adenylyl cyclase activity during construction of recombinant baculovirus, preparation of membranes, and purification of the enzyme, membranes (2-20 tzg) or appropriate aliquots of extracts and column eluates (5 Izl) are assayed for 5 min in the presence of 100 tzM forskolin and 5 mM MnClz. Purified Gsa (from Escherichia coli; see Lee et al.18) is activated by incubation with 50 mM Na-HEPES (pH 8.0), 5 mM MgSO4, 1 mM EDTA, 1 mM dithiothreitol, and 100 /zM GTPyS for 30 min at

15 E. Lee, M. E. Linder, and A. G. Gilman, this series, Vol. 237 [12].


TYPE I TYPE II

Ab Ab I A B C I D E F I

kDa

lo6

80

49.5

FIG. 2. Immunoblot analysis of membranes prepared from Sf9 cells expressing type I or type 1I adenylyl cyclase. Sf9 cell membrane proteins were alkylated, separated by electrophoresis in a 7.5% polyacrylamide gel, and transferred to a nitrocellulose membrane. Blots were processed with either anti-type I antibody C 1-1115 (lanes A-C) or anti-type II antibody C2- 1055 (lanes D-F). Samples are membranes prepared from cells expressing type I adenylyl cyclase (lanes A and D), type II adenylyl cyclase (lanes B and E), and fl-galactosidase (lanes C and F).

30 o. 19 Ac t iva ted Gsa is separa ted f rom free G T P y S by gel filtration. Bovine brain G-p ro t e in /37 subunits are purified as descr ibed by Sternweis and Robishaw. z° Calmodul in is purified by the me thod of Maune et al. z~ W h e n included in the assay , ca lmodul in , Gsa and /o r G-pro te in /37 subunits are incuba ted with m e m b r a n e s or purified adenyly l cyc lase at 30 ° for 3 rain pr ior to a s say for 7 rain.

Purification of Adenyly l Cyclase

The ability to pur i fy several his t idine-tagged r ecombinan t prote ins to near h o m o g e n e i t y by c h r o m a t o g r a p h y on resins conta in ing chela ted Ni 2÷

19 c. Dietzel and J. Kurjan, Cell (Cambridge, Mass.) 50, 1001 (1987). z0 p. C. Sternweis and J. D. Robishaw, J. Biol. Chem. 259, 13806 (1984). 21 j. F. Maune, C. B. Klee, and K. Beckingham, J. Biol. Chem. 267, 5286 (1992).


prompted us to explore the feasibility of using this methodology to purify recombinant adenylyl cyclases expressed in Sf9 cells. Unfortunately, only a modest (50-fold) enrichment of specific activity was realized. Conse- quently, we developed a rapid (2 day) two-step approach for the purification of the recombinant histidine-tagged type I enzyme: sequential chromatography using forskolin-Sepharose and nitrilotriacetic acid (NTA)-Ni 2+- agarose. Wild-type type I and type II adenylyl cyclases can be purified if lentil lectin-Sepharose is used instead of NTA-Ni2+-agarose. Protease inhibitors are included in all buffers but may be omitted from the final chromatographic step.

Preparation of Detergent Extracts. Type I adenylyl cyclase in bovine brain membranes is extracted efficiently with Lubrol PXfl Using similar conditions, recombinant adenylyl cyclases in Sf9 cell membranes are poorly extracted and appear aggregated when analyzed by gel filtration. This difference may be due to altered glycosylation of proteins expressed in Sf9 cells. We have tested a number of detergents for the capacity to extract adenylyl cyclase in an active, monodisperse form, including Lubrol PX, cholate, digitonin, 3-[(3-cholamidopropyl)dimethylammonia]-l-pro- pane sulfonate (CHAPS), octylglucoside, and dodecylmaltoside. The most efficient extraction (90%) was obtained using dodecylmaltoside in the presence of glycerol and NaCI. To prepare extracts, membranes (100 mg of protein; equivalent to roughly 800 ml of culture) are centrifuged at 100,000 g for 30 min, suspended in 30 ml of buffer A [20 mM Na-HEPES (pH 8.0), 20% (v/v) glycerol, 400 mM NaCI, 2 mM MgC12 , and 2 mM 2- mercaptoethanol], centrifuged at 100,000 g for 30 min, and suspended in 16 ml of buffer A (1.25-fold concentrated). Dodecylmaltoside (4 ml; 4%, w/v) is added dropwise while stirring. The mixture is homogenized with a motor-driven Teflon homogenizer (4 cycles of 20 strokes) and stirred for 15 rain between cycles. After centrifugation for 30 min at 150,000 g, the supernatant is removed and applied to the forskolin column. Except where noted, all steps are carried out at 4 ° .

Forskolin-Sepharose Chromatography. Forskolin-Sepharose is synthesized as described by Pfeuffer and Metzger. 22 Prior to application of the sample, the resin (2 ml) is regenerated by sequential washing with 40 ml of 20 mM Na-HEPES (pH 8.0), 7 M urea, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA, and 2 mM dithiothreitol at 4°; 2 ml of water; 20 ml of 4 M NaC1; 2 ml of water; 20 ml of 70% (v/v) methanol and 2% (v/v) acetic acid; and 2 ml of water. The column is equilibrated with 20 ml of buffer B [20 mM Na-HEPES (pH 8.0), 400 mM NaC1, 2 mM MgC1 z , and 2 mM 2-mercaptoethanol] containing 0.4% (w/v) dodecylmaltoside. The

z2 T. Pfeuffer and H. Metzger, FEBS Lett. 146, 369 (1982).


TABLE III PURIFICATION OF RECOMBINANT ADENYLYL CYCLASES FROM Sf9 CELL MEMBRANES

Specific Total Volume Protein activity activity Recovery Purification

Stage (ml) (mg) (nmol/min/mg) (nmol/min) (%) (-fold)

Type I adenylyl cyclase (hexa-His tagged)

Membranes 8.3 100 3.5 350 100 1.0 Solubilization mixture 20 100 6.3 632 181 1.8 Extract 20 76 8.3 631 180 2.4 Forskolin-Sepharose 12 0.076 1530 116 35 436 NTA-Ni 2+ 1.2 0.0036 7440 29 10 2120

Type II adenylyl cyclase Membranes 8.3 100 4.2 420 100 1.0 Solubilization mixture 20 100 6.7 672 160 1.6 Extract 20 64 8.1 516 123 1.9 Forskolin-Sepharose 12 0.072 1070 77 19 255 Lentil lectin 1.6 0.0032 2160 6.9 2 513

extract is mixed with the resin and incubated for 3 hr at 4 ° with gentle rocking. The resin is poured into a column (1.5 x 1 cm), allowed to drain, and sequentially washed with 100 ml of 0.4% (w/v) dodecylmaltoside in buffer B, 100 ml of buffer B containing 0.2% (w/v) dodecylmaltoside and 2 M NaC1, and 10 ml of buffer B containing 0.2% (w/v) dodecylmaltoside and 2% (v/v) dimethyl sulfoxide. Flow rates should not exceed 2 ml/min. Adenylyl cyclase is eluted by incubating the resin for 15 min in 0.2% (w/ v) dodecylmaltoside and 200/.~M forskolin in buffer B. The elution is repeated three times.

Nitrilotriacetic Acid-Nickel lon-Agarose Chromatography. The forskolin eluate containing histidine-tagged adenylyl cyclase is incubated with 400/xl of NTA-Ni2÷-agarose (Qiagen, Chatsworth, CA) at 4 ° with gentle rocking for 30 min. The resin is washed with 5 ml of buffer B containing 0.1% (w/v) dodecylmaltoside, 5 mM imidazole, and 5 mM 2- mercaptoethanol. The purified enzyme is then eluted three times with 400 /zl of buffer B containing 0. I% (w/v) dodecylmaltoside and 100 mM imidazole.

Lentil Lectin-Sepharose Chromatography. Forskolin eluates of wild- type type I and type II adenylyl cyclases are incubated with 400/zl of lentil lectin-Sepharose (Sigma, St. Louis, MO) for 30 min at 4 ° with gentle rocking. The resin is washed with 5 ml of 0.1% (w/v) dodecylmaltoside in buffer B, and adenylyl cyclase is eluted by incubating the resin four times for 15 min each with buffer B containing 200 mM o-methylmannoside and 0. I% (w/v) dodecylmaltoside.


A1 A2 B1 B2

kDa 106

80

49.5

FIG. 3. Silver-stained sodium dodecyl sulfate-polyacrylamide gels of adenylyl cyclases purified from Sf9 cell membranes. Samples were precipitated by the addition of trichloroacetic acid to 15% (w/v) and centrifuged. Precipitates were washed with cold acetone, resuspended in water, alkylated with N-ethylmaleimide, and electrophoresed through a 7.5% polyacrylamide gel. The gel was stained with silver as described. 22 Lane A1 contained 2.5 ~g of histidine-tagged type I adenylyl cyclase (forskolin eluate); A2, 1/xg of histidine-tagged type ! adenylyl cyclase (NTA-Ni 2+ eluate); B 1, 2.5/xg of type II adenylyl cyclase (forskolin eluate); B2, 1/xg of type II adenylyl cyclase (lentil lectin eluate). Arrows indicate the position of migration of the recombinant adenylyl cyclases. (Reprinted from Taussig et alJ 5)

Glycerol is added to the purified adenylyl cyclase preparations to a final concentration of 5% (v/v), and aliquots are stored at -80 °. Repeated freezing and thawing of the enzyme result in dramatic losses of activity.

Expected Results

A summary of the purifications of the histidine-tagged type I and wild- type type II adenylyl cyclases is shown in Table III. Recoveries from the forskolin-Sepharose column can reach 50% for the type I enzyme and 35% for type II adenylyl cyclase. This step not only permits substantial purification but also rids the preparation of nonfunctional adenylyl cyclase protein. A much higher amount of immunoreactive protein is present in the column flow-through compared to the eluate than is predicted by the recovered activities. It should be noted that the recovery of the histidine- tagged type I adenylyl cyclase from the NTA-Ni2+-Sepharose column (28%) was much better than that obtained for the wild-type type II and type I adenylyl cyclases from the lentile lectin column. Thus, the two-


A

>,09

.~,£) =>,~

15

13

11

9

7

5

3

1

B

100,

90

80

70

60

50

40

30

0 0

O

- - Purified His6-Type/.I O

...-<Pur,f,e, Type,

~0 ~ Membranes J ~ r I J / / I

0 20 40 60 80 100 400 GTPyS • rGsa (nM)

~ , , , ~ . ~ j~. Purified Type I

FI,. ~ 0 pe I

Membranes ,-/4 ~ F J

r i i 10 20 30

By (riM)

FIG. 4. Effects of G-protein subunits on recombinant type I adenylyl cyclases. (A) Stimulation by GTP3'S-activated Gsa and (B) inhibition of Gsa-activated type I adenylyl cyclase by bovine brain By. Preparations were as follows: O, membranes from Sf9 cells expressing histidine-tagged type I adenylyl cyclase; O, purified histidine-tagged type I adenylyl cyclase; and A, purified native recombinant type I adenylyl cyclase. Membranes or purified adenylyl cyclases were first incubated with activated recombinant Gsc~ and/or/33, for 3 rain and were then assayed for 7 min. (Reprinted from Taussig et al. 15)

step procedure outlined for the histidine-tagged type I adenylyl cyclase permits recovery of l0 t imes more purified protein than is achieved with the native type I enzyme.

The purities of the histidine-tagged type I and the native type I I enzymes are illustrated in Fig. 3. 23 The predominant staining proteins are the adenylyl cyclases. The type I1 enzyme migrates slightly faster than

23 W. Wray, T. Boulikas, V. P. Wray, and R. Hancock, Anal. Biochem. 118, 197 (1981).

[7] EXPRESSION OF A D E N Y L Y L CYCLASES IN S f 9 CELLS 1 0 7

A []

1.8 Membranes -.~ F q ~ O

o e 1.2 []

. ~ /E~]J Purified Type ,, 0.6 [~..O" Adenylylcyclase

0 20 40 60 80 100

GTP'~'S ° rGsc~ (riM) B

] ( ~100nM GTPyS • rGsc~

Membranes "~k ~ F ]

12

' ~ 6~.OD: ~"I Adenylylcyclase t,M

0 ~ I I I I } 0 20 40 60 80 100

BT(nM)

FIG. 5. Effects of G-protein subunits on type II adenylyl cyclase. (A) Stimulation by GTP3"S-activated Gsa and (B) activation by bovine brain /33, in the presence of 100 nM GTP3'S-G~a. Assays were performed as outlined in the legend to Fig. 4. Preparations were as follows: [], membranes from Sf9 cells expressing type II adenylyl cyclase; O, purified recombinant type II adenylyl cyclase. (Reprinted from Taussig et al. 15)

does type I, consistent with its lower predicted molecular weight. Only trace amounts of contaminants are present in the final preparations.

Regulation of the purified adenylyl cyclases by G-protein a and/33, subunits is comparable to that of the enzymes assayed in Sf9 cell membranes. As shown in Fig. 4, both the purified histidine-tagged and native type I adenylyl cyclases are activated by Go~, and the activated purified enzymes are inhibited by G-protein/33, subunits, as is the type I enzyme in Sf9 cell membranes. Gsa stimulates both the type II enzyme in Sf9 cell membranes and the purified preparation (Fig. 5A). The stimulatory effect of/33, subunits on the G~a-activated type II enzyme is markedly different

1 0 8 ADENYLYL CYCLASES [8 ]

from the inhibitory effect of 137 on type I adenylyl cyclase. Nevertheless, as shown in Fig. 5B, the effect of/33' on the purified type II enzyme mimics that seen in membranes derived from Sf9 cells expressing type II adenylyl cyclase.

The level of expression of type I or type II adenylyl cyclase in Sf9 cells is not high, approximating 0.05-0.3% of membrane protein. Nevertheless, activities of adenylyl cyclases in these membranes greatly exceed those in most cells or tissues, and the recombinant enzyme accounts for 95-99% of the total activity. Membranes prepared from I liter of Sf9 cells yield 5-I0 /zg of purified adenylyl cyclase. The scale of this procedure can easily be increased by an order of magnitude.

Acknowledgments

Work from the authors' laboratory was supported by National Institutes of Health Grant GM34497, American Cancer Society Grant BE30-O, The Perot Family Foundation, The Lucille P. Markey Charitable Trust, The Raymond and Ellen Willie Chair of Molecular Neuropharmacology, and American Heart Association Texas Affiliate Award 92G078 to W. J. T.

[8] T r a n s i e n t Expres s ion Assays for M a m m a l i a n A d e n y l y l Cyclases

By OFER JACOBOWITZ, JIANQIANG CHEN, and RAVI IYENGAR

Introduction

The cloning of eight mammalian adenylyl cyclases (EC4.6. I. 1, adenylate cyclase) over the past few years t has allowed for the study of their individual regulatory mechanisms. The properties of the cloned adenylyl cyclases can be studied by transient transfection of the cDNAs into mammalian cells. Because all mammalian cells possess endogenous adenylyl cyclase activity, high transfection efficiency or special experimental design is required in order to observe the activity of the exogenous adenylyl cyclase. A strategy for observing activities of transfected adenylyl cyclases is described in this chapter. Transient transfection assays are not always successful, but since they are relatively easy to perform, many experiments may be done within a short period of time. We have expressed cloned mammalian adenylyl cyclases in human embryonal kidney (HEK-

R. I y e n g a r , FASEB J. 7, 768 (1993).


[8] EXPRESSION OF MAMMALIAN ADENYLYL CYCLASES 109

293) cells, 2'3 as well as in C O S - 7 4 (monkey kidney) cells. Expression in each cell type has its own advantages as well as disadvantages. In this chapter we describe the conditions for observing the activity of the transfected adenylyl cyclases in both cell types.

Cell Culture

The HEK-293 cells are grown in Dulbecco's modified Eagle's basal medium (DMEM) supplemented with 10% (v/v) heat-inactivated horse serum, penicillin (I0 U/ml), and streptomycin (10 ~g/ml), The cells are grown in monolayers in T-75 or T-175 flasks. They do not attach very tightly to the flasks and lose adherence with repeated (8-10) passages such that less than 50% of the cells are flattened. The attachment status of 293 cells is important for successful transfections because well-attached cells better survive transfection conditions. Therefore, it is important to maintain frozen stocks of cells from early passages and to use cells cultured less than 6 weeks for transfection. To retain fitness for transfection and to prevent cell death, 293 cells are split 1 : 3 every 3 days or on reaching confluence. It is not advisable to maintain fully confluent bottles since confluent cells are not useful for transfections. Cells that are about 50-70% confluent are best for transfections.

The COS-7 cells are grown in DMEM supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin). In comparison to HEK-293 cells, COS-7 cells adhere much better to plates and can be repeatedly passaged without loss of transfection efficiency. Subconfluent cells are used for transfections.

Preparation of Adenylyl Cyclase cDNAs for Transfections

We have used the plasmid vector pcDNA I (Invitrogen, San Diego, CA) to express adenylyl cyclase cDNAs from the cytomegalovirus promoter/ enhancer element of the plasmid. DNA preparations of pcDNA 1 are sometimes contaminated with DNase activity from host strain E. coli MC 1061/P3. Hence, it is important to estimate the concentration of supercoiled DNA by agarose gel electrophoresis rather than from absorption spectroscopy.

2 R. T. Premont, J. Chen, H.-W. Ma, M. Ponnapalli, and R. Iyengar, Proc. Natl. Acad. Sci. U.S.A. 89, 9809 (1992).

30 . Jacobowitz, J. Chen, R. T. Premont, and R. Iyengar, J. Biol. Chem. 268, 3829 (1993).

4 j. Chen and R. Iyengar, J. Biol. Chem. 268, 12253 (1993).


The procedure for the purification of DNA we use has been adapted from Sambrook et al. 5 Briefly, transformed bacteria are lysed by the alkaline lysis method. The neutralized supernatant is precipitated with 2-propanol. The precipitate is redissolved, treated with RNase, and then extracted with phenol/chloroform. The DNA is then precipitated with ethanol, washed with 70% ethanol, and finally dissolved in TE (10 mM Tris- 1 mM EDTA pH 7.5) buffer for estimation of DNA concentration and transfection.

Transfections

We have used the DEAE-dextran method 6 to transfect adenylyl cyclase cDNAs into both HEK-293 as well as the COS-7 cells successfully. Ap- proximately 3 × 106 cells from subconfluent bottles are plated onto 10- cm dishes and grown overnight.

For 293 cells transfection is carried out for 2 hr with 5/zg supercoiled vector DNA (with or without insert) per 106 cells in MEM or DMEM supplemented with 10% fetal calf serum (GIBCO-BRL, Gaithersburg, MD), 400/.~g/ml DEAE-dextran, and 100/xM chloroquine in a final volume of 10 ml. After a 2-hr incubation of cells with the transfection mixture, the medium is removed by aspiration, and cells are shocked for 2 rain with 10% (v/v) dimethyl sulfoxide (DMSO) in phosphate-buffered saline (PBS). The duration of DMSO shock may be varied between 2 and 3 min to optimize transfection efficiency. Subsequently, the cells are gently washed with PBS and incubated overnight with DMEM supplemented with 10% (v/v) fetal calf serum and antibiotics. It is important to perform washes and aspirations gently to minimize cell detachment. After overnight incubation, cells are trypsinized and seeded at 30,000-60,000 cells/ well in a 24-well plate. To ensure accuracy in counting, cells are counted in a small volume and then diluted for plating. Adenylyl cyclase activity is assayed after an additional overnight incubation in the 24-well plates. For transfection control, a vector transfection group (no insert) is always included. In addition, it is extremely useful to include a type 2 adenylyl cyclase transfection group. The type 2 adenylyl cyclase consistently increases adenylyl cyclase activity about 3-fold in the 293 transfection system and can therefore be used to check for proper transfection protocol.

For transfection of COS-7 cells, cells are treated with 250/xg/ml DEAE- dextran and 100/zg/ml chloroquine along with the appropriate cDNAs for

5 j. Sambrook, E. F. Fritsch, and T. Maniatis (eds.), "Molecular Cloning: A Laboratory Manual," 2rid Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989,

6 W. A. Keown, C. R. Campbell, and R. S. Kucherlapati, this series, Vol. 185, p. 527.

[8] EXPRESSION OF MAMMALIAN ADENYLYL CYCLASES 1 1 1

5 hr at 37 °. The cells are then shocked for 3.5-4 rain with 10% DMSO in PBS. After an additional 24-hr incubation in DMEM plus 10% fetal bovine serum, cells are seeded in 24-well plates. To study Gi inhibition of adenylyl cyclase, we have used the [3H]adenine-prelabeling technique on COS-7 cells for cellular cAMP measurements. A priori there is no reason why adenylyl cyclase assays in COS-7 cells have to be intact cell cAMP accumulation assays and not broken cell assays described above for 293 cells.

Assays for Transfected Adenylyl Cyclases

For adenylyl cyclase assays in 293 cells, transfected cells are hypotonically lysed in individual wells of multiwell plates. The lysis medium contains 10 mM Na-HEPES (pH 8.0), 2 mM EDTA (pH 8.0), and 500 tzM phenylmethylsulfonyl fluoride (freshly prepared). Prior to lysis the culture medium is aspirated, and cells are washed with 500 izl of lysis buffer. Cells are lysed by incubation in 100 p,1 of the lysis medium for 15 rain at 37 °. Lysis is enhanced by vigorous shaking of the plates. Assay components are subsequently added in 50/A for a final volume of 150/A.

Adenylyl cyclase activity is measured by the conversion of [a-32p]ATP to [32p]cAMP. Assays are conducted for 15 rain at 32 ° in the presence of 30 mM Na-HEPES (pH 8.0), 2-3 mM EDTA (pH 8.0), 500/xM phenylmethylsulfonyl fluoride, 0.5 mM 3-isobutyl-l-methylxanthine, 20 mM creatine phosphate, 10/~g/ml creatine phosphokinase, 1/~g/ml myokinase, 0.1 mM [a-32p]ATP (-3000 cpm/pmol), 1 mM [3H]cAMP (-60,000 cpm), and the required amounts of MgCI2. Typically 12 mM MgCI 2 is used to yield approximately 10 mM free Mg 2+ . When Ca 2+/calmodulin-stimulated adenylyl cyclase activity is measured, the assay mixture also contains 0.2 mM EGTA, 0.25 mM CaCl2, and 2.5 tzM calmodulin.

The reaction is stopped by the addition of stop solution [1% sodium dodecyl sulfate (SDS), 10 mM cAMP, 40 mM ATP]. The sample is then diluted with 1 ml water prior to the isolation of the [32p]cAMP. All assays are done with triplicate wells.

For cAMP measurements in COS-7 cells the cells are labeled with [3H]adenine (2 p, Ci/ml) in DMEM and 10% fetal calf serum for 24 hr prior to measurement of cAMP accumulation. During the measurement of cAMP accumulation the cells are incubated with 1 mM 3-isobutyl-1- methylxanthine in DMEM without serum in the presence of appropriate additives. The [3H]cAMP that is formed is measured by chromatography as described by Wong. 7

; Y. H. Wong, this volume [6]; see also R. A. Johnson, R. Alvarez, and Y. Salomon, this volume [3].


0 ~O

0

CL

<

0

E Q.

50

40

30

20

10

0

350

300

250

200

150

100

50

0

A

B

A c 5 ~ A c 2

FIG. 1. Adenylyl cyclase activity in HEK-293 cells expressing the endogenous adenylyl cyclases (--), type 2, or type 5 adenylyl cyclase. HEK-293 cells were transfected with pcDNAI vector or vector containing type 2 or type 5 adenylyl cyclase cDNAs. Adenylyl cyclase activity was measured 48 hr later in the presence of 10 mM MgCI2 (A) or 10 mM MgCI2 plus 10/xM forskolin (B). All activities are means (-+S.D.) of triplicate determinations.

Results

Transfcction with vector DNA by itself moderately increases HEK- 293 cell adenylyl cyclase activity. The experiments in Fig. 1 show activities in HEK-293 cells transfected with either the vector DNA or vector containing the cDNAs for either type 2 or type 5 adenylyl cyclases. The type 2 adenylyl cyclase-transfcctcd cells have a noticeably higher basal adenylyl cyclase activity than vector-transfected cells. The increase in basal activity on expression of the type 2 adenylyl cyclase is in the range of 3- to 6-fold. Such high basal adenylyl cyclase activity is reproducible, and consequently we have used transfections with the type 2 adenylyl cyclase eDNA as a positive control for most of our transfection experiments. In contrast, increases in basal activities by other adenylyl cyclases are far more modest and typically in the twofold range (Fig. IA). Hence, increases in basal activities cannot be always taken as a measure of successful transfections for adenylyl cyclases other than the type 2 enzyme.

[8] EXPRESSION OF MAMMALIAN ADENYLYL CYCLASES 1 13

(n

(J

~D 0

{3-

2 * 50 Basal Ca I Calm0dulin

40

30

< (3

"s E c z

20

10

0 A c l ~ A c l

FIG. 2. Adenylyl cyclase activity in HEK-293 cells expressing the endogenous adenylyl cyclases (--) or type 1 adenylyl cyclase. HEK-293 cells were transfected with pcDNAI vector or vector containing the type 1 adenylyl cyclase cDNA. Adenylyl cyclase activity was measured 48 hr later in the presence of 10 mM MgCI 2 (basal) or 10 mM MgCI z plus 0.2 mM EGTA, 0.25 mM CaC12, and 2.5/xM calmodulin (Ca+2/Calmodulin). All activities are means (+--S.D.) of triplicate determinations.

80-

>, 70 [ =~ .> _~ o Q 6 0 - t~ o

® ~ so-

u ,K m >, ~ 30- > , 0 " E 20

'~ 10 I - - Ac2 Ac6

FIG. 3. Isoproterenol plus GTP-stimulated adenylyl cyclase activity in HEK-293 cells expressing the endogenous adenylyl cyclase (--), type 2, or type 6 adenylyl cyclase. HEK- 293 cells were transfected with pcDNA1 vector or vector containing type 2 or type 6 adenylyl cyclase insert. Adenylyl cyclase activity in the presence of 10 ~M isoproterenol and 10/~M GTP was measured 48 hr later. Basal activities were as follows: control cells, 1.8; type 2 transfected, 8.2; and type 6 transfected, 9.6 pmol cAMP per 106 cells. All activities are means of triplicate determinations. The coefficient of variance was less than 10%. (Adapted from Premont et al. 2)


c- o m

E

o

O.

o

6O

50

40

30

20

10

0

30

25

20

15

10

5

0

b a s a l ISO

B

z m m b a s a l hCG

Ac2 Ac2

Fie. 4. Receptor-stimulated cAMP accumulation in COS-7 cells transfected with pcDNA1 vector (--) or vector containing the type 2 adenylyl cyclase insert (Ac2). (A) Endogenous fl-adrenergic receptors were used to stimulate adenylyl cyclase. For this 10/xM isoproternol was added to the culture medium. (B) Cells were transfected with the LHR cDNA in pKNH along with pcDNAI or pcDNA1-Ac2. Stimulation was effected by the addition of 10/~g/ ml hCG to the culture medium, cAMP accumulation is given as the ratio [3H]cAMP/ ([3H]cAMP + [3H]ATP) × 10 3. Values are means (-+S.E.) of measurements from three wells.

Adenylyl cyclase activities stimulated by various agents can be used for studying the transfections of the other enzymes. Forskolin-stimulated activity is increased 2- to 4-fold in cells transfected with the type 5 enzyme. Type 6 adenylyl cyclase cDNA-transfected cells show activities very similar to that seen with type 5 enzyme-transfected cells. In contrast, the type 2 enzyme-transfected cells show only small increases in forskolin- stimulated activities (Fig. 1B) or no increases at all. Hence, forskolin stimulation of the type 2 adenylyl cyclase cannot be reliably studied in

[8] EXPRESSION OF MAMMALIAN ADENYLYL CYCLASES 115

2 0 .

=~ 15

5

0

(112" I r s n s f e c t e d + + + +

hCG In assay + + ÷ +

FIG. 5. Inhibition of the expressed type 6 adenylyl cyciase by mutant activated Gi2c~. COS-7 cells were transfected with pKNH-LHR and pcDNA1 vector or pcDNA1-Ac6. Additionally, cells were cotransfected with the mutant (Q205L) activated Gi2 a (ai2*) in pCMV or Q205L-ao (-) cDNA in pCMV. Basal and hCG-stimulated cAMP accumulation was measured, cAMP accumulation is given as the ratio of [~H]cAMP/([3H]cAMP + [3H] ATP) x 10 t . Values are means (± S. E.) of measurements from three wells. (From Chen and Iyengar. 4 )

transient transfection assays, although some experiments do show substantial stimulation of the type 2 enzyme by forskolin. 2 The expression of other adenylyl cyclases may be verified by assay of their unique properties. Transfections of 293 cells with type 1 adenylyl cyclase result in an observ- able increase in stimulation by exogenously added Ca2+/calmodulin (Fig. 2).

Stimulation by hormone receptors can also be studied in the transient transfection assays. In HEK-293 cells it is possible to use the endogenous /3-adrenergic receptors to stimulate the expressed adenylyl cyclase, Hor- monal stimulation is increased severalfold in cells transfected with type 2 or type 6 adenylyl cyclase (Fig. 3), The use of endogenous receptors to stimulate the expressed adenylyl cyclase appears to work only in HEK- 293 cells. In COS-7 cells, although the endogenous/3-adrenergic receptors stimulate endogenous adenylyl cyclase(s), it is not possible to see any enhancement of the stimulation when the type 2 adenylyl cyclase is expressed (Fig. 4A). In contrast, when type 2 adenylyl cyclase is cotransfected with luteinizing hormone receptor (LHR) cDNA, it is possible to observe stimulation of the expressed type 2 enzyme by human chorionic gonadotropin (hCG) (Fig. 4B), Although we have not explored in detail the reasons for the difference between the two transfection systems, it


appears that a much smaller fraction of the COS-7 cells take up the transfected DNA relative to the HEK-293 cells. Consequently a selection marker is required to identify the cells that are expressing the exogenous adenylyl cyclase. Because COS-7 cells do not express LH receptors, transfection with the receptors allows for the selective study of LH receptor-mediated stimulation of adenylyl cyclase in transfected cells.

Cotransfection of the hCG receptor and different adenylyl cyclases has been useful in studying Gia inhibition of adenylyl cyclases.4 A typical experiment is shown in Fig. 5, where the effect of cotransfecting the cDNA encoding a mutant activated Gi2a subunit with the LH receptor or the type 6 adenylyl cyclase was studied. Although the native adenylyl cyclase was only marginally inhibited by mutant activated G~2a, the expressed type 6 enzyme was substantially inhibited (Fig. 5).

From these experiments it can be seen that transient transfection assays can be used to study the properties of the expressed adenylyl cyclases. Whereas each of the transient expression systems has some limitations and some transfection failures, the ease of use of these systems allows for a large number of experiments to be performed within a relatively short period of time, thus making transient transfections a very useful method to study adenylyl cyclases.

Acknowledgments

This research was supported by National Institutes of Health Grants CA-44998 and DK- 38761. O.J. is a trainee of the Medical Scientist Training Program at Mount Sinai School of Medicine (New York) and the Endocrinology Training Program (DK-07645).

[9] Ident i f ica t ion of A d e n y l y l Cyclases by Ampl i f ica t ion Us ing D e g e n e r a t e P r i m e r s

By RICHARD T. PREMONT

In t roduc t ion

Adenylyl cyclases (EC 4.6.1.1, adenylate cyclase) convert ATP to 3',5'-cyclic AMP (cAMP). In most tissues in higher organisms, the activity ofadenylyl cyclase is controlled by hormones through heterotrimeric GTP- binding regulatory proteins, or G proteins. Multiple forms of adenylyl cyclase have been known for many years) Cloning efforts have led to

t E. J. Neer, Adv. Cyclic Nucleotide Res. 9, 69 (1978).


[9] DEGENERATE PRIMERS FOR ADENYLYL CYCLASES 1 17

the identification of an unexpectedly large family of G protein-regulated adenylyl cyclase enzymes. At this writing, ten distinct mammalian adenylyl cyclase forms have been identified by at least partial cloning. 2-34 Of these, six have been functionally characterized. 2-9'1~ These ten adenylyl cyclases can be grouped into six distinct structural (and, where shown, functional) classes or subfamilies. 8'9'" The diversity of adenylyl cyclase forms has great functional consequences, as the distinct adenylyl cyclase enzymes are regulated in quite different and somewhat unexpected ways. For overviews of adenylyl cyclase structure and function, see Premont et al. 15

Adenylyl cyclase enzymes have now been cloned from organisms as diverse as bacteria, yeasts, fungi, insects, and mammals. From the sequences of these enzymes, it appears that there may be structurally distinct ways of catalyzing the formation of cAMP. G-protein-regulated adenylyl cyclases from mammals are highly similar to enzymes cloned from Dro- sophi la ~6 and Dic tyos t e l ium, 17 and all appear to share a similar topological structure of a duplicated motif of six membrane spans followed by a large,

2 j. Krupinski, F. Coussen, H. A. Bakalyar, W.-J. Tang, P. G. Feinstein, K. Orth, C. Slaughter, R. R. Reed, and A. G. Gilman, Science 244, 1558 (1989). P. G. Feinstein, K. A. Schrader, H. A. Bakalyar, W.-J. Tang, J. Krupinski, A. G. Gilman. and R. R. Reed, Proc. Natl. Acad. Sci. U.S.A. 88~ 10173 (1991).

4 H. A. Bakalyar and R. R. Reed, Science 250, 1403 (1990). 5 B. Gao and A. G. Gilman, Proc. Natl. Acad. Sci. U.S.A. 88, 10178 (1991). 6 M. Yoshimura and D. M, F. Cooper, Proc. Natl. Acad. Sci. U.S.A. 89, 6716 (1992). 7 y . Ishikawa, S. Katsushika, L, Chen, N. J. Halnon, J.-l. Kawabe, and C. J. Homcy, J.

Biol. Chem. 2,67, 13553 (1992). 8 R. T. Premont, J. Chen, H.-W. Ma, M. PonnapaUi, and R. Iyengar, Proc. Natl. Acad.

Sci. U.S.A. 89, 9309 (1992). 9 S. Katsushika, L. Chen, J,-l. Kawabe, R. Nilakantan, N. J. Halnon, C. J. Homcy, and

Y. Ishikawa, Proc. Natl, Acad. Sci. U.S.A. 89, 8774 (1992). t0 R. T. Premont, O. Jacobowitz, and R. lyengar, Endocrinology (Baltimore) 131, 2774 (1992). 11 j. Krupinski, T. C. Lehman, C. D. Frankenfeld, J. C. Zwaagstra, and P. A. Watson, J.

Biol. Chem. 267, 24858 (1992), 12 j. Parma, D. Stengel, M.-H. Gannage, M. Poyard, R. Barouki, and J. Hanoune, Biochem.

Biophys. Res. Commun. 179, 455 (1991). i~ D. Stengel, J. Parma, M.-H. Gannage, N. Roeckel, M.-G. Mattei, R. Barouki, and J.

Hanoune, Hum. Genet. 90, 126 (1992). ~4 K. Hellevuo, M. Yoshimura, M. Kao, P. L. Hoffman, D. M. F. Cooper, and B. Tabakoff,

Biochem. Biophys. Res. Commun. 192, 311 (1993). t5 R. T. Premont, J. Chen, O. Jacobowitz, and R. Iyengar, in "GTPases in Biology I1" (B.

Dickey and L. Birnbaumer, eds.), H. Exp. Pharmacol. 108/II, 189. Springer-Verlag, Berlin, 1993.

16 L. R. Levin, P.-L. Han, P. M. Hwang, P. G. Feinstein, R. L. Davis, and R. R. Reed, Cell (Cambridge, Mass.) 68, 479 (1991).

17 G. S. Pitt, N. Milona, J, Bodeis, K. C. Lin, R. R. Reed, and P. N. Devreotes, Cell (Cambridge, Mass.) 69, 305 (1992).


presumed intracellular domain. The G-protein-regulated adenylyl cyclases contain regions of high similarity (in the two large intracellular domains which are presumed to form the catalytic center) with both soluble and single membrane span receptor forms of guanylyl cyclases, z,18 as well as with a single transmembrane span receptorlike adenylyl cyclase from D i c t y o s t e l i u m . 17 Both the adenylyl and guanylyl cyclase enzymes share lower sequence similarity with yeast and fungal adenylyl cyclases in this catalytic region. 2 However, bacterial cellular or exotoxin adenylyl cyclases appear quite different, with no discernible sequence similarity with the mammalian adenylyl or guanylyl cyclases. This chapter focuses on the Gcprotein-regulated forms of adenylyl cyclase.

Primers for Amplifying Adenylyl Cyclases

Many members of multigene families have been identified by polymerase chain reaction (PCR) techniques utilizing amplification from degenerate primers matching conserved regions of the family of interest. 19-2~ It is now possible to use this approach to identify the molecular forms of adenylyl cyclase present in given systems, which may help in understanding previously unexplained signal transduction differences among tissues and cells. The strategy for primer design is twofold: elements of structure conserved among known members of a family are first identified, and these conserved motifs are examined for length (generally greater than 5-6 codons), codon degeneracy of the encoded amino acid(s), and distance from other such motifs in order to determine to which regions primers should be synthesized and their relative orientation. A general discussion of designing primers for amplifying related members of multigene families is given in Wilkie and Simon. 21

By comparison of the known G-protein-regulated adenylyl cyclase sequences, many regions of high sequence conservation can be identified in the two large intracellular domains shown schematically in Fig. 1 (for an alignment of seven adenylyl cyclase subtypes, see Premont et al.~5). Thus there are many potential sites for preparing primers to amplify "all" adenylyl cyclases or members of one of the subfamilies. Although the quasi-duplicated first and second intracellular domains share many elements of sequence, 2 the differences between the two domains are sufficient

~8 D. Koesling, E. Bohme, and G. Schultz, FASEB J. 5, 2785 (1991). ~9 F. Libert, M. Parmentier, A. Lefort, C. Dinsart, J. Van Sande, C. Maenhauat, M.-J.

Simons, J. E. Dumont, and G. Vassart, Science 244, 569 (1989). 2o M. Strathmann, T. M. Wilkie, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A. 86,

7407 (1989). 21 T. M. Wilkie and M. I. Simon, Methods: Companion Methods Enzymol. 2, 32 (1991).

[9] DEGENERATE PRIMERS FOR ADENYLYL CYCLASES 119

FIFIF /

VS I LFADIVGFT V Y E / ,

Y

ECLRLLNEIIADFD F S

EKIKTIGSTYM-A T I

KWQYDVWSNDVTLANHMEAGG AGVI GARKPQYD I WGNTVNVASRMD S TGV R F H V K A QR H K LS E

FIG. 1. Predicted membrane topology of mammalian G-protein-regulated adenylyl cyclases. Hydropathy analysis of cloned adenylyl cyclase sequences indicates the potential for two sets of six putative membrane spans. Comparison of adenylyl cyclases with one another (and with guanylyl cyclases) identifies two large quasi-duplicated regions of high sequence conservation presumed to contain the catalytic center. A proposed topology of the adenylyl cyclases, 2 with these two conserved domains located within the cell, is shown. Regions of high similarity among cloned adenylyl and guanylyl cyclases are shaded, with the most highly conserved areas indicated in black. The sequences from these positions found in all presently known mammalian G~-regulated adenylyl cyclases are indicated. Portions of these sequences used for preparing PCR primers for amplifying adenylyl cyclase sequences are described in the text.

in most cases to make primers specific for one versus the other. Similarly, the domains are clearly related to the guanylyl cyclase catalytic domain, but they are distinct enough to design primers which will not amplify guanylyl cyclase sequences.

The sequences RIKILGDCYYC and WQ(Y/F)DVWS are the longest absolutely conserved stretches found in the first conserved domain of all currently known mammalian adenylyl cyclase sequences, as well as the Drosophila type 1 (rutabaga) sequence. These sequences are divergent in two recently identified Dictyostelium adenylyl cyclases, however. One of these Dictyostelium enzymes shares the two-domain, 12-membrane span structure common to the mammalian enzymes, whereas the other


contains only a single membrane span and a single "catalytic" domain as do receptor guanylyl cyclases. 17 In the second conserved domain, the sequences KIKTIGSTYMA, AGVIGA, and YDIWG(N/K)TVN are the longest candidate primer sites found in mammalian adenylyl cyclases. In the Drosophila type 1 sequence, one residue differs (KIKTVGSTYMA) from these sequences, whereas in the Dictyostelium sequences, only the KIKTIG and WG(D)TVN subsequences are conserved with the mammalian consensus.

The minimally degenerate conserved sequences for preparing PCR primers can be obtained by avoiding amino acids with 6 codons and favoring those with 1 or 2 codons. For the first domain, GDCYYC and WQ(Y/F)DVW appear best. The two sequences are separated by 57 codons in all mammalian adenylyl cyclases, which is small enough for efficient amplification, and the region contains both conserved and divergent residues allowing easy confirmation of any clones obtained. Primers based on these sequences have been used to identify the types 5, 6, 7, and 8 adenylyl cyclase sequences from various mammalian tissues.11 However, these primers would not be expected to amplify potential forms of the enzyme which are similar to sequences from Dictyostelium. For the second domain, KIKTIG or GSTYMA are possible primer sites, but the KIKTIG sequence is favored since Ser has 6 codons in two unrelated sets. KIKT- (I/V)G would be expected to recognize either Drosophila or Dictyostelium sequences as well, and thus may be preferred in lower organisms. Primers based on the complete KIKTIGSTYMA and YDIWGNTVN sequences have been used to identify the types 3, 4, and 5 adenylyl cyclase sequences from the NCB-20 neuroblastoma cell line. 6 This latter sequence can be used to prepare either YDIWSG(N/K) or WG(N/K)TVN primers. To include both 12-membrane span and 1-membrane span Dictyostelium-like sequences, the sequence WG(N/K/D)TVN can be used. Amplification from the KIKTIG to WGNTVN motifs encompasses from 72 to 85 codons owing to a variable domain whose size and sequence are characteristic of the mammalian adenylyl cyclase subfamily to which a clone belongs. This size difference is useful in screening subcloned PCR products, as they can be visually assigned to distinct classes (subfamilies) prior to sequencing.

Having found suitable primer sites, primers can be synthesized utilizing degenerate nucleotides at third base wobble positions and a neutral base (inosine) in cases of fourfold codon degeneracy {some workers prefer to replace threefold degeneracies [(a/c/t) for Ile] with inosine as well}. Thus for the second conserved domain sequence KIKTIG, the corresponding sense oligonucleotide primer sequence is 36-fold degenerate with one inosine: 5'-cggcagctcgagaa(a/g)at(a/c/t)aa(a/g)ac(i)at(a/c/t)gg, with an XhoI site underlined and a 6-bp restriction enzyme "clamp" at the extreme


TABLE I OLIGONUCLEOTIDE PRIMERS AND SEQUENCE AMPLIFICATION

Amino acid sequence, oligonucleotide Primers able to amplify orientation, and

sequence Mammalian Drosophila Dictyostelium

First conserved domain primers GDCYYC

Sense WQ (Y/F) DVW

Antisense Second conserved domain primers

KIKTIG Sense

KIKT (I/V) G Sense

WG (N/K) TVN Antisense

WG (N/K/D/E) TVN Antisense

yes yes no 5'-atcaagctcgaggg(i)ga(c/g)tg(c/t)ta(c/t)ta(c/t)tg yes yes no 5'-cacgtcctcgagcca(i)ac(a/g)tc(a/g)(c/g)a(c/t)tgcca

yes no yes 5 '-cggcagctcgagaa( a/ g)at( a/ c / t )aa( a/ g)ac( i )at( a/ c / t )gg yes yes yes 5'-cggcagctcgagaa(a/g)at(a/c/t)aa(a/g)ac(i)(a/g)t(i)gg yes yes no 5'-ccgggactcgagac(a/g)tt(i)ac(i)gt(i)tt(i)cccca yes yes yes 5'-ccgggactcgagac(a/g)tt(i)ac(i)gt(i)t(c/t)(i)cccca

5' end. XhoI is chosen in part because the sequence translates as LE, which is found in this position of many adenylyl cyclases. For the second domain sequence WG(N/K)TVN, the corresponding antisense oligonucleotide sequence is twofold degenerate with four inosines: 5'-ccgggactcgagac(a/g)tt(i)ac(i)gt(i)tt(i)cccca, also with an XhoI site and clamp. Primers corresponding to the first conserved domain sequences GDCYYC [sense primer: 5'-atcaagc.tcgal~gg(i)ga(c/t)tg(c/t)ta(c/t)ta(c/t)tg] and WQ(F/Y)- DVW [antisense primer: 5'-cacgtcctcgagcca(i)ac(a/g)tc(a/g)(c/g)a(c/t)tgcca[ are both 16-fold degenerate with one inosine and contain XhoI sites. In the following examples, the second conserved domain KIKTIG and WG(N/K)TVN primers are utilized, although the first domain primers GDCYYC and WQ(F/Y)DVW have been used successfully under the conditions which follow. These oligonucleotide primers and the sequences they would be expected to amplify are listed in Table I.

P o l y m e r a s e C h a i n R e a c t i o n M e t h o d s

R N A I so la t i on

Total RNA is isolated by the guanidinium/CsC1 gradient method 22 and poly(A) RNA selected on oligo(dT) spin columns (Pharmacia/LKB,

22 j. Sambrook, E. F. Fritsch, and T. Maniatis (eds.), "Molecular Cloning: A Laboratory Manual" 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.


Piscataway, NJ) according to the manufacturer's instructions. Alterna- tively, poly(A) RNA may be isolated directly from cell lysates by oligo(dT) spun column chromatography using the FastTrack system (Invitrogen, San Diego, CA).

cDNA Synthesis

First-strand cDNA is prepared using total or poly(A) RNA as a template for reverse transcriptase. The SuperScript Moloney murine leukemia virus (MMLV) reverse transcriptase (GIBCO/BRL, Gaithersburg, MD) has high processivity required for oligo(dT) priming and has been employed successfully in our hands. Priming of cDNA synthesis with mixtures of random hexamers or nonamers may be advantageous for extremely long mRNAs. RNase-free microcentrifuge tubes are prepared by dipping tubes individually in fresh 0.1% (v/v) diethyl pyrocarbonate (DEPC) in water (in a chemical hood) and autoclaving the dry tubes. An oligo(dT)18 primer is synthesized and resuspended at 100/.~M in DEPC- treated water. The reverse transcription reaction is performed in an RNase-free tube using 1 /xg of poly(A) RNA or 5/xg of total RNA and I/zl of 100/zM oligo(dT) primer brought to a volume of 11 ttl with DEPC- treated water. The RNA and primer are heated for 5 min at 75 ° and cooled on ice. Two microliters of 100 mM dithiothreitol (DTT), 1 ~1 (40 U) of RNasin placental RNase inhibitor (Promega, Madison, WI), 1/xl of 10 mM mixed deoxynucleoside triphosphates (dNTPs) (Boehringer Mannheim, Indianapolis, IN), 4/xl of 5X SuperScript buffer, and 1/zl of SuperScript reverse transcriptase (200 U) are added to the tube on ice. The 20-/xl reaction is incubated for 1 hr at 45 °, diluted to 100/zl with DEPC-treated water, and stored at - 2 0 ° until use. One microliter contains the cDNA prepared from 10 ng of poly(A) RNA or 50 ng total RNA. The synthesis can be monitored by adding 1 ~Ci of [o~-32p]dCTP or other dNTP tracer to the reaction.

Amplification

Amplification of DNA sequences is performed in a programmable thermal cycler using the heat-stable Taq DNA polymerase, z3 Reactions are prepared using sterile pipette tips in sterile tubes using DEPC-treated water to exclude exogenous templates from the reaction. To reduce the threat of sample contamination further, filter pipette tips are recommended (i.e,, ART tips from Fisher). Standard reactions are performed in volumes

23 M. A. Innes, D. H. Gelfand, J. J. Sninsky, and T. J. White (eds.), "PCR Protocols: A Guide to Methods and Applications." Academic Press, San Diego, 1990.

19] DEGENERATE PRIMERS FOR ADENYLYL CYCLASES 123

of 100/zl and contain I x Taq buffer with 1.5 mM MgCI2, 200/xM each dNTP, 500 nM each of the sense and antisense primers,and 10 ng of first- strand cDNA template. Reactions are covered with 100/xl of light mineral oil (Sigma, St. Louis, MO) and heated for 5 rain at 95 ° to denature the templates initially. While the samples are held above the chosen annealing temperature, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Nor- walk, CT) added to the individual tubes through the mineral oil layer, and cycling is begun immediately. For the primers given here, the standard reaction conditions are 35 cycles of 95 ° for 1 rain denaturation, 55 ° for 1 min annealing, and 72 ° for 3 min extension, followed by a final 10-rain extension at 72 ° .

The completed reaction is extracted with 175 /zl of chloroform to remove the mineral oil, and the upper aqueous phase is transferred to a clean tube and dried in a SpeedVac concentrator. The pellet is resuspended in DNA sample buffer and separated on a 2% agarose gel in TAE buffer or a 4% NuSieve agarose gel (FMC Bioproducts, Rockland, ME) in TAE buffer. 22 The products are visualized with ethidium bromide under long- wave UV illumination, and product bands of the expected size are cut from the gel. For the KIKTIG sense and WGNTVN antisense primer pair, distinct size products are expected for the various subtypes of adenylyl cyclases. Type 3 sequences yield a 312-bp band, types 2 and 4 a 291-bp band, types 9 and 10 a 288-bp band, type 7 a 285-bp band, type 1 a 282- bp band, type 8 a 276-bp band,and types 5 and 6 a 273-bp band. With the first conserved domain GDCYYC sense and WQFDVW antisense primer pair, a 227-bp band is produced for all known mammalian adenylyl cyclase subtypes.

Subcloning and Characterization

The PCR products in excised gel slices are isolated using the GeneClean kit (Bio 101, La Jolla, CA) or are spun from the gel for 2 min through a glass wool plug in a small microcentrifuge tube with a punctured bottom into a large microcentrifuge tube and then extracted with phenol. The PCR band is digested in 100/zl with excess XhoI (50-100 U) for 4 hr, and restricted ends are removed using the GeneClean procedure. Digested PCR bands are mixed with 25-50 ng of XhoI-digested, gel-purified pBlue- Script II vector and ligated overnight at 15 ° in 20-tzl reactions using 3 U of T4 DNA ligase (Promega). Ligations are used to transform competent Escherichia coli (XL1-Blue cells, Stratagene, La Jolla, CA) and plated with IPTG (isopropyl-/3-o-thiogalactopyranoside) and X-Gal (5-bromo-4- chloro-3-indolyl-/3-o-galactoside) on LB plates according to the manufacturer's protocol. White colonies are grown overnight in LB medium with 50 ~g/ml ampiciUin and DNA minipreparations prepared. 22 Clones with


appropriate size X h o I fragments are sequenced from vector (T7 and T3) primers using [a-3Zp]dATP and the Sequenase v2 T7 DNA polymerase kit (U.S. Biochemicals, Cleveland, OH). 22

Results

As an example of the ability to identify existing and novel adenylyl cyclases by this method, the adenylyl cyclase subtypes in several cell lines commonly used in studies of the adenylyl cyclase system have been examined. The murine $49 lymphoma cell line has a long history in adenylyl cyclase research, and mutants derived from this cell line played a pivotal role in identifying the G s protein 24 and in demonstrating hormonal inhibition of adenylyl cyclase activity. 2s'26 Type 6 adenylyl cyclase has been cloned from this cell line using a PCR-based approach.l° COS-7 cells, derived from African green monkey kidney, are commonly used for the expression ofadenylyl cyclase-coupled receptors. Finally, the human embryonic kidney HEK-293 cell line has been used for receptor expression and, more recently, for expression of cloned forms of adenylyl cyclases. Because of these uses, it is of some interest to know which specific subtypes of adenylyl cyclase are expressed in these cells. Poly(A) RNAs were prepared from each cell type and oligo(dT)-primed first-strand cDNAs were synthesized and used as templates in PCR amplifications using the KIKTIG and WGNTVN primers as described.

Shown in Fig. 2 are the deduced amino acid sequences of amplified adenylyl cyclase fragments from the three cell lines aligned with known adenylyl cyclase sequences. Somewhat surprisingly, all three cell lines were found to contain a novel adenylyl cyclase sequence, termed type 9, which appears to be a member of the type 2 and 4 subfamily. Hellevuo et al. have also reported this novel sequence from HEL and HEK-293 cells, ~4 and a full-length clone for this type 9 enzyme has recently been cloned (Nomura et al., unpublished, GenBank # D25538). The type 9 enzyme is very highly similar to the type 7 enzyme identified by Krupinski and coworkers in $49 cells and several rat tissues, using GDCYYC and WQYDVW primers from the first conserved domain, it As expected, $49 cells contain type 6 sequences as well. Of twelve COS-7 cell clones examined, all had the same apparent inert size, and the five which were sequenced all represented type 9. In HEK-293 cells, sixteen clones were

24 E. M. Ross and A. G. Gilman, J. Biol. Chem. 252, 6966 (1977). 25 j . D. Hildebrandt, R. D. Sekura, J. Codina, R. Iyengar, C. R. Manclark, and L. Birn-

baumer, Nature (London) 302, 706 (1983). 26 K. H. Jakobs, K. Aktories, and G. Schultz, Nature (London) 303, 177 (1983).


rat type 6

dog type 6

HEK-293 type 6

849 type 6

rat type 5

dog type b

bovine type 1

HEK-293 type 1

rat type 2

human type 2

rat type 4

human type 9

COS-7 type 9

HEK-293 type 9

$49 type 9

rat type 3

human type 8

mouse type I0

......... I ......... I ..... ....I ......... ~ .... ,...50 EKIKTIGSTYMAASGLNA ....... STYDQVGR ...... SHITALADYAM

EKIKTIGSTYMAASGLNA ....... STYDQAGR ...... SHITALADYAM

....... STYMAASGLNA ....... STYDQVGR ...... SHITALADYAH

EKIKTIGSTYMAASGLNA ....... STYDQVGR ...... SHITALADYAM

EKIKTIGSTYMAASGLND ....... STYDKAGK ...... THIKALADFAM

EKIKTIGSTYMAASGLND ....... STYDKVGK ...... THIKALADFAM

EKIKTIGSTYMAAVGLAP ....... TAGTKAKKCIS---SHLSTLADFAI ....... STYMAAVGLAP ....... TSGTKAKKSIS---SHLSTLADFAI

EKIKTIGSTYMAATGLSA ....... IPSQEHAQEPERQYMHIGTMVEFAY

EKIKTIGSTYMAATGLSA ....... VPSQEHSQEPERQYMHIGTMVEFAF

EKIKTIGSTYMAATGLNA ....... TPGQDTQQDAERSCSHLGTHVEFAV

EKIKTIGSTYMAAAGLSV ........ ASGHENQELERQHAHIGVMVEFS!

....... STYMAAAGLSV ........ ASGHENQELERQHAHIGVMVEFSI

....... STYMAAAGLSV ........ ASGHENQELERQHAHIGVMVEFSI

....... STYMAAAGLSV ........ ASGHENQELERQHAHIGVMVEFS!

TKIKTIGSTYMAASGVTPDVNTNGFTSSSKEEKSDEERWQIiLADLADFAL

EKIKTIGSTYMAVSGLSP ............ EKQQCEDHWGHLCALAD~SL

EKIKTIGATYMAASGLNT ........... AQCQEGGHPOEHLRILFEFAK

rat type 6

dog type 6

HEK-293 type 6

$49 type 6

rat type 5

dog type 5

bovine type 1

HEK-293 type 1

rat type 2

human type 2

rat type 4

human type 9

COS-7 type 9

HEK-293 type 9

$49 type 9

rat type 3

human type 8

mouse tpe i0

. . . . . . . . . I . . . . , . . . . I . . . . . . . . , [ . . . . . . . . . ~ . . . . . . . ~ t'. (; RLMEQMKH INEHS-FNNFQMK I GLNMGPVVAGVI GAEKPQY D ! WGNTV~V

RLMEQMKHINEHS-FNNFQMK I GLNMGPVVAGVi GARKPQYD I WGNTVNV

RLMEQMKH INEHS-FNNFQMKI GLNMGPVVAGV I GARKPQY D 2 ::-- =-

RLMEQMKH INEHS-FNNFQMK I GLNMGPVVAGV I GARKP QYD IWGNTVNV

KLMDQMKY INEHS- FNNFQMKI GLN I GF VVAGVI GARF~P QYD IWGNTVNV

KLMD QMKY INEH S-FNNFQMK I GLN I GP VVAGV I GARKP QY D I WGN T VNV

EMFDVLDE I N YQS- YND FVLRVG I NVGP VVAG V i G ARR ?Q Y D [ WG~(T t,':1V

EMFDVLDE INYQS-YNDFVLRVG INVGPVVAGV 1 GARRPQY D I .........

ALVGKLDAINKHS-FNDFKLRVG INHGPV IAGV I GAQKPQY D IWGNTVt~V

ALVGKLDAINKHS-FNDFKLRVG IN HGP V I AGV 1 GAQEP QYD !WG~]TVNV

ALGSKLGVINKHS-FNNFRLRVGLNHGP VVAGV i GAQKPQYD i WGNTVNV

ALMSKLDGINRHS-FNSFRLRVGINHGPVIAGV i GARKPQY D IWGNTV]q\;

ALMSKLDGINRHS-FNSFRLRVGINHGPVIAGVI 5ARKPQYD 1 . . . .

ALMSKLDP INRHS-FNSFRLRVGiNHGPV~ AGV i "~ARKpQyD i .......

ALMSKLDGINRHS-FNSFRLRVGINHGPVffAGVI3ARs<PQY[ ! -: :

AMKDTLTN INNQS-FNNFMLR I GMNKGGVLAGV ! ";AR ~:? H Y I) l WGN'?V,~JV

ALTES IQEINKHS-FNNFELRI G I SHGSVVAGTI 3ARR<PQY[" I WGKTVN L

EMMRVVDDFNNNMLWFNFKLRVGFNHG? L TAGV I ~'? T'}< i. ], ~i' ? W()~q'~ '<J I

FIG. 2. Alignment of amplified adenylyl cyclase sequences. Deduced amino acid sequences of nine cloned subtypes of mammalian adenylyl cyclase 2-I4 are shown aligned for a region of the second conserved domain encompassing the PCR primer sites KIKTIG and WG(N/K)TVN. The human type 8 partial sequence n is named following Krupinski et al.lt Sequences amplified from $49 cells, COS-7 cells, and HEK-293 cells are shown grduped with the most closely matching known adenylyl cyclase form, except for types 9 and 10, for which no sequence has been previously published for this region (see text). Primers in PCR clones, for which the actual sequence is unknown, are shown as = . In the alignment, sequences found in all mammalian adenylyl cyclase subtypes shown are indicated with * below the alignment, and residues with only conservative substitutions are indicated with/\ .

sequenced, of which one was type 1, eight were type 6, and seven were type 9. Further analysis of clones from COS and 293 cells may reveal the presence of additional subtypes; Hellevuo et al. have reported types 2, 3, 6, and 9 in HEK-293 cells} 4 The appearance of the novel type 9 adenyly!


cyclase in the three cell lines further highlights the apparently widespread distribution of this form of the enzyme. The presence of the calmodulin- stimulated type 1 adenylyl cyclase in kidney 293 cells is also notable in light of the assumed neuronal-specific distribution of this form of the enzyme. A similar PCR analysis of mouse brain adenylyl cyclases using these primers has identified another previously unknown form of adenylyl cyclase, here shown as type 10, which appears quite distinct from the five previously known subfamilies (RTP, unpublished).

Having identified a novel adenylyl cyclase fragment, the complete sequence can be obtained by using this small fragment as a hybridization probe or using alternative procedures such as rapid amplification of cDNA ends (RACE) 27 or nested PCR screening. 8'28 Additional portions of the sequence may also be obtained by amplifying the same template using the common first conserved domain primer pair GDCYYC and WD(F/Y)DVW to identify the appropriate first domain fragment and then amplifying from exact sequence primers in the first to the second domain. It is also possible to directly amplify larger fragments (1.5-2.1 kb) from domain 1 to domain 2, using degenerate GDCYYC or WQ(F/Y)DVW sense primers to KIKTIG or WG(N/K)TVN antisense primers. The $49 cell type 6 sequence was originally identified by the amplification of a 2.3- kb fragment from the first to the second domain using degenerate primers matching SILFADI and VKGKGEM (which have since been shown to not recognize all adenylyl cyclase sequences).l°

The large number of known adenylyl cyclase forms leads to the obvious question of why an organism might need so many adenylyl cyclase enzymes. At first glance, all the cloned and expressed enzymes appear to serve exactly the same function, to produce cAMP in response to hormonal activation of receptors and Gs. However, it appears that it is the secondary regulation of the distinct adenylyl cyclases, by calcium/calmodulin, 6'u'29 by fly subunits of G proteins, 3'5'3°'31 by protein kinases] °'32'33 and perhaps by unknown other cellular factors, which requires the existence of dis-

27 M. A. Frohman, M. K. Dush, and G. R. Martin, Proc. Natl. Acad. Sci. U.S.A. 85, 8998 (1988).

28 I. R. Gibbons, D. J. Asai, N. S. Ching, G. J. Dolecki, G. Mocz, C. A. Phillipson, H. Ren, W.-J. Y. Tang, and B. H. Gibbons, Proc. Natl. Acad. Sci. U.S.A. 88, 8563 (1991).

29 W.-J. Y. Tang, J. Krupinski, and A. G. Gilman, J. Biol. Chem. 266, 8595 (1990). 3~ W.-J. Y. Tang and A. G. Gilman, Science 254, 1500 (1991). 31 A. D. Federman, B. R. Conklin, K. A. Schrader, R. R. Reed, and H. R. Bourne, Nature

(London) 356, 159 (1992). 32 T. Yoshimasa, D. R. Sibley, M. Bouvier, R. J. Lefkowitz, and M. G. Caron, Nature

(London) 327, 67 (1987). 33 R. Simmoteit, H.-D. Schulzki, D. Palm, S. Mollner, and T. Pfeuffer, FEBS Lett. 285,

99 (1992).


tinct enzymes. Analysis of clonal cell lines, as shown here for $49 cells, COS-7 cells, and HEK-293 cells, by Yoshimura and Cooper 6 for NCB-20 cells, and by Hellevuo et al. 14 for HEL and HEK-293 cells, indicates that individual cells may contain several distinct subtypes of adenylyl cyclase. All appear to respond essentially equivalently to G S yet may differ markedly in their secondary regulation to produce the cell-specific character of the adenylyl cyclase stimulation by added hormones. The individual regulation of these distinct enzymes now appears to be a potentially important component in the cellular regulation of adenylyl cyclase activity.

[10] ASSAYS FOR G-PROTEIN-STIMULATED PLC ACTIVITY 131

[10] Assays for G - P r o t e i n R e g u l a t i o n of Phospho l i pa se C Act iv i ty

By M I C H A E L D E V I V O

Introduction

Phospholipase C (PLC) is the generic name for enzymes that catalyze hydrolysis of phosphoglycerides into diacylglycerols and phosphorylated alcohols (serine, choline, inositol, glycerol, and ethanolamine). In signal transduction, the minor phospholipids (< 1% of total phosphoglycerides) phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) are of special interest. Hydrolysis of PIP and PIP2 results in the formation of two second messengers: hydrophobic diacylglycerol and the hydrophilic and cytoplasmic inositol phosphates (IP 2 and IP3). The PLC that mediates hydrolysis of PIP and PIP z is a polyphosphoinositide- specific enzyme, and the name phospholipase C is not adequate to describe its function. Alternative names, such as phosphoinositidase, have been proposed, but phospholipase C is still used routinely. In this chapter, when we refer to PLC we mean the enzymes that preferentially catalyze hydrolysis of polyphosphoinositides (PIP and PIP2). In theory, either diacylglycerol or inositol phosphate formation could be measured as an index of PLC activity; in practice, it is difficult to quantitate diacylglycerol levels as precisely as inositol phosphates. This chapter describes assays for inositol phosphates only, but the interested investigator is referred to protocols for measurements of diacylglycerol levels.

Phospholipase C enzymes have been purified and cloned. The PLCs have been divided into three families: PLC-fl, PLC-7, and PLC-& z PLC- y is a substrate for growth factor receptor tyrosine kinases such as the platelet-derived growth factor (PDGF) receptor. 3 The proposed model for activation of PLC-y is as follows. Upon activation by growth factors, growth factor receptors autophosphorylate. Autophosphorylated receptors couple to intracellular substrates with appropriate tyrosine residues. On coupling, the substrate proteins (including PLC-y) are phosphorylated and, apparently, activated. In contrast, the activity of PLC-/3 is regulated by G proteins. Cell surface receptors catalyze the activation of G proteins

J. Preiss, C. R. Loomis , W. R. Bishop, R. Stein, J. E. Neidel, and R. M. Bell, J. Biol. Chem. 261, 8597 (1986).

2 S. G. Rhee and K.-J. Choi, J. Biol. Chem. 267, 12393 (1992). 3 j . Schlessinger and A. Ullrich, Neuron 9, 383 (1992).

Copyright © 1994 by Academic Press. Inc. METHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved.

132 PHOSPHOLIPASES C [10]

which results in the formation of a and fly subunits. Both BY subunits and Gqol subunits are implicated in stimulation of PLC-fl activity. 4,5

This chapter deals with measuring PLC activity in response to stimulation by G proteins. The experimental protocols differ fundamentally depending on whether the substrate pool of phosphoinositides are labeled endogenously in intact cells or whether labeled polyphosphoinositides are added exogenously as part of lipid vesicles. The type of information that can be obtained and the applicability of each method differ greatly.

Endogenous Substrates

Overview

Cells of interest are incubated with myo-[3H]inositol. Inositol is converted to phosphatidylinositol (PI) by phosphatidylinositol synthase, and PI in turn is converted to PIP and PIP2 by phosphatidylinositol kinases. After labeling of the polyphosphoinositide pool to steady state, breakdown of PIP and PIP2 is initiated by addition of appropriate stimulant (e.g., receptor agonists or, to permeabilized cells, guanylyl nucleotides) to the cell culture medium. Cells are incubated for the appropriate time, the reaction is stopped by addition of methanol or acid, and the water-soluble inositol phosphates are extracted and separated on Dowex anion-exchange columns.

Assay Procedure

The basic assay described here is used to measure PLC activity in NIH 3T3 cells. Deviations from the assay procedure may be appropriate depending on the cell type. Cells to be tested are plated at subconfluent density into wells of tissue culture dishes (24-well dishes). Cells are incubated with complete medium. We routinely use Dulbecco's modified En- gle's medium (DMEM) with 5% bovine calf serum from GIBCO, (Grand Island, NY) and in the presence of myo-[3H]inositol (I #Ci/ml) (New England Nuclear, Boston, MA; No. NET-114A) for 48 hr. (Some laboratories label cells in serum-free or inositol-free medium, but we do not.) Cells are washed twice with 1 ml of DMEM containing 10 mM LiCI, 0.1% (w/v) bovine serum albumin (BSA) and 10 mM glucose. Cells are then incubated with 0.5 ml of this solution with or without reagents. Cells are incubated for 30 min at 37 ° (blank values are determined immediately at

4 S. J. Taylor, H. Z. Chae, S. G. Rhee, and J. H. Exton, Nature (London) 350, 516 (1991). 5 M. Camps, A. Carozzi, P. Schnabel, A. Scheer, P. J. Parker, and P. Gierschik, Nature

(London) 360, 684 (1992).

[101 ASSAYS FOR G-PROTEIN-STIMULATED PLC ACTIVITY 133

this stage by adding stopping solution before the 30-min incubation). After incubation the medium is aspirated, and the reaction is stopped by addition of 0.5 ml of methanol/HCl (100 : 1, v/v). The cells are scraped and removed to glass test tubes. Chloroform (0.5 ml) and 250 tzl of 10 mM EDTA are added to the tubes. The mix is vortexed and centrifuged briefly to separate phases. The aqueous layer is washed a second time with 1 ml of preequilibrated lower phase (the lower phase of a mix of methanol/chloroform/ HCI/water/EDTA, 100 : 100 : 1 : 20 : 250 by volume). The mix is centrifuged and the aqueous phase removed. The aqueous phase is added to Dowex exchange columns for isolation of inositol phosphates.

Column Preparation. We use Sarstedt tips (Newton, NC; No. 91.787), stoppered with glass wool, for columns. We add 0.5 ml of Dowex anion- exchange resin (AG1-X8 resin, formate form; No. 140-1444 from Bio-Rad, Richmond, CA) equilibrated with 10 mM inositol and 0.1 M formic acid. The columns are regenerated with 5 ml of 3 M ammonium formate in 0. l M formic acid before every use. The resin is then equilibrated with 6 ml of 10 mM inositol in 0.1 M formic acid. The cell extract is mixed with 2 ml of 0. I M formic acid and added to the columns. The important separation step is to remove labeled inositol from the more polar inositol phosphates. Inositol is washed through the column with 5 ml of 10 mM inositol in 0.1 M formic acid. Total inositol phosphates (IP + IP 2 + IP3) are eluted by adding 5 ml of 0.8 M ammonium formate in 0.1 M formic acid. If separation of the inositol phosphates is desired, IP is eluted with 5 ml of 0.2 M, IP2 with 0.4 M, and IP 3 with 0.8 M ammonium formate/0.1 M formic acid. We find that performance of the columns is consistent after 1 year of use with regeneration of columns as described above. However, if perchloric acid (PCA) or trich|oroacetic acid (TCA) is used to stop the reaction, column performance deteriorates after only two uses. Additional information pertaining to isolation of inositol phosphates on Dowex columns is contained in Berridge et al. 6

Applicability o f Method

This method has wide applicability because any cell or tissue that can be cultured long enough (2 days) theoretically can be incubated with labeled inositol. Variations in the method occur at both the labeling step and the assay step, Some investigators use cells in suspension for labeling rather than monolayers; others label cells in monolayers and then measure inositol phosphate accumulation after suspending the cells. Cell suspensions are easier to handle when a large number of data points are being

6 M. J. Berridge, C. P. Downes, and M. R. Hanley, Biochem. J. 206, 587 (1982).


obtained (in making dose-response curves, for example). An interesting variation of the method has been described for labeling rat brain slices affixed to coverslips. 7

A variation of the method is used in some laboratories to meaure PLC activity in cell membranes. After labeling the endogenous PIP and PIP2 pool with [3H]inositol, cells are lysed and homogenized in hypotonic buffer. Crude membrane fractions are obtained, and these fractions are used to test the effects of guanylyl nucleotides or purified G proteins on PLC activity. Only some tissues can be used in this way, however, because most washed cell lysates appear to lose most of the PLC activity. The GH3 (rat pituitary tumor) cells work well in this regard, and a procedure for measuring PLC activity in GH 3 cell membranes has been published previously in this series. 8 This method has limited applicability; we have been unable to measure PLC activity in NIH 3T3 cell membranes.

Assay conditions reported from various laboratories also differ with respect to the method of stopping the reaction. Using TCA or PCA to stop the reaction obviates the extraction step, but requires that the extract be neutralized before separation on the anion-exchange columns.9 A potentially useful variation of this procedure has been described by Seuwen et al., lo who use 10 mM formic acid to stop the reaction and apply the extract to columns, eliminating both the neutralization and organic extraction steps.

Data Interpretat ion

The assay results can be standardized for amount of substrate by counting the radioactivity in the combined organic phases. Although this contains mostly PI, it is assumed that PIP and PIP 2 are proportional to the amount of PI in the assay. The substrates (PIP and PIP2) can be further analyzed by separating them chromatographically and scraping and counting the spots that run with known standards. 8 This will allow relative comparisons of incorporation of labeled inositol into PIP and PIP2] in stimulated and unstimulated cells; however, because the concentration of the polyphosphoinositides is so low, it is not possible to determine concentrations of the lipids to obtain a specific activity for the substrate.

7 p. M. Dunn, P. R. Coote, and J. N. Wood, in "Neuronal Cell Lines: A Practical Approach" (J. N. Wood, ed.), p. 114. IRL Press, Oxford, New York, and Tokyo, 1992.

8 A. Imai and M. C. Gershengorn, this series, Vol. 141, p. 100. 9 D. A. Kendall and S. J. Hill, in "Methods in Neurotransmitter Receptor Analysis"

(H. 1. Yamamura, ed,), p. 69. Raven, New York, 1990. ~ K. Seuwen, A. Lagarde, and J. Pouyssegur, EMBO J. 7, 161 (1988).


t -

O

"5 E ¢,1 cJ

e.. Q . I:n 0

J= O.

m 0

0 C

,01 8

'o 6 ,e-

E 4 0 .

'10

2

v e c t o r ~ * q

q

FIG. 1. Effect of activated aq on inositol phosphate accumulation in NIH 3T3 cells. NIH 3T3 cells were transfected with vector alone or with vector having the cDNA encoding the a subunit of Gq (Otq) or a mutant, active % subunit (Otq*). Ceils were plated at a density of 105 cells per well in 24-well tissue culture dishes and grown for 3 days in the presence of cell culture medium and 1 izCi/ml of myo-[3H]inositol. Cells were washed free of serum and incubated in serum-flee medium containing 10 mM LiCI, 10 mM glucose, 0.1% (w/v) BSA, and 10 tzM prostaglandin F2~ (PGF2~), a G-protein-coupled receptor agonist. Inositol phosphates were assayed as described in the text. Data are means of three wells (-+SE).

Because the specific activity of the substrate is unknown, results are expressed as counts per minute (cpm) or disintegrations per minute (dpm) of inositol phosphates plus or minus standard errors (SE). We plate the same number of cells per well and report the results as disintegrations per minute of inositol phosphates per well, but results can be standardized with respect to cell number, milligrams of protein, or per disintegrations per minute incorporated into the phospholipids as well. We have used the method described here to measure inositol phosphate accumulation in N I H 3T3 cells transfected with c D N A encoding a mutant, active aq subunit. I~ Receptor-stimulated PLC activity was greatly increased in mutant aq- transfected cells compared to vector-transfected control cells (Fig. 1).

Exogenous Substrates

O v e r v i e w

Labeled substrate phospholipids are obtained commercially and used with unlabeled phospholipids as a substrate. Alternatively, phospholipids

~ M. De Vivo, J. Chen, J. Codina, and R. Iyengar, J. Biol. Chem. 267, 18263 (1992).


from a given tissue can be labeled endogenously with inositol and then extracted and purified, lz The phospholipids are mixed in the presence or absence of detergent and sonicated briefly on ice. Aliquots of the substrate are mixed with a source of PLC (membranes, purified enzyme) and, often, G protein c~ subunits or 137 subunits. The assay is run for a predetermined time and stopped by addition of chloroform/methanol/HCl (CMH) (100: 100: 1, by volume). Then EDTA is added, phases are separated, and aliquots of the aqueous phase removed and counted.

Assay Procedure

We use an assay volume of 100/zl. A typical assay consists of the following: 25/A of a 4 x assay buffer, 25/zl of a source of [3H]PIP2, 25 /zl of a solution of CaCI2, 10/zl of G protein a or f17 subunits, and 15/zl of a source of PLC (usually either purified enzyme or cell cytosol). Final components of the assay are I0 mM HEPES (pH 6.8), 100 mM KC1, 125 mM NaCI, 1 mM EDTA, 1 mM dithiothreitol (DTT), 6 mM MgSO4, 2.75 mM EGTA, 25/zM CaCI2, 25,000 dpm of labeled PIP z, and 500 pmol of unlabeled polyphosphoinositides (see below for preparation of substrate and calculation of free Ca z+ concentration). We start the reaction by addition of either [3H]PIP2 or PLC. The assay is incubated for 10 min at 30 ° and stopped by addition of I ml of CMH. Then 250/A of 10 mM EDTA is added, the mix is centrifuged, and 400/zl of the aqueous phase is removed and counted. Note that because no labeled inositol is used, and the substrate is PIP2, it is not necessary to separate inositol phosphates on Dowex columns because the substrate is presumably removed in the organic extract and the sole product should be IP3. However, it is a good idea to check at least once that the reaction products do migrate correctly on the anion-exchange columns.

Preparation ofSubstrate. We use [3H]PIPz from New England Nuclear (No. NET-895; 8.8 mCi/mmol) as a source of labeled PIP2 and a mixture of phosphoinositides from Sigma (St. Louis, MO, No. P 6023) as a source of unlabeled PIP and PIP2. The unlabeled phosphoinositides are dissolved in 2 ml of chloroform and kept at - 2 0 °. We use enough labeled PIP2 to yield approximately 25,000 dpm in the final assay tube. This corresponds to 1 pmol of PIP:. We use enough of the unlabeled phosphoinositides to yield 500 pmol in the final assay. Typically, we use 40/A of labeled and 40/zl of the unlabeled solutions as prepared above. The mix is dried under nitrogen and resuspended in 10 mM HEPES, pH 7.0 (we do not use detergent to resuspend lipids, but some researchers use deoxycholate or

12 G. L. Waldo, A. J. Morris, and T. K. Harden, this volume [15].

[10 l ASSAYS FOR G-PROTEIN-STIMULATED P L C ACTIVITY 137

cholate). The solution is kept on ice and sonicated briefly (3 pulses) with a probe sonicator (some researchers use bath sonicators).

The specific activity is calculated by counting on aliquot of the mix and dividing the number of disintegrations per minute of the solution by the number of moles of the unlabeled PIP and PIPz (because the unlabeled phosphoinositides are in great molar excess over the labeled phosphoinositides). To determine the number of moles of unlabeled PIP and PIP2, we first multiply the number of milligrams by 0.2 to take into account that only 20% of the phosphoinositides are PIP and PIP z according to the manufacturer's specifications. We estimate the molecular weight to be 950 (the molecular weights of PIP and PIP z vary because of different fatty acids at the first and second carbons). A typical assay includes 25,000 dpm and 500 pmol of substrate, for a specific activity of 50 dpm/pmol.

Note that in many of these experiments, a significant (up to 30%) percentage of the exogenous substrate is used up in the course of an assay. Therefore, interpretation of the results are complicated by the fact that substrate concentration is not constant and the velocity of the enzyme reaction is not maximal. These conditions are acceptable for qualitative experiments (i.e., detecting PLC activity during purification or testing whether a G protein subunit is stimulating PLC activity); however, they are not acceptable for rigorous quantitative characterization of the various PLC enzymes.

Applicability of Method

This procedure is used to test the effects of pure G proteins directly on either crude or pure preparations of PLC. It is also used to add exogenous substrates to permeabilized tissues and cells. 13,x4 In contrast to assays of endogenous labeled substrates, in which the assay conditions are relatively straightforward to work out but performing the assay can be somewhat laborious, determining assay conditions for exogenous assays is somewhat difficult, but once the assay conditions have been established the assay is relatively simple. The reason the assay conditions are difficult to determine is that a significant amount of the substrate is used up during the assay. There is a relatively narrow window in which to work, usually from 1000 to 10,000 dpm for a 30,000 dpm assay. Thus, all the factors that determine activity must be very narrowly controlled or the effects of regulators of PLC activity will not be measurable. Some of these factors are discussed below.

t3 M. A. Wallace, E. Claro, H. R. Carter, and J. N. Fain, this series, Vol. 197, p. 183. 14 G. M. H. Thomas, E. Cunningham, and S. Cockcroft, this volume [13].


Detergents. Because many of the PLC and G-protein purification procedures involve solubilization with Lubrol, cholate, or octylglucoside, these detergents may end up interfering with a PLC assay. For example, we find that cholate stimulates PLC activity and can obscure stimulation by G proteins. We keep cholate concentration under 0.1% (w/v) in the final assay.

Temperature. We find that a partially purified PLC from Drosophila that we use in our studies is more active at 30 ° than at 37 ° (Fig. 2). This may not be true for all PLCs, however, and must be tested on an individual basis.

Enzyme Concentration. Optimization of enzyme and free Ca 2+ (see below) concentrations is the key element to developing a useful PLC assay. For crude sources of PLC, such as rat brain cytosol, high enzyme concentrations result in a decrease in observed PLC activity (Fig. 3A). The data suggest that inhibitors of PLC activity are present in cytosol, possibly phospholipids that were not removed at the low speeds (10,000 g) used to prepare the crude cytosol. For pure PLCs, this inhibition is not observed (Fig. 3B). However, high enzyme concentrations tend to increase basal PLC activity to such an extent that any stimulation by G proteins is not measurable. An enzyme concentration must be chosen to allow a sufficient window to detect stimulation by an appropriate regulator. We find 2/~g per 100-/A assay works well, with low basal and sufficiently

A :: e - " ' ( l)

.> '~

oE' 5

.~ E 4

m ~ 0 0

1

3 7 ° 3 0 °

Fla. 2. Effect of assay temperature on PLC activity. Partially purified PLC from Drosoph- ila was assayed, and the PLC activities were compared at 30 ° and 37 °. Data are means of three determinations (+SD).


2 5 > ,

A 20

• ~ 15

O J= 0 .

5

,A

1 10 50 1 5 2 5

Protein Concentration (pg/assay)

FIG. 3. Effect of enzyme concentration on PLC activity. Crude rat brain cytosol (A) and purified PLC from Drosophila (B) were assayed at the indicated concentrations of protein per 100-txl assay. Data are means of three determinations a(-SD).

high stimulated PLC activity. Most laboratories use much lower concentrations of pure PLCs, usually from 1 to 10 ng per assay tube.

Free Calcium Ion. As mentioned above, both enzyme and free Ca2* concentrations must be determined together to develop a useful PLC assay. For example, in Fig. 4, we varied total Ca 2+ from no added Ca ,-+

, '2

= o 4

O O

A

25 I~M 250 ~.M

CaCI 2 (total)

FIG. 4. Effect of Ca 2+ on/3y-stimulated PLC activity. Enzyme activity was assayed as described except that added CaCI2 was either none (0), 25, or 250/xM. Assay buffer contained 2.75 mM EGTA. Assays were performed in the absence (open bars) or presence (filled bars) of 0.5 tzM/33, subunits. Data are means of three determinations (-+SD).


(Fig. 4A), to 25 tzM (Fig. 4B), and 100/ ,M (Fig. 4C) added, all in the presence of 2.75 mM EGTA, and stimulation by fly subunits was observed only with 25/xM added Ca 2+ . This yields approximately I0 nM free CaC12 using the calculations of Harafuji and Ogawa. 15 However, others use much higher Ca 2+ concentrations (usually I00 nM to 1 #M), and the optimal concentration must be determined empirically. A high free Ca 2+ concentration (> 1/.,M) is appropriate for detecting PLC activity during a purification procedure, but a lower concentration is more useful when assaying for stimulation by G proteins. Because different laboratories use different formulas for calculating free Ca 2 + concentrations, it is preferable to include the total concentration of Ca 2+ used in the assay procedure.

Interpretation of Results

Because the specific activity of the substrate is known, the results from an assay can be expressed in terms of a rate of production of inositol phosphates. We subtract blanks (assays with no enzyme or inactivated enzyme) from the 10-min assay results and divide the results by specific activity, by assay time, and by milligrams protein. Final results are usually expressed as nanomoles IP3/per minute per milligram protein with standard deviations (SD) as shown in Fig. 4.

Acknowledgments

The author is an Aaron Diamond Fellow, and work was supported in part by a grant from the Aaron Diamond Foundation and National Institutes of Health Grant DK-38761.

15 H. Harafuji and Y. Ogawa, J. Biochem. (Tokyo) 87, 1305 (1980).

[1 1] C h l o r i d e C u r r e n t A s s a y for P h o s p h o l i p a s e C

in X e n o p u s O o c y t e s

By EMMANUEL M. LANDAU and ROaERT D. BLITZER

Introduction

Activation of phospholipase C can indirectly be measured in oocytes by recording a CaE+-dependent chloride current (Icl(Ca)). 1-3 This method

I K. Kusano , R. Miledi, and J. St innakre, Nature (London) 270, 739 (1977). 2 N. Dascal , E. M. Landau , and Y. Lass , J. Physiol. (London) 352, 551 (1984). 3 N. Dascal , B. Gillo, and Y. Lass , J. Physiol. (London) 366, 299 (1985).


[11] CI- CURRENT IN OOCYTES 141

is very effective in identifying receptors which are coupled to the phosphoinositide (PI) second messenger system 4 and may also be useful in identifying G proteins and lipases in this pathway. 5 The oocyte has also been extensively used to clone receptors, using Ic~(ca) to monitor receptor expression. 6 The advantage of the oocyte approach lies in the relative ease both of expressing a variety of genes in the cell and of injecting second messengers and proteins into these very large cells. An example of the power of this approach is the discovery of the metabotropic glutamate receptor, which was initially seen in oocytes injected with total brain RNA 7 and was finally isolated by sib selection, using Xenopus oocytes as an expression system)

This chapter discusses technical points regarding the maintenance of healthy frogs, the securing of healthy oocytes, the injection of mRNA into the cells for receptor expression. Also covered are the technique of voltage clamping, evidence for the chloride current being a good measure of phospholipase C activation, and pitfalls and dangers in this approach.

Frog Oocytes

The ovary of adult female Xenopus laevis flogs contain 24 lobes and a large number of oocytes in different stages of development. These have been divided into six stages (I to VI). 9 As oocytes mature from stage to stage their size increases and their appearance changes. Only the largest oocytes (stages V and VI) are used for experimental purposes. These have diameters of 1000 to 1300 tzm (volume 0.7 to 1.3 tzl) and have clearly delineated hemispheres. One of the hemispheres is brown (light brown in stage V and darker brown in stage VI) and is named the animal pole. The other hemisphere is yellow and is called the vegetal pole. In stage VI there appears an unpigmented equatorial band.

The oocytes are surrounded by four layers. Closest to the oocyte membrane is the acellular vitelline envelope, next comes a layer of small follicular cells, next the theca (a connective tissue layer containing blood vessels and fibrocytes), and finally the surface epithelium which is continu-

4 y. Oron, N. Dascal, E. Nadler, and M. Lupu, Nature (London) 313, 141 (1985). 5 T. M. Moriarty, E. Padrel, D. J. Carty, G. Omri, E. M. Landau, and R. Iyengar, Nature

(London) 343, 79 (1990). 6 H. Lubbert, B. J. Hoffman, T. P. Snutch, T. van Dyke, A. J. Levine, P. H. Hartig,

H. A. Lester, and N. Davidson, Proc. Natl. Acad. Sci. U.S.A. 84, 4332 (1987). 7 C. B. Gundersen, R. Miledi, and I. Parker, Proe. R. Soc. London B 221, 127 (1984). 8 M. Masu, Y. Tanabe, K. Tsuchida, R. Shigemoto, and S. Nakanishi, Nature (London)

349, 760 (1991). 9 j. N. Dumont, J. Morphol. 136, 153 (1972).


T A B L E I

SOLUTIONS FOR OOCYTE RECORDING

Concentration (raM)

Component ND96 Ringer's OR2 b NaHCO3-Ringer's Barth's c

N a C I 96 116 82 .5 115.6 88

K C I 2 2 2 2 1

MgC12 1 1 1 - - - -

CaCI2 1.8 '~ 1.8 1.8 1.8 0.41

N a - H E P E S 5 - - 5 - - - -

T r i s - H C l - - 5 - - - - 2

Ca(NO3)2 . . . . 0.41

M g S O 4 . . . . 0 .82

N a H C O 3 - - - - - - 2 .4 - -

CaC12 is omitted from Ca2+-free ND96. b Modifiedy0,si c Modified.52

ous with the epithelium covering the ovary. An oocyte with all the layers intact is called a follicle. The three outermost layers can be removed by treatment with collagenase, whereas the vitelline layer can be removed mechanically from oocytes submerged in a hypertonic solution. 9a The collagenase treatment yields defolliculated or denuded oocytes, which are most often used for our purpose. Removal of the vitelline membrane is useful if one wishes to record single-channel currents and is not discussed further here.

Oocyte Physiology

The oocyte membrane is semipermeable and contains receptors, various transporters, and ion channels. The oocyte resting potential (Er) is determined by the intra- and extracellular ionic composition and by the relative membrane permeability. The ions which determine the resting potential are Na*, K +, and CI-. The respective equilibrium potentials in frog Ringer's solution (Table I) are ENa, +40 mV; EK, --102 mV; and Ec~, -24 inV. 2,~° The permeability ratios between the ions, that is, PNa : PcI : PK are 0.12 : 0.4 : 1 in follicles and 0.24 : 0.39 : 1 in denuded oocytes.2 Denuded oocytes are thus more permeable to Na +. The Er value in follicles is approximately -45 mV, but it tends to be more negative in denuded

9~ C. M e t h f e s s e l , V. W i t z e m a n n , T. T a k a h a s h i , M. M i s h i n a , S. N u m a , a n d B. S a k m a n n ,

Pfluegers Arch. 407, 577 (1986). 10 K . K u s a n o , R . Mi led i , a n d J . S t i n n a k r e , J. Physiol. (London) 328, 143 (1982).


oocytes, owing to the activation of an electrogenic Na+-K + exchange pump) The input impedance of the oocyte is 0.6 Mf~ (range 0,1 to 3 M~) and the specific resistance of the order of 30 kft cm2. z~° The fact that Ec~ is more positive than Er indicates that the intracellular CI- concentration is higher than would be the case if the ion were passively distributed. This is probably due to an inward directed C1- pump. The CI- current (/cO flowing through the C1- conductance (gc0 is Icl = g c l (Er - Ecl), which for an oocyte clamped at -50 mV will be -gc~ (26). The negative sign for the current indicates that it is inwardly directed, which is equivalent to an outward flow of CI-.

Calcium-Activated Chloride Current

The oocyte possesses a Ca2+-activated CI- conductance gcl(c~) which is strongly modulated by voltage. The single-channel conductance is 3 pS and it becomes activated by C a 2+ concentrations in the range of 10 -7 to 10 -6 M. t~ In the presence of sufficient and stable intracellular C a 2+

concentration, the C1- conductance increases 8-fold when the membrane is stepped from -130 to +50 mV (B. Gillo, M. Jafri, and E. Landau, unpublished). More complex voltage effects are seen when Ca z+ influx determines gc~(c,). ~2,~3 When gc~(ca) is activated, it permits the efflux of CI- down its electrochemical gradient. The efflux of CI- is equivalent to an inward-directed electric current (see above).

Mechanism of Action of Receptors Coupled to Phospholipase C

When activated by a transmitter, receptors coupled to phospholipase C generate a typical inward current with a relatively fast initial phase (D~) and a slower second phase (D2) with superimposed current oscillations, z This current can be obtained by activating native receptors (muscarinic, angiotensin II receptor; see Fig. 1, first trace) or receptors expressed by injecting the appropriate messenger RNA (for a comprehensive list see below). Figure 1 (second trace) shows the response of an expressed adrenergic a~b receptor. The receptors are coupled to a pertussis toxin-sensitive G protein (probably Go). The activated G protein stimulates a membrane phospholipase C which hydrolyzes phosphatidylinositol bisphosphate to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. IP3 liberates Ca 2+ from intracellular stores which in turn activates gct(ca) (see above) to

t~ T. Takahashi, E. Neher, and B. Sakmann, Proc. Natl. Acad. Sci. U.S.A. 84, 5063 (1987). z2 R. Miledi, Proc. R. Soc. London B 215, 491 (1982). 13 M. E. Barish, J. Physiol. (London) 342, 309 (1983).


ANG II NE IP 3 Ca 2+

:E

FIG. 1. Four different oocyte responses. The first trace shows the response of an "angiotensin supeffrog" to application of 10 -6 M angiotensin II (transmitter application marked by a horizontal bar). Note the sharp initial response (Dr) and the slower secondary wave (D,). Also, note the typical current fluctuations (F). The second trace shows the response of an oocyte, expressing the cloned adrenergic am receptor, to the application of 10 -5 M norepinephrine. Note that Dz and F are missing, which often happens with expressed receptors. The third trace gives the response to the intraceUular injection of 0.2 pmol of IP3, with the times of injection being indicated by arrows. Note that the second injection of IP3 did not elicit any response. The fourth trace shows the response to intracellular injection of 60 pmol of CaC1 z. Note the increase in size of the second response, probably caused by the filling of intracellular Ca 2+ stores IN. Dascal and R. Boton, FEBS Left. 267, 22 (1990)]. The current (vertical) calibration bars indicate (from left to right) 1000, 1000, 100, and 200 nA. The corresponding time calibrations (horizontal) are 2, 2, 1, and 0.5 rain.

produce the inward current seen in Fig. 1 (first and second traces). Each step in the cascade of events has been confirmed.

The activation of the G protein can be directly induced by injecting oocytes with the GTP analog GTPyS (100-150 pmol). This procedure evokes a CI- current similar to that evoked by transmitters, except that it is very prolonged, the time to peak of the early phase being 3-5 times that of the transmitter-induced current and the late phase being very prolonged, lasting many minutes. In addition, injection of GTPyS occludes the responses to transmitters. Similarly, injection of GDPflS (100-500 pmol/oocyte) inhibits the responses to transmitters.14

The G protein which is activated by transmitters is sensitive to pertussis

~4 N. Dascal, C. Ifune, R. Hopkins, T. P. Snutch, H. Lubbert, N. Davidson, M. I. Simon, and H. A. Lester, Mol. Brain Res. 1, 201 (1986).


toxin (PTX), which places it in the Gi class of G proteins.~4a However, high concentrations of PTX (2-10/xg/ml) and prolonged exposure (24-72 hr) are necessary to obtain an effect. Also, the inactivation of the G protein by PTX is rarely complete, t4:5 The G protein involved is probably the native Go since transmitter action is augmented by injection into the oocyte of bovine holo-G o (2 fmol/oocyte) and is mimicked by injection of activated bovine o~ o (0.3 fmol/oocyte)) In addition, coupling between receptors and the PI system can be disrupted by injecting the oocytes with antisense oligonucleotides directed against the Xenopus Go. 16 However, the PTX- induced labeling of recombinant Xenopus Go is much weaker than that of the bovine protein, which may account for the reduced sensitivity of oocytes to treatment with PTX. It is intriguing to note that receptors that couple to Gq in the native tissue ~7'~8 regularly couple to the Xenopus G O . Although differing in amino acid sequence, both proteins may be similar in terms of the receptor binding site. Because Go does not activate mammalian phospholipase C-/3 (PLC-/3),I6a the Xenopus Go probably interacts with a unique Xenopus PLC enzyme, which has been cloned recently. Isa Some coupling through Gq/ll m a y also occur. 18b

The formation of IP 3 by receptor activation is well documented. Thus, in oocytes prelabeled with [3H]inositol, receptor activation leads to the accumulation of phosphatidylinositol 4,5-bisphosphate breakdown products: inositol 1-phosphate, inositol 1,4-bisphosphate, and IP3. In addition, direct injection of IP 3 (0.1-2 pmol/oocyte) produces a C1- current which is very similar to the one caused by receptor activation. In addition, the amplitude of the CI- current increases with the amount of IP 3 injected, albeit not in a linear fashion: The response to IP 3 often shows a rapid and a slow component (slow component not seen in Fig. 1). When IP3 is injected more deeply into the cell (>100/xm) the rapid phase diminishes and the slow phase becomes larger. ~9 A noteworthy feature of IP 3 injection

~4a M. I. Simon, M. P. Strathmann, and N, Gautam, Science 252, 802 (1991). ~5 T. M. Moriarty, S. C. Sealfon, D. J. Carty, J. L. Roberts, R. Iyengar, and E. M. Landau,

J. Biol. Chem. 264, 13524 (1989). z6 R. D. Blitzer, G. Omri, M. G, Caron, R. J. Lefkowitz, S. Coteccia, E. M. Landau, and

R. lyengar, J. Biol. Chem. 268, 7532 (1993). 16a S. D. Kroll, J. Chen, M. De Vivo, D. J. Carry, A. Buku, R. T. Premont, and R. lyengar,

J. Biol. Chem. 267, 23183 (1992). 17 A. V. Smrcka, J. R. Hepler, K. O. Brown, and P. C. Sternweis, Science 251, 804 (1991). J8 S. J. Taylor, H. Z. Chae, S. G. Rhee, and J. H. Exton, Nature (London) 350, 516 (1991). ~8a H.-W, Ma, R. D. Blitzer, E. C. Healy, R. T. Premont, E. M. Landau, and R. Iyengar,

J. Biol. Chem. 268, 19915 (1993). JSb D. Lipinsky, M. C. Gershengorn, and Y. Oron, FEBS Lett. 307, 237 (1992). f9 B. Gillo, Y. Lass, E. Nadler, and Y. Oron, J. Physiol. (London) 392, 349 (1987).

146 PHOSPHOLIeASES C [11]

is that the response to consecutive injections diminishes markedly, probably owing to the depletion of internal Ca 2+ stores 5,2° (Fig. I, third trace).

The next step, namely the release of Ca z+ from internal stores, has been documented with the Ca2+-sensitive dye Fura-2. When transmitter is applied, the intracellular concentration of Ca ~+ rises from about 0.1 to above 1/zM, resulting i,a a CI- current.r1 Finally, the C1- current can be elicited directly by intracellular injection of Ca 2+ (0.5-100 pmol/oocyte; Fig. I, fourth trace). Notably, repeated injections of Ca 2+ do not result in diminution of the response, indicating that the Ca2+-dependent C1- conductance does not become inactivated. 3

Finally, the identity of the ionic mechanism can be proved by studying the current-voltage (I-V) relationship of the currents induced by transmitters, G proteins, etc. This is done by plotting the current flowing through the membrane against the membrane voltage (I-V plot). When I-V plots in the control and in the test situation are superimposed, the crossing point defines the equilibrium potential for the induced current. This potential was found to be identical to the CI- equilibrium potential and to be shifted with changes in bathing C1- according to the Nernst relationship, 2 indicating that the induced current flows through a Cl--selective channel.

In summary, the pathway leading from receptor via IP 3 to increased CI- conductance is well established in the oocyte.

Receptors Coupled to Oocyte Phosphoinositide Second Messenger System

It is likely that all receptors which couple to the PI system in mammalian cells are able to couple to this system in the oocyte. However, the coupling in the oocyte is somewhat unusual, utilizing Go as well as Gq and activating a different kind of phospholipase C. For this reason caution should be exercised when applying lessons from studies of receptor coupling in oocytes to mammalian cells. The oocyte is most useful as a receptor expression system and has been for this purpose in cloning the serotonin (5-HT~ 21 and 5-HT222), metabotropic glutamate, 8'23 endothelin, 24

2o M. J. Berridge, Proc. R. Soc. London B 238~ 235 (1989). 2t D. Julius, A. B. MacDermott, R. Axel, and T. M. Jessel, Science 241, 558 (1988). 22 D. B. Pritchett, A. W. Bach, M. Wozny, O. Taleb, R. Dal Toso, J. C. Shih, and P. H.

Seeburg, EMBO J. 4135 (1988). 23 K. M. Houamed, J. L. Kuijper, T. L. Gilbert, B. A. Haldeman, P. J. O'Hara, E. R.

Mulvihill, W. Almers, and F. S. Hagen, Science 252, 1318 (1991). 24 H. Arai, S. Hori, I. Aramori, H. Ohkubo, and S. Nakanishi, Nature (London) 348,

730 (1990).


substance K , 25 neuromedin K , 26 substance p,27 platelet-activating factor (PAF),28 prostaglandin F 2 ,29 bradykinin B2, 3° neurotensin, 3t cholecystoki- nin A,32 thyrotropin-releasing hormone,32a and gonadotropin-releasing hormone 33 receptors. Additional PI-coupled receptors also evoke the typical C1- current in the oocyte. These include the muscarinic ,34 o~ lb-adrenergic, 16 thromboxane A235 and interleukin 8 36 receptors, as well as the receptor for bombesin. 3v

The oocyte has been used to study the role of Ca 2+ and IP3 in the generation of intracellular Ca 2+ oscillations. 38-43 The oocyte is a suitable preparation for such studies because it is a large cell in which both electrical and optical changes can be readily recorded. Also, the size allows the injection of substance such as IP 3 analogs which can induce oscillations.

25 Y. Masu, K. Nakayama, H. Tamaki, Y. Harada, M. Kuno, and S. Nakanishi, Nature (London) 329, 836 (1987).

26 R. Shigemoto, Y. Yokota, K. Tsuchida, and S. Nakanishi, J. Biol. Chem. 265,623 (1986). 27 y. Yokota, Y. Sasai, K. Tanaka, T. Fugiwara, K. Tsuchida, R. Shigemoto, A. Kakizuka,

H. Ohkubo, and S. Nakanishi, J. Biol. Chem. 264, 17649 (1989). 28 Z. Honda, M. Nakamura, I. Miki, M. Minami, T. Watanabe, Y. Seyama, H. Okado, H.

Toh, K. lto, T. Miyamoto, and T. Shimizu, Nature (London) 349, 342 (1991). 29 K. Sakamoto, T. Ezashi, K. Miwa, E. Okuda-Ashitaka, T. Houtani, T. Sugimoto, S. Ito.

and O. Hayaishi, J. Biol. Chem. 269, 3881 (1994). 30 A. E. McEachern, E. R. Shelton, S. Bhakta, R. Obernolte, C. Bach, P. Zuppan, J.

Fujisaki, R. W. Aldrich, and K. Jarnagin, Proc. Natl. Acad. Sci. U.S.A. 88, 7724 (1991). 3~ K. Tanka, M. Masu, and S. Nakanishi, Neuron 4, 847 (1990). 3-~ S. A. Wank, R. Harkins, R. T. Jensen, H. Shapira, A. de Weerth, and T. Slattery, Proc.

Natl. Acad. Sci. U.S.A. 89, 3125 (1992). 32a R. E. Straub, G. C. Frech, R. H. Joho, and M. C. Gershengorn, Proc. Natl. Acad. Sci.

U.S.A. 87, 9514 (1990). 33 M. Tsutsumi, W, Zho, R. P. Millar, P. L. Mellon, J. L. Roberts, C. A. Flanagan, K.

Dong, B. Gillo, and S. C. Sealfon, Mol. Endocrinol. 6, 1163 (1992). 34 Y. Kubo, K. Fukuda, A. Mikami, A. Maeda, H. Takahashi, M. Mishina, T. Haga, K.

Haga, A. Ichiyama, K. Kangawa, M. Kojima, H. Matsuo, T. Hirose, and S. Numa, Nature (London) 323, 411 (1986).

35 M. Hirata, Y. Hayashi, F. Ushikubi, Y. Tokota, R. Kageyama. S. Nakanishi, and S. Narumiya, Nature (London) 349, 617 (1991).

36 p. M. Murphy and H. L. Tiffany, Science 253, 1280 (1991). 37 T. M. Moriarty, B. Gillo, S. Sealfon, J. L. Roberts, R. D. Blitzer, and E. M. Landau.

Mol. Brain Res. 4, 75 (1988). 38 W. C. Taylor, W. J. Berridge, K. D. Brown, A. M. Cooke, and B. V. L. Potter, Biochem.

Biophys. Res. Commun. 150, 626 (1988). 39 S. DeLisle, K.-H. Krause, G. Denning, B. V. L. Potter, and M. J. Welsh, J. Biol. Chem.

265, 11726 (1990). 40 j. Lechleiter, S. Girard, D. Clapham, and E. Peralta, Nature (London) 350, 505 (1991). 4J j. Lechleiter, S. Girard, E. Peralta, and D. Clapham, Science 252, 123 (1991). ~2 M. S. Jafri, S. Vajda, P. Pasik, and B. Gillo, Biophys. J. 63, 235 (1992). 4.~ G. Brooker, T. Seki, D. Croll, and C. Wahlestedt, Proc. Natl. Acad. Sci. U.S.A. 87,

2813 (1990).


Finally, many receptors can be expressed in the oocyte and their effect on the oscillations studied. 4°

Maintaining Healthy Frogs

The health and well-being of the frogs is a major factor in the success of oocyte experiments. Frogs can be obtained from Xenopus One (Ann Arbor, MI) or Nasco (Ft. Atkinson, WI). When ordering, large females should be specified (90-100 g), nonprogesterone treated. On arrival they should be housed in large tanks made of dark plexiglass (not white or transparent) at a density of not more than 1 frog per gallon of water. The tanks should be covered with a mesh to prevent the flogs from escaping. The depth of the water should be such that the flogs can touch bottom. Tap water should be dechlorinated by letting it stand uncovered for 3 days before adding it to the maintenance tanks. Frogs should be maintained at 18 ° to 20 °. A climate-controlled room is ideal for this purpose. In addition it is useful to have the light-dark cycle in the room regulated (15 hr light/ 9 hr dark cycle; e.g., light from 7 am to 10 pm). This may help minimize the seasonal variation in oocyte function which is often observed) 4

Frogs should be fed twice a week with diced liver. The liver (beef or chicken, 10 g per frog) should be put into the tank and left there for 5-10 hr. It is convenient to feed the frogs in the morning and replace the water in the afternoon or evening of the same day. Be sure to clean the tanks thoroughly after each feeding. Care should be taken to discover and eliminate frogs which develop the "red leg disease," when their limbs become reddened and stiff, since this may be transmitted to other frogs.

Obtaining Healthy Oocytes

Frogs should be anesthetized by submerging them in a 0.2% tricaine sulfate (3-aminobenzoic acid ethyl ester; MS-222) solution until they lose the righting reflex (20-30 min). When anesthetized, a small incision (<1 cm) is made in the abdomen (left or right lower quadrant), and a section of the ovary is pulled out with embryonic forceps. A small piece of ovary is then excised and put in Ca2÷-free ND96 medium (for composition see above). The skin is then sutured and the frog returned to the tank after a period of recovery in a shallow bath. The instruments used for dissection should be prewashed in ethanol (70 or 100%).

The tissue thus obtained is transferred to Ca2÷-free ND96 medium containing 2 mg/ml of collagenase (we use coUagenase B from Boehringer

44 N. Dascal and E. M. Landau, Life Sci. 27, 1423 (1980).

[11] C1- CURRENT IN OOCYTES 149

Mannheim, Indianapolis, IN) and gently shaken for 30 min to 1 hr. When doing this, it is useful to cut the ovary into clusters of 10-20 oocytes each, since this will make the coUagenase treatment more efficient. Let the oocytes stay in collagenase for 30 min and inspect them. If they are not sufficiently defolliculated, put the oocytes in a fresh collagenase solution and let them stay there for an additional 10 to 30 min. Usually, no more time is required. The end point of the process occurs when the oocytes have fully separated from the original clusters and about half show the halo typical of fully denuded oocytes (see below). It is not advisable to proceed until most oocytes are completely denuded because many of them turn out to be damaged. At the end of the process oocytes are washed with ND96 and the largest and healthiest looking are transferred into ND96 medium. By the next day, the follicular cell sheath will have sloughed off the nondenuded oocytes. Healthy oocytes will have an intact animal pole, whereas damaged oocytes will have a mottled or spotted animal pole. Also, damaged oocytes appear soft when prodded with a glass rod. The defoniculated oocyte has a luminous corona (halo) when observed with transmitted light (Fig. 2).

This procedure will defolliculate the oocytes and make them ready for RNA or protein injections. Oocytes from different donors vary in ease of

FIc. 2. Defolliculated oocytes. Note the two poles (animal, dark, vegetal, light). Also visible is the corona, a sliver of light on the left aspect of most cells. Bar, 1 ~m.


defolliculation, as do different batches of collagenase. It is advisable to purchase from the supplier samples from several stocks of collagenase and try them out. Once a suitable stock is identified, purchase a large quantity of it for use over an extensive period of time. One should avoid frequent changes in the collagenase lots, because different lots will have widely varying effectiveness. The defolliculation step is one of the more crucial ones in preparing viable oocytes. Underexposure to collagenase will leave the follicular cells largely intact, which will make it difficult to penetrate them with injection pipettes. However, overexposure will damage the oocytes. Constant observation is required to master this stage effectively. Sometimes oocytes are defolliculated manually (without the use of collagenase) by using a pair of fine forceps. However, this is a very labor-intensive procedure and can also result in damage to the oocytes.

After defolliculation the oocytes can be maintained for 7 and sometimes up to 10 days. The medium should be changed daily, and dead cells should be discarded.

It is important to mark the frog from which a certain batch of oocytes has been obtained. This can be achieved by noting the physical characteristics of the frog or housing it alone. Being able to identify frogs is useful because oocytes from different donors vary in viability, ability to express injected mRNA, and ability to keep expressing native receptors after defolliculation. Frogs which yield oocytes with the latter characteristic are dubbed "superfrogs." To date we are aware of two types of superfrog. The first is a muscarinic superfrog, which is very rare (1/25 to 1/100 frogs), and the second is a superfrog whose oocytes express the native angiotensin II receptor. The latter frogs are much more common (about 4/20). The angiotensin receptors here are located on the oocyte and not on the follicular cell membrane, since the angiotensin response remains viable for up to 5 days, when follicular cells would already have withered away. Oocytes from superfrogs have the advantage of yielding very stable, predictable responses, and they allow the study of the PI signaling pathway without requiring a preparatory injection of mRNA.

Once a frog is identified, oocytes may be harvested from it on repeated occasions at 3- to 4-week intervals. If more than 300 oocytes had been harvested, a 6-week interval is recommended. During this period the incision heals completely and the ovary has time to regenerate. This offers the advantage of obtaining oocytes with known and predictable characteristics.

Injecting Substances into Oocytes

Injection of Large Quantities. For injecting large quantities of substances into oocytes, use the Drummond lO-tzl displacement pipettor

[1 l ] CI- CURRENT IN OOCYTES 151

(Drummond, BroomaU, PA). Fit it with a micropipette drawn from Drum- mond glass (10-~1 replacement tube); for RNA injection bake the tube at 250 ° for 2 hr. The tip of the pipette should be broken by hitting it against the side of a fire-cracked glass slide until it has a diameter of 12 to 15 ~m. The pipette is first backfilled to the tip with extra heavy mineral oil (e.g., Swan's), and then the tip is filled with the solution to be injected. This is done by immersing the tip of the pipette in a drop of the required solution placed on top of a sheet of Parafilm. The Drummond pipettor, prefilled with mineral oil, is then made to draw the solution up into the pipette. A volume of up to 7 ill can be fitted into the pipette tip. For this step, as well as for oocyte injection, the pipettor should be mounted on a micromanipulator, and the drop or oocytes should be placed under a dissecting microscope, Oocytes are placed on a nylon mesh (mesh opening 0.5 ram; e.g., Small Parts, Miami, FL, Cat. No. CMP-500) in the bottom of a culture dish and arranged in a row (15-20 oocytes at a time). The tip of the pipette is inserted about 200 ixm into the oocyte, and 50 nl of solution is injected into the cell. Always inject at the same spot, namely, at the center of the animal pole or the border between the poles.

This method can be used to inject cloned messenger RNA (depending on the clone, 10 to 200 pg RNA/celI) or total tissue RNA (50 to 200 ng/ cell). To perform hybrid arrest experiments, that is, to eliminate a certain species of RNA using a specific antisense probe, incubate the RNA with the antisense probe for 10 rain in a buffer containing 200 mM NaCI and 5 mM Tris-HC1 (pH 7.4, 37 °).33 Also, to remove a native protein, antisense nucleotides (2-jxg/ml) can be directly injected into the cells. 45-47 Finally, proteins, for example, By or activated a subunits of G proteins, can be injected in this manner. ~,48

For expression of receptor proteins, wait 48 to 72 hr after the injection. For studying an effect of antisense injection wait 4 to 48 hr, depending on the protein targeted.16,45,46 For protein studies wait 30 to 60 rain after the injection. 5,49

Injection of Small Quantities. To study the effect of acute injections of substances such as IP 3 or Ca 2+, oocytes are impaled with two pipettes for voltage clamping (see below) and a third pipette to inject the substance.

45 S. D. Kroll, G. Omri, E. M. Landau, and R. Iyengar, Proc. Natl. Acad. Sci. U.S.A. 88, 5182 (1991).

46 A, Davidson, G. Mengod, N. Matus-Leibovitch, and Y. Oron, FEBS Lett. 284, 252 (1991 ). 47 p. Dash, I. Lotan, M. Knapp, E. R. Kandell, and P. Goelet, Proc. Natl. Acad. Sci. U.S.A.

84, 7896 (1987). 48 T, M. Moriarty, B. Gillo, D. J. Carry, R. T. Premont, E. M. Landau, and R. Iyengar,

Proc. Natl. Acad. Sci. U.S.A. 8$, 8865 (1988). 49 E. Padrell, D. J. Carty, T. M. Moriarty, J. D. Hildebrandt, E, M. Landau, and R. Iyengar,

J. Biol. Chem. 266, 9771 (1991).


The tip of the injection pipette is broken to a diameter of 5/~m under microscopic control. It is then connected to a pressure system which delivers pulses of compressed nitrogen for defined time periods (Pico- spritzer II, General Valve Corporation, Fairfield, NJ). The amount of pressure is adjusted by injecting a drop of solution from the pipette into oil and measuring the diameter of the drop. We employ a pressure of 40 psi and vary the length of the pressure pulse between 20 and 100 msec. The output of the pipette should be measured again when the pipette is withdrawn from the oocyte to ensure that no plugging has occurred. In this type of experiment, small amounts of solution are injected and the resulting currents recorded via the voltage clamp amplifier. It is advisable not to inject more than 2 nl at a time, to avoid stretch artifacts. When larger volumes are required, it is advisable to inject a series of smaller drops (e.g., 0.5 nl repeated 10 times in rapid succession). The total volume injected will usually be less than 1% of the volume of the oocyte. The injection is done very superficially, so that the injected drop can be observed microscopically as it appears just below the surface of the membrane.

Recording Electric Currents from Xenopus Oocytes

Tissue Bath. Oocytes are put in a small tissue bath made with suitable input and output posts and a side well for the ground wire. The posts hold tubes which carry the incoming medium and remove excess medium through suction. The tubes are inserted into the bath from above, and care should be taken to ensure a smooth flow through it. The floor of the bath is made from the cut-off end of a plastic bulb (e.g., Elkay Co., Shrewsbury, MA, Cat. No. 127@511-000)with paraffin in the bottom. A small depression is made in the paraffin to hold the oocyte. The bath floor should be changed frequently to avoid the accumulation of debris which interfere with oocyte survival. The oocyte is completely submerged in the bathing solution which can be exchanged by flowing medium in through the input tube and drawing excess medium off through the output tube. Solutions can be switched by using a six-way valve. A ground AG[AgCI wire is put into a separate well, and an agar bridge is used to connect it to the main bath.

Composition of Bathing Medium. A number of different frog Ringer's solutions are used for recording. The most commonly used solution is ND96 s° (see Table I) which gives somewhat better stability than classic Ringer's solution. 5° When the need arises to keep oocytes for longer

5o R. Boton, N. Dascal, B. Gillo, and Y. Lass, J. Physiol. (London) 408, 511 (1989).


periods of time, some authors use OR 5~ or Barth's solution 52 with even lower osmolarity. However, we find that ND96, fortified with sodium pyruvate (2.5 raM), penicillin (100-1000 units/ml), and streptomycin (0.1 mg/ml), serves well for keeping oocytes alive for up to l0 days. The buffers used for recording are Na-HEPES or Tris-HC1, although NaHCO3 buffer also has been used.l° The solutions are maintained in the range of pH 7.2 to 7.5. The various solutions are shown in Table I.

Micropipettesfor Recording. Micropipettes can be pulled on any puller including the Brown and Flaming P-80 or P-87 horizontal puller (Sutter, San Francisco, CA) and the Narishige (Narishige, U.S.A., Greenvale, NY) or Kopf (David Kopf, Tujunga, CA) vertical pullers. We use thin wall glass capillaries (1.5 mm o.d., without a filament; World Precision Instruments, Sarasota, FL). When backfilled with 3 M KCI the resistance of the recording pipettes should be low (0.5-2 Mft). We find that it is easier to obtain this type of pipette with the vertical pullers. The pipettes often plug because of yolk seeping into them. Plugging of the current pipette leads to a loss of the voltage control or to oscillations.

Recording Setup. The oocyte is placed in the tissue bath. The recording pipettes are held by suitable micro-manipulators (e.g., Narishige) and are connected to the recording amplifier via AglAgCl wires. For voltage clamping one pipette serves to measure the oocyte membrane potential, whereas the other is utilized to pass the clamping current. The amplifier most frequently used for oocyte experiments is the Dagan-8500 (Dagan Corp., Minneapolis, MN) which can pass very large currents (>10/xA). Alternatively use the GeneClamp amplifier by Axon Instruments (Burlin- game, CA).

Data Storage. Data are stored on a hard disk (e.g., IBM PC/AT) using the TL-1 interface and pClamp software from Axon Instruments. The computer is also employed to set the holding voltage and to apply voltage steps if required. Data are also recorded on a paper chart using a Gould (Saddle Brook, N J) or Yokogawa (Tokyo, Japan) recorder.

Alternative Methods for Monitoring Phosphoinositide Second Messenger System

Two additional methods have been used to monitor changes caused in oocytes by the activation of PLC. One of these is the measurement of 45Ca2÷ efflux from preloaded oocytes) ° Oocytes are preloaded by incuba-

sl R. A. Wallace, D. W. Jared, J. N. Dumont, and M. W. Sega, J. Exp. Biol. 184, 321 (1973). ~2 L. G. Barth and L. J. Barth, J, Embryol. Exp. Morphol, 77, 210 (1959).


tion groups of 40 to 50 cells at 20°C for 18-24 hr in OR medium containing 1 mM sodium pyruvate as an energy source and 20 tzCi of the radioactive Ca 2+. For the experiment the oocytes are placed in a 250/A laminar flow chamber and perfused with OR medium at a constant rate of 1 ml/min. Fractions of the perfusate are collected every 20 sec, after removal of the rapidly exchangeable Ca 2+ fraction. An experiment may comprise 40 to 50 data points.

A second alternative method is to monitor the changes in intracellular Ca 2+ using an optical method. A detailed exposition of this method is outside the scope of this review, and the reader is referred to appropriate references. 11,40,41,43

Acknowledgments

This work was supported by National Institute of Mental Health Grant PO1 MH45212 and a Veterans Administration Merit Grant to E.M.L.

[12] U s e o f C y t o s o l - D e p l e t e d H L - 6 0 Cel ls for R e c o n s t i t u t i o n

S t u d i e s o f G - P r o t e i n - R e g u l a t e d P h o s p h o i n o s i t i d e - S p e c i f i c

P h o s p h o l i p a s e C-fl I s o z y m e s

By S H A M S H A D COCKCROFT, G E R A I N T M. H. THOMAS,

E M E R C U N N I N G H A M , a n d A N D R E W B A L L

Introduction

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme that is responsible for generating two intracellular messengers (diacylglycerol and inositol 1,4,5-triphosphate, IP3) on cell surface stimulation of receptors. Phospholipase C is present in most mammalian cells, and multiple forms of PLC enzymes have been purified from both particulate and soluble fractions of a variety of mammalian tissues. ~'2 Three families of PLC (fl, 3/, and ~) have been identified by protein purification and the amino acid sequences deduced by cloning. 1 Other PLC isoforms have also been identified at the protein level, but their sequences are not yet available] For the study of these enzymes, it is essential to set up assays where it is possible to study their regulation by cell surface receptors and their modulation by other cellular factors.

I S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992). 2 S. Cockcroft and G. M. H. Thomas, Biochem. J. 288, 1 (1992).

Copyright © 1994 by Academic Press. Inc. METHODS IN ENZYMOLOGY. VOL. 238 All rights of reproduction in any form reserved.

[12] RECONSTITUTING PLC IN CYTOSOL-DEPLETED CELLS 155

Phospholipase C enzymes can be activated by G proteins or by tyrosine phosphorylation.~,2 Moreover, PLC regulation can occur with G proteins which are pertussis toxin-sensitive (e.g., G i o r G O family) or pertussis toxin-insensitive (e.g., Gq family). 1,2 We have established a reconstitution system to assay for G-protein-coupled PLCs. 3 The most significant advantage of this reconstitution system is that the substrate for the phospholipase is presented in its native environment, namely, the membrane. This method of reconstitution complements the use of exogenous substrates, where the system can be controlled more precisely but which presents the PLC to its substrate in a nonphysiological environment. 4'5 Although a reconstitution system using exogenous substrate provides a powerful tool for studying the regulation of PLC by both receptors as well as G proteins, it is not designed to identify contributions made by other unknown factors.

The development of the reconstitution system that we have established was based on the observation that regulation of PLC by G proteins or the agonist-receptor interaction was better preserved in permeabilized cells compared to membrane preparations 6,7 In addition, if loss of cytosol was allowed to occur prior to phospholipase C activation by the agonist or the G protein, this led to loss of responsiveness) The results suggest that cytosolic components were essential for sustained signaling.

Experimental Procedures

To study the role of the cytosolic components in signaling, the following procedure for a reconstitution assay was developed (Fig. 1). Basically, cells are permeabilized to release their cytosolic contents and then washed. These cells become refractory to GTPTS stimulation of PLC. The cytosol- depleted cells are then incubated with cytosol (or cytosol-derived proteins) to restore the GTPyS-mediated activation of the PLC.

Streptolysin O, used as the permeabilizing agent for making the cytosol-depleted cells, generates sufficiently large lesions in the plasma membranes of cells to allow the efflux of cytosolic proteins including the endogenous phospholipases C. However, the G proteins are membrane- associated and therefore remain within the cells. The membrane also

3 G. M. H. Thomas, B. Geny, and S. Cockcroft, EMBO J. 10, 2507 (1991). 4 G. Berstein, J. L. Blank, A. V. Smrcka, T. Higashijima, P. C. Sternweis, J. H. Exton,

and E. M. Ross, J. Biol. Chem. 267, 8081 (1992). 5 G. Berstein, J. L. Blank, D.-Y. Jhon, J. H. Exton, S. G. Rhee, and E. M. Ross, Cell

(Cambridge, Mass.) 70, 411 (1992). 6 S. Cockcroft and J. Stutchfield, Biochem. J. 256, 343 (1988). 7 j. Stutchfield and S. Cockcroft, Eur. J. Biochem. 197, 119 (1991).


Step I

Step 2

• o O f ~ 0 • ~ O •

)°

1

• t." £ / ' .

1

Prelabeled cellr,

Petmeabllize wllh strept~ysJn 0 for 30mln to release Cytor~41C prot~r~

Cytosol-dep/eled oells washed to remove reli~;ed proteins from ~e ex~acellulat medium

Incubate wflh reconstituting factor e , e.g, cytosd, column fracJJom;, pullfied ~osphol iw, e

Assay for released inos~tol phosphates

FIG. 1. Scheme representing the steps required for a reconstitution assay. In Step 1, intact HL-60 cells prelabeled with [3H]inositol are washed, and the permeabilizing agent streptolysin O is added for 30-45 rain. In Step 2, the permeabilized cells are washed to remove the released proteins. In Step 3, the cytosol-depleted cells are then incubated with the reconstituting factors (e.g., cytosol, column fractions, purified PLC-fll) in the presence and absence of a G protein activator such as GTPTS or the receptor-directed agonist in the presence of GTP. After 20 rain the reaction is terminated, and after centrifugation at 2000 g at 4 ° the supernatant is assayed for released inositol phosphates.


contains the substrate for the PLC. The pool of phosphatidylinositol 4,5- bisphosphate (PIP2) is normally replenished from the much larger pool of phosphatidylinositol (PI) by sequential phosphorylation by phosphatidylinositol 4-kinase and phosphatidylinositol-4-phosphate 5-kinase. These enzymes also remain membrane-associated. We have used HL-60 (human (pro-myelocytic) cells (either undifferentiated or differentiated to neutrophils) for most of our studies, but the protocol is generally applicable for any cells in suspension. Figure 1 graphically illustrates the permeabilization protocol followed by reconstitution with cytosol-derived components.

Time Course of Efflux of Cytosolic Proteins

It is first necessary to establish the time course of the efflux of cytosolic proteins from the cells. For this HL-60 cells (108 cells) are washed and resuspended in 4.5 ml of buffer A (pH 6.8) comprising Na-PIPES (20 raM), NaCI (137 mM), KCI (2.7 raM), Glucose (5.6 raM, 1 mg/ml), and Bovine serum albumin (BSA, 1 mg/ml). Permeabilization is initiated by addition of a 10 times concentrated cocktail (500/zl) made up in buffer A to give final concentations of 100 nM Ca 2+, 2 mM MgATP, and 0.6 IU/ml streptolysin O:

Preparation of Ca2+/EGTA buffers is carried out essentially as previously described. 8 The final EGTA concentration is maintained at 3 mM.

MgATP is made up as a stock solution of 100 mM and can be kept at - 2 0 ° for months. ATP is purchased as a disodium dihydrogen salt. To prepare I0 ml of a 100 mM stock solution of MgATP, dissolve 605 mg of ATP in 10 ml of a solution containing 2 ml of 1 M Tris and 1 ml of 1 M MgCI z . The use of 200 mM Tris effectively results in a neutral solution (pH 7). This should be checked with a pH electrode and adjusted accordingly. Stock solutions of MgATP are very Stable over a period of I-2 years providing that the solution is neutral.

Streptolysin O is purchased from Wellcome Diagnostics (Kent, UK) and is supplied in powder form. The powder is suspended in 2 ml of distilled water to give a stock solution of 20 International Units (IU)/ml. This solution can be kept at 4 ° for 1-2 weeks. It does get cloudy with time and can be partially clarified on warming at 37 ° . However, the cloudiness does not affect the permeabilization.

8 B. D. Gomperts and P. E. R. Tatham, this series, Vol. 219, p. 178.


100

80

60

40

20

/0--0 LDH @--------Q PLC

0 I I I I f , I i I

0 10 20 30 40 50 60

Time (Min)

F=o. 2. Comparison of the time course of leakage of macromolecules and loss of OTPTS- stimulated phospholipase C activation after treatment with streptolysin O. Intact cells were incubated with streptolysin O, and at the indicated times the reaction was quenched, the cells sedimented, and the supernatant assayed for lactate dehydrogenase (LDH) activity and phospholipase C (PLC) activity with the appropriate in vitro assay. Also shown is the loss of GTPTS-stimulated PLC activation. In this case, labeled cells were prepermeabilized for the indicated times and subsequently stimulated with GTPTS for 10 min. Data are expressed as the percentage of maximal response [i.e., GTP~/S-stimulated PLC activity or total enzyme in lysed cells (LDH and PLC)].

Streptolysin O is a bacterial (streptococcal) cytolysin which generates persistent membrane lesions sufficiently large to permit the flux (in choles- terol-containing liposomes 9) of proteins of molecular weight up to 400,000. The cocktail containing streptolysin O is added to the cells to initiate permeabilization. The concentration of streptolysin O required for this step has to be arrived at empirically. We have found that a concentration of 0.6 IU/ml is sufficient when using HL-60 cells and neutrophils to deplete them completely of cytosolic contents. Aliquots of cells (100/~I) are removed at timed intervals and the cells diluted into ice-cold 0.9% saline and centrifuged at 1700 g for 5 min at 4 °. The supernatant (50/zl) is assayed for the release of lactate dehydrogenase (LDH, 140 kDa). Enzyme activity is monitored by observing the consumption of NADH following addition of pyruvate as substrate, which is converted to lactate. Within 5 min of streptolysin O addition, the majority of the LDH is found in the supernatant. Release of PLC is also monitored, and it takes 40-60 min for maximal release to occur (see Fig. 2).

9 L. Buckingham and J. L. Duncan, Biochim. Biophys. Acta 729, 115 (1983).


In Vitro Assay for Phospholipase C

The amount of PLC-fl present in the cytosol-depleted cell preparation greatly influences the extent of any subsequent reconstitution. It is important, therefore, to characterize any new system of this kind with respect to the loss of these enzymes. The assay for measuring PLCs vary from laboratory to laboratory, and different assays appear preferentially better for detecting any one isoform over another. We employ two methods in our laboratory, one that uses phosphatidylinositol as substrate and another which uses phosphatidylinositol bisphosphate. Although many of the known PLC isozymes are able to utilize all three inositol-containing lipids, namely, phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2), there are ill-defined PLCs that seem only to utilize the polyphosphoinositides as substrates. In our experience the PLC-yl enzyme appears to prefer phosphatidylinositol whereas PLC-fll appears to prefer PIP2. 3

Assay for Determining Phospholipase C Activity against Phospha tidyl- inositol in Vitro. The concentration of PI used in the assay is 1 mM (i.e., 50 nmol per assay).

Reagents PI (Sigma, St. Louis, MO), which comes as a chloroform solution

(10 mg/ml) [3H]PI (Amersham Radiochemical Centre, Amersham, UK) Stock solution of 0.2 Tris-maleate (2.42 g Tris plus 2.32 g maleic acid

in 100 ml)

Twenty-five milliliters of 0.2 M Tris-maleate is diluted to 100 ml to obtain a 50 mM Tris-maleate buffer, the pH is adjusted to 5.5 using NaOH. Add 2 mM CaC12. This is the buffer required for suspending the substrate.

Preparation of substrate 1. Add into a glass tube 250 t~l of PI (10 mg/ml stock solution) and

20 ~1 of 3H-labeled PI (50 t~Ci/ml), that is, 2.2 × 10 6 disintegrations per minute (dpm).

2. Dry the sample under a stream of nitrogen. 3. Immediately add 2.5 ml of 50 mM Tris-maleate buffer, pH 5.5,

containing 2 mM CaCI2. 4. Sonicate and count the aqueous phase to check solubilization. (Four

bursts of 20 sec each at the lowest setting is generally sufficient.) 5. The substrate is ready for use and can be prepared in large quanti-

ties, divided into aliquots in Eppendorf tubes, and kept frozen at - 2 0 ° until required.


Enzyme assay 1. Incubate 25/zl of enzyme solution or column fraction after chroma-

tography with 25/zl of substrate for 10-30 rain at 37 °. 2. Quench with 250/zl of chloroform/methanol (I : 1, v/v). 3. Add 75/zl of 1 M HCI. Mix vigorously to form an emulsion. 4. Centrifuge to separate the aqueous and organic phases. We rou-

tinely centrifuge for 5 min at 4 ° at 2000 g. 5. A 100-/A portion of the upper aqueous phase (total volume -250

/zl) is removed for liquid scintillation counting.

Comments. The PI hydrolyzing activity of many PLC isozymes is maximal at millimolar concentrations of Ca 2+.

Assay for Determining Phospholipase C Activity against Phosphatidyl- inositol 4,5-Bisphosphate in Vitro

Preparation of substrate I. Stock solutions of the following are prepared and can be kept at

- 20°: sodium cholate 20% (w/v); calcium chloride 10 raM; 2-mercaptoethanol 250 mM; PIPES, pH 6.8, 1 M; NaC1 2.5 M; PIP2 (purchased from Sigma as 5 mg of solid) dissolved in 1.25 ml of chloroform/methanol (I : 1, v/v); [3H]PIP 2 (Amersham, 10/xCi/ml).

2. For preparation of the reaction mixture stock (14 ml), mix the following: 4.2 ml stock sodium cholate, 1.4 ml stock CaCIE, 1.4 ml stock 2-mercaptoethanol, 1.4 ml stock PIPES, 2.8 ml stock NaCI, and 2.8 ml distilled water.

3. The solution is then diluted 1 : 1 with distilled water to give a final volume of 28 ml. This reaction mixture can be kept at - 20 ° for several months.

4. For preparation of substrate/reaction mixture cocktail, in a glass tube add 1.25 ml of PIP z solution and 1.25 ml of [3H]PIP2 solution.

5. Dry the lipid mixture under a stream of nitrogen and add 28 ml of reaction mixture. Sonicate extensively on ice several times, allowing the solution to clear between sonications.

6. Dispense 1-ml aliquots into Eppendorf tubes and freeze at - 2 0 ° until required. Assay reactions are initiated by the addition of 20/zl of the substrate/reaction mixture cocktail, and the final concentrations of the individual components in the assay are as follows: 0.6% (w/v) sodium cholate, 200 ~M CaClz, 5 mM 2-mercaptoethanol, 20 mM PIPES, 120 mM NaC1, 60/zM PIP2, and 10 6 dpm/ml [3H]PIPz.

Enzyme assay 1. Incubate up to 30/zl of a column fraction with 20 ~1 of substrate

in an Eppendorf tube for up to 10 min at 37 °. Sample volumes less than 30 txl must be made up to this volume with buffer.

[12] RECONSTITUTING P L C IN CYTOSOL-DEPLETED CELLS 161

2. Quench the reaction with 250/zl of ice-cold chloroform/methanol/ concentrated HC1 (50 : 50 : 1, v/v).

3. Add 75/zl of 1 M HC1. Mix vigorously to form an emulsion. 4. Centrifuge for 5 min at 4 ° at 2000 g to separate the aqueous and

organic phases. 5. A 100-/xl portion of the upper aqueous phase out of a total of approxi-

mately 250/zl is removed for liquid scintillation counting.

Monitoring Phospholipase C Release by Immunoblotting

Because it is enzymes of the PLC-/3 family that have so far been identified as G protein-regulated, the specific release of this family can also be monitored. A typical experiment is presented in Fig. 3. Release of PLC-/32 into the supernatant from HL-60 cells is observed over a period of 45 rain. In addition, the amount of PLC-/32 associated with the membranes is also monitored for comparison.

To assay for released phospholipases in the supernatant by immunoblotting, HL-60 cells are permeabilized for varying lengths of time and

. . . . . . . . . . . . . . . :

PLC-132

0 5 10 15 20 25 30 45 m

Time (min) FIG. 3. Time course of phospholipase C-/32 release on permeabilization with streptolysin

O. HL-60 cells were permeabilized with streptolysin O, and samples were removed at the indicated times after permeabilization and centrifuged. The supernatant was analyzed for the presence of PLC-/32 after separation of the proteins by SDS-PAGE and detected by Western blotting with antibodies against PLC-/32. The lane labeled m shows HL-60 membranes obtained after freeze-thawing packed HL-60 cells and preparing a crude membrane fraction from the particulate material. The supernatant was derived from the equivalent of 2.4 x 10 6 cells, whereas the membrane fraction represented an equivalent of 4 x 10 6 cells. Two proteins at approximately 150 and 100 kDa were detected with the antibody against PLC-/32. The 100-kDa band may represent a proteolytic fragment of the 150-kDa protein.


the medium tested for the presence of the phospholipases by Western blotting. Approximately 3 x 108 HL-60 cells are washed in PIPES buffer (buffer A), then treated with diisopropyl fluorophosphate for 10 min prior to permeabilization in a total volume of 4.5 ml. After the addition of the cocktail containing streptolysin O (0.6 IU/ml final), Ca 2÷ (100 nM final), and MgATP (2 mM final) to the cells, a 250-/,1 sample is removed and the cells pelleted by centrifugation in a bench- top microcentrifuge. Two hundred microliters of the clear supernatant is then removed from the tube and immediately treated with an equal volume of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Further samples are removed and similarly treated at time points of 5, 10, 15, 20, 25, 30, and 45 min. The samples of supernatant are then run on a polyacrylamide gel, blotted, and probed as described below.

In principle, the amount of phospholipase C retained in the cytosol- depleted cells should also be analyzed. However, we have found that the nuclear contents of the cytosol-depleted cells interfere during the separation of the proteins on SDS-PAGE. To circumvent this problem, we use membranes to establish whether any PLC is retained. HL-60 cell membranes are prepared from 108 cells. The cells are washed three times with phosphate-buffered saline (PBS), and the pelleted cells are freeze-thawed three times. The volume of the cells is made up to 1 ml with PBS and the mixture centrifuged at 2000 g to sediment any nuclei/ undisrupted cells. The supernatant is spun at 17,000 g in a microcentrifuge for 15 min at 4 ° to pellet the membranes. The membranes are resuspended in PBS, and washed two more times, and solubilized with SDS-PAGE sample buffer.

Samples (80 tzl) are loaded onto 8% cross-linked polyacrylamide mini- gels (1.5 mm thickness). The material is then run at 150 V for 1 hr. At the end of this time the gel is blotted onto PVDF membranes (polyvinyl- difluoride; immobilon-P, Millipore, Bedford, MA), at 30 V overnight. The membrane is then stained with Ponceau S to determine adequate transfer and to mark the position of the accompanying molecular weight markers. Subsequently, the membrane is blocked with 5% dried low- fat milk in PBS-Tween 20 (0.1%) for 1 hr, washed to remove excess milk protein, incubated with the appropriate antibodies (against phospholipase C-/31, -/32, -/33), rinsed at the end of 1 hr, and again blocked as before. After the final blocking step the blots are washed with PBS-Tween 20 and then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies at a dilution of 1/4000 for 1 hr. At the end of the incubation the blot is washed repeatedly for a total period of 1 hr

[ 1 2 ] RECONSTITUTING P L C IN CYTOSOL-DEPLETED CELLS 163

and visualized using the Amersham ECL (enhanced chemiluminescence) system.

Monitoring Loss of Responsiveness to GTPyS after Permeabilization with Streptolysin 0

To establish the time interval for the loss of the PLC response to GTPyS [or the receptor-directed agonist formylmethionylleucylphenyl- alanine (fMetLeuPhe)], the cells have to be incubated with [3H]inositol to label the endogenous pool of inositol phospholipids. This is accomplished by growing the cells in the presence of [3H]inositol for 2-3 days to label the cellular lipids to equilibrium. We normally grow HL-60 cells in RPM! 1640 medium with heat-inactivated 12.5% (v/v) fetal calf serum (FCS), 4 mM glutamine, 500 IU/ml penicillin, and 500 ~g/ml streptomycin. Because RPMI 1640 medium contains a high level of inositol, the cells are labeled in Medium 199 supplemented with glutamine, penicillin, and streptomycin and labeled with 1 ~Ci/ml [3H]inositol for 48 hr. Fetal calf serum is also excluded as this contains high levels of inositol, and instead the Medium 199 is supplemented with insulin (5 t~g/ml) and transferrin (5 t~g/ml) as growth factors. If cells are to be differentiated the labeling medium also contains 300 ~M dibutyryl-cAMP for the 48-hr period. Dibu- tyryl-cAMP is dissolved in dimethyl sulfoxide (DMSO) as a stock solution of 60 mM which can be stored at - 20 ° indefinitely. HL-60 cells grow to a density of 1-2 × 10 6 cells/ml, and generally we use between 50 and 70 ml of cells per experiment. This allows for about 50-70 incubations containing approximately 105 dpm in the inositol lipids per incubation.

A cocktail containing streptolysin O (0.6 IU/ml final), MgATP (2 mM final), and Ca 2+ (100 nM buffered with 100 ~M EGTA final) is added to the labeled cells, and at timed intervals an aliquot of cells (100 t~l) is transferred to tubes containing Ca z+ (1 ~M buffered with 3 mM EGTA), LiC1 (10 raM), MgATP (2 mM), MgCI2 (2 mM), and an activator either GTPyS (10/~M final) or fMetLeuPhe (1 p~M final), plus GTP (10 ~M final). The cells are incubated at 37 ° for 10 min to monitor the extent of G-protein-stimulated PLC activity. At the end of the incubation, the reactions are quenched with 1 ml of ice-cold 0.9% NaC1. The cells are sedimented at 2000 g for 5 min at 4 °. Then 0.9 ml of the supernatant is removed and used for the analysis of released inositol phosphates as described below. Figure 2 shows the results of a typical experiment. As the time of incubation with streptolysin O is extended (and therefore more cytosolic components have leaked out of the cells), the subsequent addition of GTPyS to stimulate PLC is impaired. [An important distinction needs to


be made regarding the permeability of macromolecules and molecules smaller than 1 kDa. Molecules such as GTPyS equilibriate into streptolysin O-permeabilized within 20 sec. In contrast, lactate dehydrogenase leaks out over a period of 5 min (see Fig. 2). This difference is attributed to the size of the molecule.]

Reconstitution of G-Protein-Stimulated Phospholipase C by Cytosolic Factors in Cytosol-Depleted Cells

The time of lactate dehydrogenase release and the loss of stimulation by GTPyS or fMetLeuPhe coincide very well, and generally speaking we use 30-45 min of prepermeabilization to ensure that the endogenous response to GTPyS has declined sufficiently (Fig. 2). The endogenous response never declines to zero (see Fig. 2), however, presumably because the cytosol-depleted cells still contain some PLC that cannot be removed by this procedure. Indeed, membranes prepared from HL-60 cells are found to retain some phospholipase C-fl2 (Fig. 3).

Assay for Reconstituting Activity

1. Approximately 50-70 ml of [3H]inositol-labeled HL-60 cells ( - 5 x 10 7 cells) are centrifuged at 450 g for 5 min at room temperature and the medium removed. The cells are resuspended in 40 ml of buffer consisting of 20 mM PIPES, 137 mM NaC1, 3 mM KC1, 1 mg/ml glucose, 1 mg/ml albumin, pH 6.8, and recentrifuged. This is repeated once more. The washed cells are resuspended in 4.5 ml of the same buffer.

2. Permeabilization is initiated by addition of 0.5 ml buffer supplemented with streptolysin O, MgATP, and Ca 2+ buffered to 100 nM such that the final concentration of the individual components are 0.6 IU/ml streptolysin O, 2 mM MgATP, and 100 nM Ca z+ when added to the cells. (The Ca 2+ is buffered with 100/~M EGTA at this stage.) The cell suspension (5 ml) is incubated for 30-45 min at 37 ° with occasional mixing.

3. The permeabilized cells are then diluted with 40 ml of cold buffer and sedimented at 2000 g at 4 ° for 5 min. Because the cells are depleted of cytosolic contents, the centrifugation speed is increased so that all the cell "ghosts" are sedimented. (Nonetheless some loss of cells does occur at this stage.) The cytosol-depleted cells are resuspended in the same buffer, generally in a volume of 1-2 ml, the buffer being supplemented with a cocktail of 4 mM MgATP, 4 mM 2,3-diphosphoglycerate (inhibitor of inositol phosphate phosphatases), 20 mM LiC1, 4 mM MgCI2, pH 6.8. Free calcium concentration is maintained with CaZ+/EGTA buffers with 6 mM EGTA. We generally work with a free calcium concentration of 1 ~M for the experiment. Activation of PLC by GTPyS is sensitive to the

[ 1 2 ] RECONSTITUTING P L C IN CYTOSOL-DEPLETED CELLS 165

Ca 2+ concentration, and this can be varied between 0.1 and 10 I~M. We compromise at 1 ~M generally.

4. Twenty microliters of the cytosol-depleted cells is transferred to Eppendofftubes containing 20 t~l of fractions to be tested for reconstituting ability and 5 tzl of GTPyS, bringing the total incubation volume to 45/~l. The final concentration of GTP~,S is 10 I~M. This is all done on ice, and the last addition to the tubes is the cytosol-depleted cells. In principle, the cytosol or fractions derived after chromatography should be dialyzed into 20 mM PIPES, 137 mM NaC1, 3 mM KC1, pH 6.8, prior to use in the reconstitution assay. However, we have found that the system is quite tolerant of changes in salt concentration, and we generally use column fractions directly. However, changes in pH influence Ca 2+ buffering by EGTA and should therefore be taken into account. Note that the assay volumes chosen for the reconstitution are dictated by the availability of material. The reactions are initiated by transferring the tubes from ice to a 37 ° water bath. Incubations proceed for 20 rain, after which the tubes are transferred to ice and the reactions quenched with 1 ml of ice-cold 0.9% NaC1. The cells are sedimented at 2000 g (3000 rpm) for 5 rain at 4 °. Then 0.9 ml of the supernatant is removed and used for the assay of released inositol phosphates.

5. Inositol phosphates are separated from free inositol and glycerophosphoinositol by passage through Dowex l-X8 anion-exchange resin (formate form). We purchase the resin as the chloride form. The following procedure is used to convert it to the formate form.

a. Add the Dowex resin (100 g) into a beaker. b. Add 1 M NaOH (400 ml) and stir with a glass rod. c. Allow the resin to settle (1-2 hr). d. Carefully decant the NaOH solution. e. Add 400 ml of 1 M formic acid and stir with a glass rod. f. Allow the resin to settle and decant the formic acid. g. Wash the resin 5 times with 400 ml of distilled water. h. Leave the resin as a 50% slurry in distilled water at 4 ° and use

as required.

We generally make up the Dowex resin in Pasteur pipettes (0.5 ml bed volume) equipped with a plug of glass wool. The columns can be recycled indefinitely providing that the columns are washed with 2 M ammonium formate/0.1 M formic acid followed by extensive washing with water (15-20 ml) afer use. The sample is loaded on the column, [3H]inositol is washed off with 6 ml water, and glycerophosphoinositol is removed with 6 ml of 5 mM sodium tetraborate/5 mM sodium formate. The total inositol


phosphates are eluted together with 3 ml of 1 M ammonium formate/0.1 M formic acid directly into scintillation vials. The radioactivity is measured after addition of a scintillation cocktail that is able to accommodate 1 M salt. We usually use PCS (phase combining system) from Amersham.

Results from a typical experiment are shown in Fig. 4. When cytosol- depleted cells are stimulated with GTPyS, a small activation of PLC is observed. Addition of PLC-/31 (purified from bovine brain membranes) increases the basal activity, which is stimulated in the presence of GTPyS. In contrast, PI-TP (phosphatidylinositol transfer protein) has little effect in the absence of GTPyS, but in its presence, PI-TP reconstitutes the inositol-lipid signaling pathway.

Calculation of Data

All determinations are carried out in duplicate. The increase in inositol phosphates can be expressed as a function of the total radioactivity (dpm) incorporated in the inositol lipids. This allows the results to be calculated as a percentage hydrolysis of the total inositol lipids and allows compari-

3500 , , , ,3.5

3000

o

2 5 0 0

o 2 0 0 0

o

= 1 5 0 0

E

1 0 0 0

i

[ - - I c o n t r o l

czP~-s 3.0

5' o

2.5 ~

" o

2 . 0 F,'

1.5 S N

g_ 1.0

500 ~ 0 .5 0 1 2 3

cont ro l PLC- 131 PI-TP

FIG. 4. Reconstitution with PI-TP and PLC-/31 in cytosol-depleted HL-60 cells. HL-60 cells were depleted of cytosolic contents by incubation with streptolysin O for 40 min. After the cytosol-depleted cells were washed, 20 /xl of cells was incubated with 20 /xl phosphatidylinositol transfer protein (PI-TP) or PLC-/31, both purified from bovine brain. Five microliters of GTPyS or buffer was added as indicated. After 20 min at 37 °, the incubations were quenched and the released inositol phosphates were analyzed.

[12] RECONSTITUTING P L C IN CYTOSOL-DEPLETED CELLS 167

sons to be made from different experiments, as the level of radioactivity in the inositol lipids can vary from experiment to experiment. The total lipids from a pair of test aliquots are extracted by adding 1.5 ml of a mixture of 1 : 2 (v/v) chloroform/methanol acidified with concentrated HCI (1 ml per 100 ml of mixture) to the cell suspensions after they have been diluted to a volume of 400/xl with water. This gives a single-phase solution. After vigorous mixing, 0.5 ml of chloroform and 0.5 ml of 1 M HC1 are added to obtain a two-phase system. After vigorous mixing, the samples are centrifuged for 5 min at 1000 g. The lipids are present in the lower chloroform phase (approximate volume 1 ml), and the top aqueous phase contains the water-soluble components including any inositol phosphates, free inositol, etc. The lipid phase is carefully removed with a syringe via side puncture of the tube. The lipids are suspended in 500 ~1 methanol, and after addition of scintillation fluid the radioactivity is quantified by liquid scintillation counting.

R e m a r k s

Although we have used streptolysin O as the reagent for permeabilization, in principle, other permeabilizing agents (e.g., digitonin) can be used provided the lesions are large enough for proteins to leak out of cells. Using the techniques outlined above, we have successfully identified two cytosolic proteins that reconstitute G-protein-mediated inositol lipid signaling. One protein was identified as PLC-fll and the other as the lipid transfer protein, PI-TP. 3'1° The use of cytosol-depleted cells is not restricted to reconstitution of G-protein-regulated PLC but can be extended to the study of other phospholipases. We have used this method successfully to reconstitute phospholipase D activity and have identified a low molecular weight GTP-binding protein, ARF, required for regulated phospholipase D activity. 11'12 In this case the cells are prepared such that phosphatidylcholine is labeled, and the same protocol is applied except that the product of the phospholipase D pathway is monitored, namely, phosphatidate (or phosphatidylethanol if ethanol is also added during the assay) if the cells are labeled with [3H]lyso-platelet-activating factor (PAF). ~3 Alternatively, the choline-containing lipids can be labeled to equilibrium if the cells are grown in [methyl-3H]choline for 48 hr. 11 Choline

~0 G. M. H. Thomas, E. Cunningham, A. Fensome, A. Ball, N. F. Totty, O. Troung, J. J. Hsuan, and S. Cockcroft, Cell (Cambridge, Mass.) 74, 919 (1993).

II B. Geny, A. Fensome, and S. Cockcroft, Eur. J. Biochem. 215, 389 (1993). ~2 S. Cockcroft, G. M. H. Thomas, A. Fensome, B. Geny, E. Cunningham, I. Gout, I.

Hiles, N. F. Totty, O. Truong, and J. J. Hsuan, Science in press (1994). t3 B. Geny and S. Cockcroft, Biochem. J. 284, 531 (1992).


release then provides a sensitive assay for phospholipase D activity, although this should be rigorously demonstrated to correlate with lipid turnover in any new system and shown not to be a product of water- soluble choline-metabolite turnover.

Functional responses can also be reconstituted. In the case of the neutrophils and HL-60 cells, the cytosol-depleted cells can be used to study the requirement for cytosolic components in exocytosis and also in the activation of the NADPH oxidase. This technique has been used to identify the G protein (Ge) that regulates exocytosis (A. Fensome, G. M. H. Thomas, and S. Cockcroft, unpublished, 1993).

Acknowledgments

We thank the Leukemia Research Fund, Medical Research Council, and the Wellcome Trust for support. We thank Peter Parker for antibodies to PLC-fll, -/32, and -/33. E.C. is supported by a studentship from Science and Engineering Research Council.

[13] Pur i f ica t ion of Phospha t idy l inos i to l T r a n s f e r P ro t e in f rom Bra in Cytosol for Recons t i t u t i ng G - P r o t e i n - R e g u l a t e d

Phospho inos i t ide -Spec i f i c Phospho l i pa se C-/3 I sozymes

By GERAINT M. H. THOMAS, EMER CUNNINGHAM,

and SHAMSHAD COCKCROFT

Introduction

On binding to cell surface receptors, hormones, neurotransmitters, chemoattractants, and growth factors elicit intracellular responses by activating inositol phospholipid-specific phospholipase C (PLC). Activated phospholipase C catalyzes the hydrolysis of a membrane phospholipid, phosphatidylinositol bisphosphate, to form two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate (IP3). From direct protein isolation and molecular cloning studies, the existence of multiple phospholipase C isoforms has been established. 1 Known members of the PLC family are PLC-fl, PLC-7, and PLC-8, for which the amino acid sequences have been deduced from nucleotide sequences. In addition, there are PLCs which have been identified only by protein purification. 2

1 S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992). 2 S. Cockcroft and G. M. H. Thomas, Biochem. J. 288, 1 (1992).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY. VOL. 238 All rights of reproduction in any form reserved.

[131 PURIFICATION OF PI TRANSFER PROTEIN 169

We have noted that regulation of phospholipase C by G proteins or the agonist-receptor interaction is better preserved in permeabilized cells compared to membrane preparations? '4 In addition, in permeabilized cell preparations, loss of cytosolic proteins coincides with loss of GTP~/S- stimulated phospholipase C activity. 5 These studies strongly indicated that cytosolic components, possibly the cytosolic phospholipases C, are required for signaling.

To identify cytosolic components which are required for G-protein regulation of phospholipases, we have devised a protocol that screens for proteins that can reconstitute GTPyS-stimulated phospholipase C activity in cytosol-depleted cells. 6 The reconstitution system was developed as a result of the observation that permeabilization with streptolysin O leads to the loss of cytosolic proteins, including lactate dehydrogenase and phospholipase C, and this matches with loss of phospholipase C activation by GTPTS. 6 The cytosol-depleted cells still contain receptors and G proteins, and they have been used to show that cytosol prepared from a number of sources can reconstitute G-protein-driven phospholipase C activation. 5

Using the reconstitution assay, we had initially reported that in rat brain cytosol two partially purified phospholipases, PLC-fll andPLC-e, could reconstitute G-protein-driven phospholipase C activation.5 Although reconstitution with a homogeneous preparation of PLC-fll has confirmed the observation, further studies have indicated that the reconstituting factor present in the partially pure preparation of PLC-e is not the phospholipase C but is another protein identified as the phosphatidylinositol transfer protein (PI-TP). 7

The phosphatidylinositol transfer protein was originally identified by its ability to transfer PI from one membrane compartment to another in vitro, and its transfer activity was the original basis for its purification (for a review see Wirtz8). Depending on the ratio PI/PC (phosphatidylcholine) in both donor and acceptor compartments, PI-TP could catalyze a net transfer of either PI or PC. The PI-TP has 16-fold greater affinity for PI compared to PC. Thus, depending on the relative concentrations of the two lipids in a membrane, PI-TP becomes loaded with either PI or PC, its binding site being always occupied by one of these lipids.

3 S. Cockcroft and J. Stutchfield, Biochem. J. 256, 343 (1988). 4 j. Stutchfield and S. Cockcroft , Eur. J. Biochem. 197, l l9 (1991). 5 G. M. H. Thomas, B. Geny, and S. Cockcroft, EMBO J. 10, 2507 (1991). 6 S. Cockcroft, G. M. H. Thomas, E. Cunningham, and A. Ball, this volume [12]. 7 G. M. H. Thomas, E. Cunningham, A. Fensome, A. Ball, N. F. Totty, O. Troung, J. J.

Hsuan, and S. Cockcroft, Cell (Cambridge, Mass.) 74, 919 (1993). 8 K. W. A. Wirtz, Annu. Rev. Biochem. 60, 73 (1991).


This chapter deals with the purification of PI-TP from rat brain cytosol as well as from bovine brain cytosol (outlined in Fig. 1). Purification from rat brain cytosol provides a relatively clean preparation of PI-TP within 2-3 days. This is a convenient source as enough material can be made for several reconstitution experiments very quickly. If larger amounts of protein are required, purification from bovine brain cytosol is recommended; this requires 1 week, and approximately 500/zg of pure protein can be prepared from one brain.

Purification of Phosphatidylinositol Transfer Protein from Rat Brain

Preparation of Rat Brain Cytosol

Rat brains are generally stored frozen at - 8 0 ° until required. Five or six rat brains ( -10 g) are thawed on ice and homogenized in a Potter homogenizer with 20 strokes in 25 ml of ice-cold buffer (20 mM PIPES, 2.7 mM KCI, pH 6.8) containing 5 mM EGTA, 5 mM EDTA, 10 mM benzamidine, 1 mM dithiothreitol (DTT), 1/~g/mi soybean trypsin inhibitor, 5/zg/ml aprotinin, 2/zM pepstatin A, 100/zM 1-chloro-3-tosylamido- 7-amino-2-heptanone hydrochloride (TLCK), 0.1 mM leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF).

The homogenate is centrifuged for I hr at 150,000 gay, 4°, to pellet the membranes. The supernatant (14-20 ml) is passed through a filter (0.45

A Bovine brain cytosol B Rat brain cytosol

1 1 Ammonium sulfate Hepadn sepharose

fractionation (40-60%)

1 1 DE52 weak anion exchange Superdex 75

1 1 Hepadn sepharose Phenyl superose

1 Superdex 75

1 Phenyl superose

FIG. ]. Scheme of purification of PI-TP from rat and bovine brain ¢ytosol.

[13] PURIFICATION OF PI TRANSFER PROTEIN 171

/xm) and loaded onto a heparin-Sepharose column equilibrated in 20 mM PIPES, 3 mM KC1 (pH 6.8). Heparin-Sepharose (Pharmacia, Piscataway, N J) has a binding capacity of approximately 1.5 mg protein per milliliter of swollen gel, and we use a column (1 x 50 cm) which contains 40 ml of heparin-Sepharose. After loading, the column is washed with the equilibration buffer until the bulk of the proteins that have not stuck to the column are washed off. The adsorbed proteins are eluted with a 500-ml linear gradient of NaCI (0-600 mM) in the same buffer at a flow rate of 0.2 ml/min. (The flow rate can be increased to 1 ml/min if desired.) Fractions (6 ml) are collected, and an aliquot of each column fraction is assayed for phosphatidylinositol transfer activity.

Assay for Phosphatidylinositol Transfer Protein Activity in Vitro

In the PI-TP assay, transfer of [3H]PI from donor microsomes to acceptor liposomes [98 : 2 (w/w) PC/PI] is monitored. 9 The ratio ofliposomes to microsomal protein is 1 /zmol lipid/1.25 mg protein.

Preparation of Microsomes

1. Three rat livers are homogenized in a Waring blender in 150 ml of SET buffer (0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HC1, pH 7.4) at 4 ° .

2. The homogenate is centrifuged at 2000 g at 4 ° for 5 min to sediment unbroken cells.

3. The supernatant is retained and centrifuged at 10,000 g, 4 °, for 15 min to pellet the mitochondria.

4. The supernatant is further centrifuged at 100,000 g for 1 hr at 4 ° to pellet the microsomes.

5. The microsomal pellet is resuspended in 40 ml of labeling buffer (50 mM Tris, 2 mM MnCI2, pH 7.4) and rehomogenized in a glass homogenizer. Then 300/xCi of [3H]inositol is added and the microsomes incubated for 1.5 hr at 37 °. (They can be left at 4 ° overnight at this stage.) Addition of MnC12 is essential as it promotes the head group exchange of inositol from PI.

6. The microsomes are spun at 100,000 g for 90 min at 4 °, and the pellet is resuspended in 100 ml of 10 mM Tris-HCl, 2 mM inositol, pH 8.6.

7. After recentrifugation of the microsomes, they are resuspended in 100 ml of 1 mM Tris-HCl, 2 mM inositol, pH 8.6, and recentrifuged.

9 G. M. Helmkamp, Jr,, M. S. Harvey, K. W. A. Wirtz, and L. L. M. van Deenen, J. Biol. Chem. 249, 6382 (1974).


8. The microsomal pellet is resuspended in 20 ml of SET buffer and the protein concentration adjusted to 25 mg/ml. The microsomes are divided into aliquots (500/zl per tube) and frozen at - 8 0 ° until required.

Preparation of Liposomes

Liposomes are prepared by drying down 784 ~g of PC with 16/zg PI in a glass tube, adding 1 ml of SET buffer, and sonicating prior to use. The liposomes can be stored at 4 ° for up to 1 week without any deterioration.

Assay for Phosphatidylinositol Transfer Protein

1. The microsomes are diluted 1/20 to obtain a protein concentration of 1.25 mg/ml. Then 100/zl of microsomes is mixed with I00/zl of liposomes, and the reaction is initiated by addition of 50/zl of the fraction under investigation.

2. The samples are incubated at 25 ° for 30 min. 3. To terminate the reaction, 50/zl of 0.2 M sodium acetate in 0.25 M

sucrose at pH 5 is added to aggregate the microsomes. After vigorous mixing, the tubes are centrifuged at 15,000 g, 15 min at 4 °, to pellet the microsomes.

4. One hundred microliters of supernatant is removed, and after addition of scintillation fluid the radioactivity transferred to the liposomes is determined. The percent transfer is calculated from the amount of radioactivity transferred from the microsomes to the liposomes as a fraction of the total microsomal counts. This is measured by counting a sample of the microsome preparation. Over 90% of the radioactivity in the microsomal preparation is in phosphatidylinositol, and this can be established by extraction of the lipids and analysis by thin-layer chromatography (TLC). The reaction is approximately linear for up to 20% transfer of PI.

Figure 2 illustrates the elution of PI-TP from the heparin-Sepharose column. The peak of activity is pooled and concentrated to 200-400/zl in an Amicon (Danvers, MA) ultrafiltration cell fitted with a YM10 membrane. Alternatively, the fractions can be concentrated in a Centricon-10 centrifugal concentrator (Amicon). The concentrated samples can then be used immediately for reconstitution or can be left at 4 ° for at least a week before the ability to reconstitute PLC activity begins to decline.

The final amount of material that can be made from a single run on heparin-Sepharose is about 200-400/zl, which is enough to do several reconstitution experiments. However, the preparation is relatively impure and is contaminated with phospholipase C activity (named PLC-e). 5


t 2 0

n 1 a

3 1

I I I

0 30 60

o

- - 0 . 6

i 0

F r a c . N o .

FIG. 2. Elution profile of PI-TP activity of rat brain cytosol from a heparin-Sepharose column on elution with a gradient of NaCI.

For further purification, the sample is loaded onto a gel-filtration column. We use Superdex 75 or Superose 12 (Pharmacia), and the samples are run on a fast protein liquid chromatography (FPLC) system at 4 °. Figure 3 illustrates the analysis of such a run on Superdex 75. On calibration of the gel-filtration column, the apparent molecular mass of the protein is calculated as approximately 20 kDa. However, the calculated molecular mass of PI-TP is 35 kDa, and it runs as a 35-kDA protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 3). This apparent anomaly greatly aids the purification of this protein by this method. The fractions are analyzed for (1) in vitro PLC activity (as described elsewhere in this volume6), (2) PI transfer activity, and (3) reconstitution. 6 The PI transfer activity and reconstituting activity match with a 35-kDa protein on SDS-PAGE (Fig. 3). We have further confirmed the identity of the protein as PI-TP by Western blotting with an affinity- purified polyclonal antibody raised against recombinant PI-TP. At this stage the protein is 80-90% pure (Fig. 3). If required, an essentially pure protein can be obtained if the fractions from the gel-filtration column are pooled and further chromatographed on phenyl-Superose as described later for the bovine brain preparation.

For reconstitution studies the samples obtained from gel filtration are in 20 mM PIPES, 137 mM NaC1, 2.7 mM KCI (pH 6.8) and can therefore be used directly for the reconstitution assays. If further purified on phenyl- Superose, the active fractions are pooled and then dialyzed into this buffer before use. To obtain more material for reconstitution experiments, we have scaled up the preparation by introducing two initial stages (see Fig.


I 2 o

v

8- 1 t:3

20 40 60

Frac. No.

o I

- PI-TP

4-0 41 42 43 44 4-5 46 47 48 49

F rac . No.

FIG. 3. Chromatography of rat brain PI-TP on a gel-filtration column followed b y SDS-PAGE of active fractions. Active fractions obtained after chromatography on heparin- Sepharose were pooled and concentrated to 2 ml. The sample was loaded onto a Superdex 75 column and eluted with 20 mM PIPES, 137 mM NaC1, and 2.7 mM KC1, pH 6.8. Fractions were assayed for PIP2-hydrolyzing activity in vitro (©), PI transfer activity (O), as well as reconstitution of PLC activity in cytosol-depleted HL-60 cells (V) as described elsewhere in this volume. 6 For the reconstitution assay, GTPTS (10/zM), MgATP (1 mM), and Ca 2÷ (1 /zM) were present. Fractions 40-49 were electrophoresed on a 12% polyacrylamide gel in the presence of SDS, and the gel was stained with Coomassie blue. The position of PI-TP is marked on the gel. The molecular weight markers (× 10 -3) used were 66, 44, 36, 29, 24, 20, and 14 (outside lanes of the gel).


1A) prior to running the sample on heparin-Sepharose. Additionally, instead of using rat brains, bovine brains have been used and similar results obtained.

Purification of Reconstituting Activity from Bovine Brain Cytosol

Step I: Preparation of Ammonium Sulfate Precipitates

One bovine brain is used per preparation. Frozen brains are thawed overnight at 4 °, cut into small pieces with a pair of scissors, and homogenized in a Waring blendor after the addition of approximately 2 ml/g tissue of Tris buffer (20 mM Tris, 3 mM KC1 at pH 7.6). The buffer also contains the following components so that the final concentration is 1 mM PMSF, l mM DTT, 5 mM EDTA, 5 mM EGTA, 10 mM benzamidine, 2 /zM pepstatin A, 0.1 /zM leupeptin, 37 tzg/ml TLCK, and 5/zg/ml aprotinin. The homogenate is centrifuged (10,000 gav, 15 hr, 4°). The supernatant is decanted and fractionated with solid ammonium sulfate and centrifugation (10,000 gay, 30 min, 4°). The precipitate between 40 and 60% saturation is redissolved in 100 ml Tris buffer and dialyzed overnight against a further 100 volumes at 4 °. The final conductivity of the solution is equivalent to an ammonium sulfate concentration of less than 15 mM.

Step 2: Weak Anion-Exchange Chromatography

The dialyzed solution (180 ml) is pumped onto a column of DE-52 (Whatman, Clifton, NJ) weak anion-exchange resin (200 ml; 50 x 230 mm column) previously equilibrated in Tris buffer. The column is then washed with a further 250 ml of buffer. Proteins are eluted with a 2-liter linear gradient of 0 to 300 mM NaC1 in the same buffer at 2 ml/min. Fractions (10 ml) are collected and assayed for PI-TP activity. Active fractions from a single peak of activity are pooled (Fig. 4A).

Step 3: Heparin-Sepharose Affinity Chromatography

The fractions containing the PI-TP activity are pooled and concentrated by ultrafiltration to 25 ml (the conductivity of the solution must be -<90 mM NaC1). This is chromatographed on immobilized heparin (100 ml Hi- Trap, Pharmacia-LKB custom column) by elution isocratically with 45 mM NaCI in Tris buffer (300 ml). Alternatively, the material can be purified as five batches on a column of four serial 5-ml Hi-Trap heparin columns (Pharmacia). Resolution can be increased further by excluding the salt if required. Fractions (10 ml) are collected and analyzed for PI-TP activity (Fig. 4B). Fractions containing a peak of PI-TP activity are pooled and concentrated on a YM10 membrane in an Amicon ultrafiltration cell to


1 0 x v

Q_ E3

0 I

o

J ?

" ' " ' " . . . . . . . . / . .

I I I

25 50 75

F r o c . No.

2 ~ s.o

I ~ z

I ° 1 - - I

I ~ 0 . 0

B

2 I O

v

IX. t ~

/

I " . . . . . . . . . . . . . . . . . . . 0

I I

10 20 30

Froc. No.

.> n n O

FIG. 4. Chromatography of PI-TP on DE-52 (A) followed by heparin-Sepharose (B). Bovine brain cytosol was initially fractionated by ammonium sulfate precipitation, and the proteins precipitated at 40-60% were chromatographed on DE-52. Fractions were assayed for PI-TP activity. The fractions containing the peak of PI-TP activity were pooled and, after dialysis, chromatographed isocratically on heparin-Sepharose.

approximately 2 ml. The pooled fractions can be tested for reconstitution to confirm that the reconstituting activity copurifies with the PI-TP activity. Alternatively, the individual column fractions can also be directly assayed by recOnstitution.

Step 4: Gel-Permeation Chromatography

The pooled and concentrated preparation is passed through a Super- dex-75 16/60 gel-permeation column (Pharmacia). The column is equilibrated in 20 mM PIPES, 3 mM KC1, 137 mM NaCI, pH 6.8, and elution


is at 0.12 ml/min. Fractions (0.8 ml) are collected after the void volume (40 ml). Both PI-TP and reconstituting activity can be monitored in the fractions, and both activities coelute. Figure 5 illustrates that the PI transfer activity corresponds to the 35-kDa protein.

Step 5: Chromatography on Phenyl-Superose

A concentrated solution of 3.4 M ammonium sulfate is added to the pooled active fractions to a final concentration of 340 mM before loading

I o 2

v

n 1 a

o I

o

2 .-, •

1

!i .......... , ...................... ~, o i I

/

3O

F r a c . No.

60

o

P I - T P

3 8 40 42 4-4- ¢ 6 4 8 50

FIG. 5. Chromatography of PI-TP by gel filtration. The active fractions obtained after chromatography on heparin-Sepharose were pooled, concentrated, and chromatographed on Superdex 75. The fractions were assayed for PI transfer activity, and the active fractions (38-51) were analyzed by SDS-PAGE. The gel was stained with silver only. The position of PI-TP is marked on the gel. The molecular weight markers (× 10 -3) used were 66, 44, 36, 29, 24, 20, and 14 (last lane on the left). Dotted lines, A280 ; filled circles, PI transfer activity.


the sample onto a Phenyl-Superose HR 5/5 hydrophobic interaction column (Pharmacia) equilibrated in a Tris buffer with the same final salt concentration as the sample. After washing to remove unretained material, the remaining protein is eluted with salt-free Tris buffer. Fractions (2.5 ml) are collected, desalted by dialysis or passage through NAP-10 columns (Pharmacia), and analyzed for PI-TP activity (Fig. 6). The majority of the PI-TP elutes in a single fraction.

The extent of purification is monitored by SDS-PAGE on a 15% cross- linked polyacrylamide gel with silver staining followed by staining with

I o 2

X v

a

..... , . _ / \ L I

o 5 1o

F r o c . N o .

1,0 I z0"34

- i -

o.5 g o

o.o I o.o

- - PI-TP

1 2 3 4 5 6 789 FIG. 6. Chromatography of PI-TP on Phenyl-Superose. The active fractions obtained

after chromatography on Superdex 75 were pooled, concentrated, and chromatographed on Phenyl-Superose. The fractions were assayed for PI transfer activity, and the active fractions (1-9) were analyzed by SDS-PAGE. The gel was stained with silver followed by Coomassie blue. The position of PI-TP is marked on the gel. The molecular weight markers (× 10 -3) used were 66, 44, 36, 29, 24, 20, and 14 (last lane on the left).


Coomassie blue. We have observed that the main impurity present after the gel-filtration step does not stain well with silver but is clearly visible after staining with Coomassie blue. Column fractions are analyzed after gel filtration (Fig. 5) and Phenyl-Superose chromatography (Fig. 6).

Step 6: Separation of Phosphatidylinositol and Phosphatidylcholine Forms of Phosphatidylinositol Transfer Protein by Anion-Exchange Chromatography and Analysis by Isoelectric Focusing

The PI-TP can be loaded with either phosphatidylcholine (PC) or with phosphatidylinositol (PI), and the difference in charge between PI and PC accounts for their separation on Mono Q.l° When the purified protein is analyzed on isoelectric focusing gels, PI-TP resolves into two major bands, one corresponding to the PC-loaded form (pI 5.6) and the other to the PI-loaded form (pI 5.3) (Fig. 7). In general, approximately 70% of the molecules contain PI and 30% of the molecules contain PC, although this may vary among preparations.

The PI and PC forms of PI-TP can be resolved on Mono Q.7,10 Active desalted fractions from the Phenyl-Superose column are pooled, and the material is loaded onto a Mono Q HR 5/5 anion-exchange column equilibrated in Tris buffer at pH 7.8. Protein is eluted with a linear gradient of NaCI (0-300 raM, 20 ml) in the same buffer. Fractions (1 ml) are collected at a 1 ml/min flow rate. On Mono Q, PI-TP elutes as two peaks at 85 and 90 mM NaC1 at pH 7.8. 7'1° The early eluting peak contains the PC form of PI-TP, and the second peak contains the PI form of PI-TP. Both forms of PI-TP are active in PI transfer activity assays in vitro as well as in reconstitution assays.

Conversion of Phosphatidylinositol Transfer Protein to Phosphatidylcholine or Phosphatidylinositol Form

The PI-TP can be converted to the PC or the PI form by incubation of the pure protein with appropriate lipid vesicles. When the purified PI-TP is incubated with PI vesicles, the PC form of PI-TP is converted predominantly to the PI form. On the other hand, incubation of the protein with PC-containing vesicles converts only a portion to the PC form. We have studied reconstitution after incubating the protein with PI or PC vesicles and found that the protein is effective in reconstitution te-

l0 p. A. van Paridon, A. J. W. G. Visser, and K. W. A. Wirtz, Biochim. Biophys. Acta 898, 172 (1987).


pl

5.6

5.3

PC-loaded PI-TP

Pl-loaded PI-TP

FIG. 7. Analysis of PI-TP by isoelectric focusing. The PI-TP obtained after Phenyl- Superose chromatography was analyzed by isoelectric focusing. The isoelectric points of the separated proteins are marked. The protein running at an isoelectric point of 5.6 is the PC-loaded PI-TP, and the protein running at an isoelectric point of 5.3 is the PI-loaded PI-TP. Protein was visualized by silver staining.

gardless of the lipid constituent. 7 We find that the method of Van Paridon et al. 1° is most effective for the interconversion, modified as detailed below.

The PI-TP (45/~l) is incubated with 5/.~l of PC/phosphatidic acid (PA) (70:30 mol%; 40/~M final) or PI (120/.~M final) in 20 mM Tris buffer containing 5 mM MgCl2 and 60 mM NaCI (pH 7.4). The solution is incubated at room temperature for 15 rain. Then 20/~l of DE-52 (50% slurry) equilibrated in the same buffer is added and the samples mixed. (The DE52 removes the unbound lipids.) After 3 min with regular shaking, the samples are centrifuged, and the supernatant is analyzed by isoelectric focusing on 0.8-mm-thick, 5% cross-linked acrylamide/piperazine diac- rylamide gels containing Bio-Lytes (Bio-Rad, Richmond, CA) with a pH 4-6 gradient. Proteins are detected by silver staining.

[14] 13y SUBUNIT STIMULATION OF PHOSPHOLIPASE C 181

Remarks

The PI-TP is best used flesh for reconstitution assays. Generally, purified PI-TP can be left at 4 ° for 3-4 days before a fall in reconstituting activity becomes apparent. When stored in 50% (v/v) glycerol, PI-TP has to be transferred back into the assay buffer (PIPES, NaC1/KC1, pH 6.8), and this is generally done by using a Centricon concentrator. In our experience, storage of PI-TP in 50% glycerol at - 20 ° leads to considerable loss of reconstituting activity (but not necessarily a decline in in vitro PI transfer activity).

Acknowledgments

We thank the Medical Research Council and the Wellcome Trust for support. E.C. is supported by a studentship from Science and Engineering Research Council.

[14] S t i m u l a t i o n o f P h o s p h o l i p a s e C b y

G - P r o t e i n f l y S u b u n i t s

By PETER G I E R S C H I K a n d MONTSERRAT CAMPS

Introduction

The generation of the second messengers inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol by phosphoinositide-specific phospholipases C (PLCs) is involved in the regulation of a wide variety of cellular functions, t'2 Many extraceUular signaling molecules like hormones, neurotransmitters, and growth factors exert their action by stimulating the PLC effector moiety. To date, at least eight distinct PLC isozymes have been identified in mammalian tissues. 3 The amino acid sequences of PLC isozymes share considerable similarity within two regions of about 170 and 260 residues. These regions, designated domains X and Y, are believed to be involved in the catalytic function of these molecules.

According to sequence similarity outside domains X and Y, PLCs are divided into three groups designated PLC/3, PLCy, and PLCS. Three

I M. J. Berridge, Nature (London) 361, 315 (1993). 2 y . Nishizuka, Science 258, 607 (1992). 3 S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992).

Copyright © 1994 by Academic Press, lnc, METHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved,


PLCfl isozymes referred to as PLC/31,4'5 PLCfl2,6 and PLCfl37'8 have been identified in mammalian cells. All three PLC/3 isozymes have been shown to be stimulated in a pertussis toxin-resistant manner by members of the aq subfamily of G-protein a subunits (PLCfll --- PLCfl 3 >> PLCfl2). 7-15 The aq subfamily comprises aq, ~N, a~4, and O/15/16 .16 Members of the PLCy and PLC8 subfamilies appear to be resistant to stimulation by aq.9 A PLC purified from turkey erythrocytes has been shown to be activated by an all subunit purified from the same system. 17'~8

We have demonstrated that a PLC present in soluble fractions of HL-60 cells is activated by G-protein fly subunits. 19 We have subsequently shown that this stimulation is PLC isozyme-selective and have identified PLCfl22° and PLCfl321 as prime targets of fly subunit stimulation. The fact that purified PLCfl3 was stimulated by purified fly subunits suggested that fly stimulation of PLCfl isozymes is achieved by direct protein-protein interaction. 21 Similar results have been reported by other laboratories. 13'18'22-24 The observation that the concentrations of fly subunits required for PLCfl stimulation are substantially higher than those of acti-

4 M. Katan, R. W. Kriz, N. Totty, R. Philp, E. Meldrum, R. A. Aldape, J. L. Knopf, and P. J. Parker, Cell (Cambridge, Mass.) 54, 171 (1988).

5 P.-G. Suh, S. H. Ryu, K. H. Moon, H. W. Suh, and S. G. Rhee, Cell(Cambridge, Mass.) 54, 161 (1988).

6 D. Park, D.-Y. Jhon, R. W. Kriz, J. L. Knopf, and S. G. Rhee, J. Biol. Chem. 267, 16048 (1992).

7 A. J. Carozzi, R. W. Kriz, C. Webster, and P. J. Parker, Eur. J. Biochem. 210, 521 (1992). 8 D.-Y. Jhon, H.-H. Lee, D. Park, C.-W. Lee, K.-H. Lee, O.-J. Yoo, and S. G. Rhee, J.

Biol. Chem. 268, 6654 (1993). 9 S. J. Taylor, H. Z. Chae, S. G. Rhee, and J. H. Exton, Nature (London) 350, 516 (1991). l0 A. V. Smrcka, J. R. Hepler, K. O. Brown, and P. C. Sternweis, Science 251, 804 (1991). II S. J. Taylor and J. H. Exton, FEBS Lett. 286, 214 (1991). 12 C. H. Lee, D. Park, D. Wu, S. G. Rhee, and M. I. Simon, J. Biol. Chem. 267, 16044 (1992). 13 A. V. Smrcka and P. C. Sternweis, J. Biol. Chem. 268, 9667 (1993). 14 D. Wu, C. H. Lee, S. G. Rhee, and M. 1. Simon, J. Biol. Chem. 267, 1811 (1992). 15 G. Berstein, J. L. Blank, A. V. Smrcka, T. Higashijima, P. C. Sternweis, J. H. Exton,

and E. M. Ross, J. Biol. Chem. 267, 8081 (1992)~ 16 M. I. Simon, M. Strathmann, and N. Gautam, Science 252, 802 (1991). 17 G. L. Waldo, J. L. Boyer, A. J. Morris, and T. K. Harden, J. Biol. Chem. 266, 14217 (1991). 18 j. L. Boyer, G. L. Waldo, and T. K. Harden, J. Biol. Chem. 267, 25451 (1992). 19 M. Camps, C. Hou, D. Sidiropoulos, J. B. Stock, K. H, Jakobs, and P. Gierschik, Eur.

J. Biochem. 206, 821 (1992). 20 M. Camps, A. Carozzi, P. Schnabel, A. Scheer, P. J. Parker, and P. Gierschik, Nature

(London) 360, 684 (1992). 21 A. J. Carozzi, M. Camps, P. Gierschik, and P. J. Parker, FEBS Left. 315, 340 (1993). 22 j . L. Blank, K. A. Brattain, and J. H. Exton, J. Biol. Chem. 267, 23069 (1992). 23 A. Katz, D. Wu, and M. I. Simon, Nature (London) 360, 686 (1992). 24 D. Park, D.-Y. Jhon, C.-W. Lee, K.-H. Lee, and S. G. Rhee, J. Biol. Chem. 268, 4573

(1993).

[14] fl-/SUBUNIT STIMULATION o r PHOSPHOLIPASE C 183

vated aq subunits, together with the fact that pertussis toxin-sensitive G proteins are generally more abundant in membranes than members of the Gq class, has led us 19'2° and o t h e r s 13A8'22-24 to suggest that stimulation of PLC/3 isozymes, in particular of PLC/32 and PLC/33, may represent the mechanism by which pertussis toxin-sensitive G proteins stimulate phospholipase C.

In this chapter we describe methods for the purification of/3y subunits of bovine retinal transducin and for the preparation of soluble and membranous phospholipases C, as well as recombinant PLC/3 isozymes suitable for stimulation by these and other G-protein 137 subunits. The conditions used to assay/37 subunit stimulation of native or recombinant phospholipases C are specified and discussed in detail.

Purified/3y Subunits of Retinal Transducin

To avoid potential artifacts arising from the detergents present in fly subunit preparations purified from membranous sources,Z5 we use hydrophilic, detergent-free/37 subunits of retinal transducin (/3%) to activate PLC. The ease of preparing large amounts of/3% in high purity is another reason to use this/37 dimer. The/3t polypeptide is identical in structure to the 36-kDa/31 subunit found in nonretinal cellsfl 6 The % polypeptide is related to the nonretinal y subunits but structurally distinct. 27'28 Stimula- tion of PLC is also observed, however, with nonretinal/37 subunit preparations. 13'18'19'22'24 Purification of nonretinal /33, subunits is described by Camps et al. w and elsewhere in this series. 29

Preparation of Heterotrimeric Transduein

Retinal rod outer segment membranes are prepared from bovine eyes as described? ° The method we use for purification of heterotrimeric transducin (Gt) is a modification of the procedure developed by Kiihn 31 and is described by Baehr et al. 32 and elsewhere in this series. 33

25 A. Yatani, K. Okabe° L. Birnbaumer, and A. M. Brown, Am. J. Physiol. 258, H1507 (1990). 26 j. Codina, D. Stengel, S. L. C. Woo, and L. Birnbaumer, FEBS Lett. 207, 187 (1986). 27 p. Gierschik, J. Codina, C. Simons, L. Birnbaumer, and A. M. Spiegel, Proc. Natl. Acad.

Sci. U.S.A. 82, 727 (1985). z8 N. Gautam, J. K. Northup, H. Tamir, and M. I. Simon, Proc. Natl. Acad. Sci. U.S.A.

87, 7973 (1990). 29 j. Dingus, M. D. Wilcox, R. Kohnken, and J. D. Hildebrandt, this series, Vol. 237 [36]. 3o D. S. Papermaster and W. J. Dreyer, Biochemistry 13, 2438 (1974). 3~ H. KiJhn, Nature (London) 283, 587 (1980). 32 W. Baehr, E. A. Morita, R. J. Swanson, and M. L. Applebury, J. Biol. Chem. 257,

6452 (1982). 33 j. Bigay and M, Chabre, this series, Vol. 237 [11].


Comments. We routinely prepare transducin from rod outer segment membranes collected from about 700 eyes. Transducin (-25 mg protein) is eluted from the membranes with buffer containing 100/zM GTP and is used for the subunit preparation procedure without delay.

Purification of Transducin Subunits

Transducin can be readily resolved into subunits by chromatography on Blue S e p h a r o s e . 34,35 The solvent G t is changed to buffer B [10 mM Tris-HCl, pH 7.5, 20% (v/v) glycerol, 6 mM MgCIE, 1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride] using prepacked Seph- adex G-25 columns (PD-10, Pharmacia LKB, Piscataway, NJ) according to the protocol supplied by the manufacturer. Fractions containing G t a r e

pooled and applied to a column (HR 16/10, Pharmacia LKB) of Blue Sepharose CL-6B (20 ml, Pharmacia LKB) which has been equilibrated with buffer B using a Pharmacia FPLC (fast protein liquid chromatography) system. The flow rate is 0.5 ml/min. Fractions of 2.5 ml are collected. After application of the sample, the column is washed with 85 ml of buffer B followed by 85 ml of buffer B containing 500 mM KCI. The ~t subunits are eluted isocratically from the resin in the absence of KC1; o~ t dissociates from the resin at increasing concentrations of KCI. Fractions containing either ott or/3% subunits are pooled and concentrated about lO-fold by pressure filtration in a stirred cell equipped with an Amicon (Danvers, MA) PMIO membrane. The flow-through solutions generated in the concentration steps are used as control buffers in the reconstitution experiments. The yield of purified a t and/37 t subunits is typically around 8 mg of each protein. The purified proteins are snap-frozen in liquid nitrogen and stored at -70 ° .

Preparation of Soluble Phospholipases C for fly Subunit Stimulation

Preparation of Soluble Phospholipase C from HL-60 Cells

Human promyelocytic HL-60 ce l l s 36 (ATCC, Rockville, MD, CCL 240) are grown in suspension culture in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (FCS) (for culture volumes <100 ml) or horse serum (culture volume >100 ml), 44 mM NaHCO3, 5.5 mM glucose, 5

34 H. Shichi, K. Yamamoto, and R. Somers, Vision Res. 24, 1523 (1984). 35 C. Kleuss, M. Pallast, S. Brendel, W. Rosenthal, and G. Schultz, J. Chromatogr. 407,

281 (1987). 36 S. J. Collins, R. C. Gallo, and R. E. Gallagher, Nature (London) 270, 347 (1977).

[14] /37 SUBUNIT STIMULATION OF PHOSPHOLIPASE C 185

mM L-glutamine, minimal essential medium (MEM) nonessential amino acids (1 x, GIBCO Grand Island, NY), 1 mM sodium pyruvate, 50 U/ml penicillin, and 50/xg/ml streptomycin in a humidified atmosphere of 90% air and 10% CO2. The cell density is maintained at approximately 1 x l06 cells/ml. Cells (1 x 10 l° cells in l0 liters of medium) are harvested by centrifugation in Beckman (Palo Alto, CA) type JA-10 rotors (ray 98 mm) at 3500 rpm for 20 rain at room temperature. The pellets are resuspended in 50 ml of l0 mM triethanolamine hydrochloride, pH 7.4, and 140 mM NaCl. Cells are sedimented again by centrifugation in a Beckman type JA-20 rotor (ray 70 mm) at 4300 rpm for 20 rain at 4 °.

The final pellet is resuspended in 80 ml of lysis buffer containing 20 mM Tris-HCl, pH 7.5,250 mM sucrose, 1.5 mM MgCl 2, l mM ATP, 3 mM benzamidine, 1/xM leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 2/zg/ml soybean trypsin inhibitor. The cells are then pressurized with nitrogen at 2.8 MPa (400 psi) for 30 min at 4 ° in a nitrogen bomb (No. 4635, Parr, Moline, IL) and slowly collected into 1 ml of 100 mM EGTA, adjusted to pH 7.4 with NaOH. This treatment results in disruption of approximately 85% of the cells. The cavitate is then centrifuged in a JA- 20 rotor at 4300 rpm for 45 sec at 4 ° to remove unbroken cells and nuclei, then filtered through two layers of cheesecloth. The fraction containing soluble PLC activity is prepared from the postnuclear supernatant by sequential centrifugation in a JA-20 rotor at 15,000 rpm for 20 min at 4 ° and in a Beckman type 60 Ti rotor (rav 63.4 ram) at 40,000 rpm for 60 min at 4 °. The supernatant is concentrated about 6-fold in a stirred cell equipped with an Amicon PM 10 membrane. The concentrate is passed through 0.45- /xm pore size Millipore (Bedford, MA) Millex HA filters, snap-frozen in liquid nitrogen, and stored at - 70 ° .

Preparation of Soluble Phospholipase C from Bovine Tissues

Bovine tissues are freshly obtained from the slaughterhouse. Following the removal of connective tissue, blood vessels, fat, or white matter (from brain samples), the tissues are cut into small cubes ( -1 x 1 x 1 cm), suspended in an equal volume of ice-cold lysis buffer (see above) containing 1.25 mM EGTA, and homogenized in a Waring blendor at medium speed using five consecutive 15-sec bursts separated by 1-min intervals. The homogenates are centrifuged in a JA-20 rotor at 5000 rpm for 10 min at 4 ° to remove unbroken cells and debris. The supernatant is filtered through two layers of cheesecloth and then cleared of residual particulate matter by two consecutive centrifugations in a JA-20 rotor at 15,000 rpm for 40 min at 4 ° and in a Beckman 70 Ti rotor (ray 65.7 mm) at 50,000 rpm


for 60 min at 4% The supernatants are passed through 0.45-~m pore size Millipore Millex HA filters, snap-frozen in liquid nitrogen, and stored at - 70 ° .

Preparation of Membranous Phospholipase C for fly Subunit Stimulation

Preparation o f Membranes from HL-60 Cells

HL-60 cells are grown and homogenized as specified above. The membranes are prepared from the postnuclear supernatant by centrifugation in a JA-20 rotor at 15,000 rpm for 20 min at 4 °. The membrane pellets are washed three times with buffer containing 20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM DTT, 3 mM benzamidine, 1 mMphenylmethylsulfonyl fluoride, 10/zM leupeptin, and 2/zg/ml soybean trypsin inhibitor, resuspended to 10 mg protein/ml with this buffer, and stored at - 70 °. The yield of membrane protein is approximately I00 mg from 101° cells.

Preparation of Recombinant Phospholipase C-fl Isozymes in COS-1 Cells

Construction o f Expression Vectors

The cDNAs encoding bovine PLCfll 4 and human PLCfl26 are ligated into the unique EcoRI restriction site of the mammalian expression vector pMT2. 37 Plasmid DNAs are produced according to standard molecular biological protocols 38 and purified on Qiagen columns (Qiagen, Chatsw- orth, CA) prior to transfection according to the protocol supplied by the manufacturer.

Cell Culture and Transfection

African green monkey kidney COS-1 cells 39 (ATCC CRL-1650) are propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FCS (DMEM/FCS) at 37 ° and 8% (v/v) CO 2 . For DNA transfection, 1 × 106 cells are seeded onto 10-cm dishes 60 hr before transfection. About 30 min before transfection, cells are rinsed three times with phosphate-buffered saline (PBS) followed by the addition of 1.5 ml DMEM without FCS per dish. Fifteen micrograms of DNA is mixed with 75/.tg of Transfectam [both in 1.5 ml of DMEM without FCS; Transfectam

37 R. J. Kaufman, this series, Vol. 185, p. 487. 38 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual,"

2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 39 y. Gluzman, Cell (Cambridge, Mass.) 23, 175 (1981).

[14] /37 SUBUNIT STIMULATION OF PHOSPHOLIPASE C 187

is taken from a 2.5 /zg//~l stock in 10% (v/v) ethanol; Transfectam is obtained from Promega, Madison, WI] and added to the dishes. Cells are incubated for 8 hr at 37 ° and 8% CO 2. Subsequently, the transfection medium is replaced with fresh DMEM/FCS, and the incubation is continued for 40 hr.

Preparation of Cell Extracts

Forty-eight hours after transfection the dishes are rinsed three times with PBS followed by the addition of 500/~l of ice-cold extraction buffer containing 20 mM Tris-HC1, pH 7.5, 5 mM EDTA, 10 mM EGTA, 37 mM sodium cholate, 3 mM benzamidine, 43 mM 2-mercaptoethano[, and 100 p~M phenylmethylsulfonyl fluoride. Cells are scraped on ice into extraction buffer, incubated for 1 hr at 4 ° with gentle vortexing every 10 min, and then centrifuged in a JA-20 rotor at 15,000 rpm for 20 min at 4 °. Supernatants containing the extracted proteins ( -10 mg protein/ml) are divided into aliquots, snap-frozen in liquid nitrogen, and kept at -70 ° until assayed for PLC activity.

Immunochemical Analysis of Phospholipase C-fl Isozyme Expression

Expression of the PLCfl isozyme is determined by immunoblotting. Antibodies are prepared by coupling oligopeptides representing the carboxyl-terminal regions of PLCfll (NHz-GENPGKEFDTPL-COOH) and PLCfl2 (NHz-QDPLIAKADAQESRL-COOH) to keyhole limpet hemocyanin using glutaraldehyde 4° and then immunizing rabbi t s f Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting are performed as described, z7 except that immunoreactive proteins are visualized using the ECL (enhanced chemiluminescence) Western blotting detection system from Amersham (Arlington Heights, IL).

Assay of Soluble Phospholipase C Using Exogenous Radiolabeled Substrate

Preparation of Lipid Substrate

A mixture of phosphatidylethanolamine, phospha~idylinositol 4,5-bisphosphate [PtdIns(4,5)P2], and [3H]PtdIns(4,5)P2 in chloroform is evaporated to dryness under a stream of NE in a 14 x 100 mm Pyrexglass tube

40 M. Reichlin, this series, Vol. 70, p. 159. 41 j. L. Vaitukaitis, this series, Vol. 73, p. 46.


at room temperature. The lipids are then resuspended by continous vortex mixing for 30 min in buffer (40/~l/assay) containing 87.5 mM Tris-maleate (taken from a 1 M stock adjusted to pH 7.5 at 20 ° with NaOH42), 17.5 mM 2,3-bisphosphoglycerate, 17.5 mM LiCI, 17.5 mM EDTA, and 1.6 mM sodium deoxycholate. After vortexing, the lipid suspension is sonicated for 15 min in a bat.h-type sonicator (Sonorex RK 102; Bandelin, Berlin, Germany).

Comments. We have compared lipid suspensions vortexed and sonicated at 4 ° and at 20 ° and have found no difference in the ability to serve as a PLC substrate. 2,3-Bisphosphoglycerate and LiC1 are present in the incubation medium to prevent degradation of inositol phosphates. 43

Phospholipase C Assay

The activity of PLC is assayed at 25 ° in a mixture (70/~1) containing 40/zl of the lipid substrate, 5/zl of the sample containing PLC, 5-10/zl of the fly subunit preparation, and 5-10/zl of CaCI 2 to adjust the concentration of free Ca 2+ to 0.1 p.M. The final assay concentrations of phosphatidylethanolamine and [3H]PtdlnsP2 are 280 and 28/xM (5 Ci/mol), respectively. The reaction is started by the addition of the lipid substrate and terminated by adding 350/zl of chloroform/methanol/concentrated HC1 (500 : 500 : 3, by volume, stop solution I) followed by vortex mixing. Sam- ples are then supplemented with 100/zl of 1 M HC1 containing 5 mM EGTA (stop solution II). Phase separation is accelerated by centrifugation (30 sec) in an Eppendorf microcentrifuge. The formation of water-soluble inositol phosphates is measured by supplementing a 200-/zl portion of the aqueous (upper) phase (total volume -325 ~1) with 4 ml of scintillation fluid (Quicksafe A; Zinsser Analytic, Frankfurt, Germany) followed by liquid scintillation counting.

CommOnts. Half-maximal and maximal stimulation of soluble PLC in HL-60 cells are observed at approximately 0.3 and 2 tzM flYt (Fig. 1A). GDP-liganded o~ t blocks stimulation of PtdlnsP2 hydrolysis by f'Yt (Fig. 1B). The methodology outlined above has been used to assay fly subunit stimulation of crude soluble PLC prepared from a variety of bovine tissues (Table I) and of purified phospholipases C, for example, PLCf3 purified from human HeLa $3 cells. 21

To achieve the desired concentrations of fly subunits, PLC, or other assay constituents, it may be necessary to adjust the volumes specified above. The volume of the lipid suspension has been varied from 25 to 50 /zl without affecting the results. We have used either EDTA or EGTA (3

42 G. Gomori, this series, Vol.1, p. 138. 43 M. J. Berridge, Annu. Rev. Biochem. 56, 159 (1987).

[14] /3-/SUBUNIT STIMULATION OF PHOSPHOLIPASE C 189

1000 A

g 800

o_ 600

° ~ 400

0--~1 . . . . . . . . . . . . . - m -7 -6

B'It (log M)

' HI--- -m

at (log M)

I 5g \ - ~ 3

i00 = ~, -~- ~

0 -6 -5

FIG. 1. Stimulation of the soluble phospholipase C of HL-60 cells by/37 t and reversal of stimulation by GDP-liganded a t. (A) HL-60 cell cytosol (80 p,g protein/assay) was incubated as described in the text in the presence of increasing concentrations of/37t with phospholipid vesicles containing [3H]PtdInsP2. (B) HL-60 cell cytosol was incubated in the presence (filled circles) or absence (open circles) of 0.7 p,M/3% with increasing concentrations of GDP-liganded ott and phospholipid vesicles containing [3H]PtdInsP2. Reactions were terminated by the addition of chloroform/methanol/HC1 and analyzed for inositol phosphates as specified in the text.

TABLE I EFFECT OF ~'Yt ON SOLUBLE PHOSPHOLIPASE C

OF BOVINE TISSUES a

Inositol phosphate formation (pmol/min x mg of protein

Tissue Control /3"yt

Brain (cortex) 1079 -+ 211 2513 -+ 235 (2.3) Brain (cerebellum) 533 +-- 138 1878 - 195 (3.5) Brain (striatum) 342 -+ 14 1526 - 156 (4.5) Lung 132 -+ 38 388 -+- 30 (2.9) Kidney 465 -+ 7 688 _+ 103 (1.5) Heart 184 -+ 85 1081 +_ 55 (5.9) Spleen 235 -+ 27 704 _+ 3 (3.0) Liver 1021 -- 477 2385 _+ 231 (2.3)

a Soluble fractions of several bovine tissues were incubated in the absence (Control) or presence of /3yt (2 /zM) with phospholipid vesicles containing PtdInsP 2. Values correspond to the means -+ SD of triplicate determinations. The stimulation (-fold) of inositol phosphate formation caused by the addition of ]~'Yt is given in parentheses.


or 10 mM in both cases) as divalent cation chelators and have found no changes in basal or fly-stimulated PLC activity. The concentration of CaCI 2 required to adjust the concentration of free Ca 2+ to the desired value is calculated as described by O'Sullivan 44 using the stability constants for EDTA and EGTA given by Bartfai. 45 A computer program written in BASIC to perform these calculations is available from the authors on re- quest.

Analysis of lnositol Phosphates

Incubations are performed and terminated as described above, except that stop solution II is composed of 1 M HC1, 5 mM EGTA, and 23% (v/v) of a liquid pH indicator (Riedel de Hahn, Seelze, Germany, Cat. No. 36803). Using the indicator at higher proportions compromises phase separation. Two hundred microliters of the aqueous phase obtained by phospholipid extraction is then neutralized with approximately 40/xl of 1.5 M KOH containing 75 mM HEPES and applied to columns (0.6 x 1.5 cm) of Dowex 1 resin (C1- form; Sigma, St. Louis, MO, 1-X8-200; converted batchwise to the formate form by washing two times with an equal volume of 1 M NaOH, followed by two washes with an equal volume of 1 M formic acid and washing with water until the pH of the supernatant is at least 6.5.46 Inositol is eluted with 3 ml of water, glycerophosphoinositol with 3 ml of 60 mM sodium formate/5 mM sodium tetraborate, InsP with 3 ml of 200 mM ammonium formate/100 mM formic acid, InsPz with 3 ml of 400 mM ammonium formate/100 mM formic acid, and InsP3 with 3 ml of 1 M ammonium formate/100 mM formic acid.

Comments. The formate form of the resin is kept at 4 ° in distilled water (1 : 1, by volume) until the columns are filled. Although the columns can be reused after regeneration by sequential washing with 8 ml of 2 M ammonium formate/0.1 M formic acid and 10 ml of water, we normally use fresh resin.

Parameters Affecting fly Stimulation of Soluble Phospholipase C Activity

Calcium Ions

Phosphoinositide-specific phospholipases C are dependent on and markedly stimulated by Ca 2÷. Using the conditions outlined above, the

44 W. J. O'Sullivan, in "Data for Biochemical Research" (R. M. C. Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones, eds.), 2nd Ed., p. 423. Oxford Univ. Press, Oxford and New York, 1969.

45 T. Bartfai, Adv. Cyclic Nucleotide Res. 10, 219 (1979). 46 Bio-Rad Ion Exchange Manual, p. 14. Bio-Rad Laboratories, Richmond, California, 1982.

[14] fly SUBUNIT STIMULATION OF PHOSPHOLIPASE C 191

basal soluble PLC activity of HL-60 cells is activated half-maximally and maximally at 300 nM and 10/zM free Ca 2+ , respectively (Fig. 2). In the presence of/37t, half-maximal and maximal activation are observed at 30 nM and 1/zM free Ca z+, respectively. We routinely use 100 nM free Ca 2+ to assay [33 ̀subunit stimulation of soluble phospholipase C.

Salts

MgCI: inhibits both basal and /37t-stimulated PLC activity. The /33' subunit stimulation is inhibited half-maximally at approximately 10 mM and nullified at concentrations of 30 mM or above. NaCI and NaESO4 do not affect basal activity, but they reduce/33' subunit stimulation. Stimulation is half-maximally reduced at approximately 30 mM NaCI and 15 mM Na2SO4 and completely lost at concentrations of 100 mM NaC1 or above and 50 mM NazSO 4 or above.

Detergents

Sodium deoxycholate affects basal PLC activity in a biphasic manner, with stimulation occurring at concentrations of 2.3 mM or below (Fig. 3) and inhibition at higher concentrations (not shown). Stimulation of soluble PLC by/37 subunits is lost at concentrations of deoxycholate of 1.5 mM or higher (Fig. 3). Sodium cholate does not affect basal PLC activity (in the presence of 0.9 mM sodium deoxycholate) at concentrations up to 4.5 mM, but it inhibits/33, subunit stimulation. Half-maximal and maximal inhibition are observed at approximately 2 and 4.5 mM, respectively.

20O0 g

o o 1500

looo

500

o - -~ -8 -7 -6 -5

Ca 2÷ (log M)

FIG. 2. Concentration dependence on Ca 2+ of the stimulation of soluble phospholipase C by [3y subunits. HL-60 cell cytosol (110 tzg protein/assay) was incubated at increasing concentrations of free Ca 2+ in the absence (open circles) or presence (filled circles) of 3.4 izM flYt with phospholipid vesicles containing [3H]PtdlnsP2.


200C

~'~" 15oo ,,.,. +a

~ ,< 1000

500 c :

/ q__oJO

, i , i i

05 1.0 1.5 2.0 Sodium deoxycholate (mM)

21s

FIG. 3. Effect of sodium deoxycholate on basal and/37t-stimulated activity of soluble phospholipase C. HL-60 cell cytosol (190/zg protein/assay) was incubated at increasing concentrations of sodium deoxycholate in the absence (open circles) or presence (filled circles) of 1.7 t~M/3yt with phospholipid vesicles containing [3H]PtdlnsP2. The phospholipid substrate was prepared in the presence of 1 mM deoxycholate. The incubation medium thus contained a minimum of 0.46 mM deoxycholate. Additional deoxycholate was added to the incubation medium to obtain the final assay concentrations given.

Incubation Time

The/3y subunits stimulate PLC without delay. Stimulated PLC activity is linear with time for at least 20 min when assayed at 25 °.

Assay of Particulate Phospholipase C Using Exogenous Radiolabeled Substrate

/3y Stimulation of Phospholipase C Activity from HL-60 Membranes

Frozen HL-60 membranes are thawed and pelleted by centrifugation in a microcentrifuge tube in a JA-20 rotor at 15,000 rpm for I0 min at 4 °. The membranes are then resuspended to approximately 7 mg protein/ml with buffer containing 10 mM triethanolamine hydrochloride, pH 7.5, and homogenized by several passes through a 0.5 × 25 mm needle. Five microliters of this suspension is then added to the PLC assay, which is performed essentially as described above for soluble PLC preparations except that the incubation mixture contains 1.15 mM sodium deoxycholate and is supplemented with 80 mM KCI, 1 mM ATP, and 5 mM MgCI2. The assay is terminated, and the formation ofinositol phosphates is determined as described above.

[14] /3y SUBUNIT STIMULATION OF PHOSPHOLIPASE C 193

C o m m e n t s . Stimulation by /37 t leads to a 2.4- and 14-fold increase in PtdlnsP 2 hydrolysis by HL-60 membranes at 0.3 and 2 .7/zM, respectively (Fig. 4A). Note that saturation of the effect of/37t on inositol phosphate formation is not achieved even at the latter concentrat ion of/3yr. GDP- liganded at blocks stimulation of PtdlnsP2 hydrolysis by /37t (Fig. 4B). The basal particulate PLC activity of HL-60 cells is activated half-maximally and maximally at 3 and 100/zM free Ca 2+, respectively (Fig. 5). In the presence of/37t , half-maximal and maximal activation are observed at 1 and 10 /zM free Ca 2+, respectively. We routinely use 100 nM free Ca 2+ to assay/37 subunit stimulation of particulate PLC.

Although we have not exhaustively studied the application of the protocol to assaying other particulate PLC preparations, this methodology has worked well with membranes of GH4C 1 pituitary cells or L tk- fibroblasts. Membranous PLC can be solubilized using buffer containing 23 mM sodium cholate and then stimulated by /37 subunits as specified by Camps et a l J 9

Assay of Recombinant Phospholipase C-fl Isozymes Using Exogenous Radiolabeled Substrate

Detergent extracts of transfected COS-1 cells are diluted approximately 200-fold with buffer containing 20 mM Tris-HC1, pH 7.5, 10 mM

A 8 125

o o I00

~× 75 ,~_e - - ~ 50 o o

~ & 25

150 B

, II, . . . . . . . . . . . . I~ ~ .~ ..........

120 ~ o "o__ . o__o_..

×-~

4o ~

- m -7 -6 - m -6 -5

Bit (log M) a t (log M)

FIG. 4. Stimulation of the particulate phospholipase C of HL-60 cells by/]Yt and reversal of the stimulation by GDP-liganded a t. (A) HL-60 cell membranes (35/zg protein/assay) were incubated in the presence of increasing concentrations of/3yt with phospholipid vesicles containing [3H]PtdlnsPz (see text for details). (B) HL-60 membranes were incubated in the presence (filled circles) or absence (open circles) of 2/zM/]Yt with increasing concentrations of GDP-liganded at and phospholipid vesicles containing [3H]PtdlnsPz.

2000 o ~

E

o o 1500

× 1000 o c ~ . ~

o o

~ ,~-~ 500

o = "~._c E o~ ~ . ¢ . J O O

~ x

-6"6

- m -8 -7 -6 -5 -4

Ca 2. (log M)

FIG. 5. Concentration dependence on Ca 2+ of stimulation of particulate phospholipase C by fly subunits. HL-60 membranes (35/zg protein/assay) were incubated at increasing concentrations of free Ca 2+ in the absence (open circles) or presence (filled circles) of 3.4 I.~M flYt with phospholipid vesicles containing [3H]PtdlnsP2.

EGTA, 3 mM benzamidine, 43 mM 2-mercaptoethanol, and 0.1 mM phenylmethylsulfonyl fluoride. Five microliters of the preparation is assayed for PLC activity as described above for soluble PLC preparations.

2C

16

1.~

pMT2

I I • pMT2-PLCB I pMT2-PLCB 2


FIG. 6. Effect of f l ) t t o n inositol phosphate formation by recombinant PLC/31 and PLCfl2 expressed in COS-1 cells. COS-1 cells were transfected as indicated with the mammalian expression vector pMT2 without insert, with pMT2-PLCflI, or with pMT2-PLC/3 z. Cell lysates were prepared as described in the text and incubated (0.3/~g protein/assay) in the absence (open bars) or presence (hatched bars) of 2 txM fl'Yt with phospholipid vesicles containing [3H]PtdInsP2.

[15] TURKEY ERYTHROCYTE PHOSPHOL1PASE C 195

Comments. The/37t subunits have no significant effect on the inositol phosphate formation by lysates of cells transfected with vector containing no insert but clearly stimulate the formation of inositol phosphates by lysates of cells transfected with pMT2-PLC/31 and pMT-PLC/32 (Fig. 6). The degree of stimulation is considerably higher for extracts containing PLC/3 2 than for extracts containing PLC/31 (see Camps et al.19).

Acknowledgments

The expert technical assistance of Elke Strohmaier and Susanne Gierschik is greatly appreciated. Studies performed in the authors' laboratory reported herein were supported by grants from the Deutsche Forschungsgemeinschaft and Fritz Thyssen Stiftung. M.C. received a grant from the Commission of the European Communities.

[15] Pur i f ica t ion of G - P r o t e i n - R e g u l a t e d Phospho l ipa se C f rom T u r k e y E r y t h r o c y t e s

By GARY L. WALDO, A N D R E W J. MORRIS,

and T. KENDALL HARDEN

Introduction

Extracellular signaling molecules including a broad range of hormones, neurotransmitters, peptide growth factors, and chemoattractants produce their physiological effects at least in part by activation of an inositol lipid-specific phosphodiesterase, phospholipase C (PLC). l,z The minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate [Ptdlns (4,5)P2] is hydrolyzed by PLC to two intracellular second messenger molecules 1,2-diacylglycerol and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which activate protein kinase C and mobilize intracellular Ca 2+ .

Phospholipase C enzymes have been purified to homogeneity and the cDNAs cloned from a variety of tissues and species, and approximately 16 PLC isozymes have been identified. 3,4 Structural comparison of the proteins led to division into three families designated PLC-/3, -y, and -8. 3`4 Each family has been further divided into subtypes, for example, 131,/39, etc.

t M. J. Berridge, Annu. Rev. Biochem. 56, 159 (1987). z R. M. Michell, A. H. Drummond, and C. P. Downes, "Inositol Lipids in Cell Signalling."

Academic Press, London, 1989. 3 S. G. Rhee, P. G. Suh, S. H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 4 S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992).


196 PHOSPHOLIPASES C [15l

Two categories of cell surface receptors are known to activate PLC by distinct molecular mechanisms .4 Receptors for epidermal growth factor and platelet-derived growth factor span the plasma membrane once and contain a cytoplasmic tyrosine kinase domain. These receptors promote phosphoinositide hydrolysis through a mechanism(s) that involves receptor-catalyzed tyrosine phosphorylation of the PLC-y family of isozymes. Guanine nucleotide binding protein (G-protein)-coupled receptors are proteins that exhibit a seven-transmembrane-spanning motif; a large subset of these regulate inositol lipid hydrolysis through activation of PLC-/3 family isozymes.

The G proteins are heterotrimeric proteins consisting of an a, /3, 7 subunit structure, and four distinct G-protein classes have been identified. 5'6 The function of certain members of the o~-subunit classes (G s and Gi/Go) can be modified by cholera toxin or pertussis toxin, whereas other classes (G~2 and Gq) are not substrates for the bacterial toxins. Evidence has accumulated implicating both pertussis toxin-sensitive and toxin-insensitive pathways in PLC regulation. 7 The identity of the pertussis toxin- sensitive G protein(s) coupled to PLC and its mechanism of regulation are unknown. Receptors that activate PLC through pertussis toxin-insensitive G proteins employ members of the Gq class of G proteins in a mechanism similar to that for G s and hormonally regulated adenylyl cyclase (EC 4.6. I. 1, adenylate cyclase) as well as transducin and retinal cGMP phosphodiesterase. 6,7 Although receptor-promoted activation of PLC apparently primarily involves direct interaction of o/q, all , 0/14, or 0/16 with PLC, other mechanisms may prove important. For example, Camps e t

al . 8 and Boyer e t al . 9 have reported direct activation of specific PLC isozymes by G-protein/37 subunits. Release of/3y-subunits from G proteins that are pertussis toxin substrates likely accounts for pertussis toxin- sensitive activation of PLC.

The turkey erythrocyte offers many advantages as a model system for study of the components of a G protein-regulated PLC. This is a homogeneous cell type widely available in large quantities that expresses a well-characterized PLC activity that is markedly activated by PEv-purinergic receptors in a GTP-dependent manner, l°'H Turkey erythrocytes

5 A. G. Gilman, Annu. Reo. Biochem. 56, 615 (1987). 6 M. I. Simon, M. P. Strathmann, and N. Gautam, Science 252, 802 (1991). 7 T. K. Harden, Ado. Second Messenger Phosphoprotein Res. 26, 11 (1992). s M. Camps, C. Hou, D. Sidiropoulos, J. B. Stock, K. H. Jakobs, and P. Gierschik, Eur.

J. Biochem. 206, 821 (1992). 9 j . L. Boyer, G. L. Waldo, and T. K. Harden, J. Biol. Chem. 267, 25451 (1992). l0 T. K. Harden, L. Stephens, P. T. Hawkins, and C. P. Downes, J. Biol. Chem. 262,

9057 (1987). ii j. L. Boyer, C. P. Downes, and T. K. Harden, J. Biol. Chem. 264, 884 (1989).

[15] TURKEY ERYTHROCYTE PHOSPHOLIPASE C 197

synthesize phosphatidylinositol (Ptdlns) de novo, which then is phosphorylated to phosphatidylinositol 4-phosphate [Ptdlns(4)P] and Ptdlns(4,5)P2. Thus, [3H]inositol can be used to label substrates for study of the PLC in situ. Further, Ptd[3H]Ins(4,5)Pz and Ptd[3H]Ins(4)P can be inexpensively prepared by neomycin affinity chromatography of lipid extracts from [3H]inositol-labeled turkey erythrocytes. 12'13 Three component proteins involved in the inositol phospholipid signaling pathway of turkey erythrocytes have been purified to homogeneity: Gall ,14-16 G-protein fl~/ subunits,9'17 and G-protein-regulated PLC.12'18 The purified PLC, when reconstituted with [3H]inositol-labeled turkey erythrocyte ghosts or plasma membranes devoid of endogenous PLC activity, exhibits the same capacity for receptor and guanine nucleotide regulation as does the enzyme in s i t u ) 8A9 This PLC is a member of the PLC-fl class of isozymes based on the internal amino acid sequence and immunoreactivity) 9 Furthermore, an amino acid sequence deduced from 1722 base pairs of two overlapping turkey erythrocyte mRNA-polymerase chain reaction products beginning 5' of the X region of the enzyme and extending to a sequence that is 3' to the Y region is consistent with a peptide sequence from the purified PLC. This predicted sequence is 79% homologous with mammalian PLC- flZ. Purification of the protein is straightforward, and turkey erythrocytes therefore provide an excellent source for purification of G-protein- regulated PLC.

P r e p a r a t i o n o f 3 H - L a b e l e d P o l y p h o s p h o i n o s i t i d e S u b s t r a t e s

Neomycin affinity resin for the purification of Ptd[3H]Ins(4,5)P2 and Ptd[3H]Ins(4)P from lipid extracts of [3H]inositol-labeled turkey erythrocyte membranes is prepared from glyceryl-coated controlled pore glass beads (pore size 240 ,~, 200-400 mesh, Sigma Chemical Co., St. Louis, MO) by a modification of the method of Schacht. 13 Oxidize 50 ml of glass beads under vacuum with 200 ml of 20 mM NalO4 while stirring constantly

t2 A. J. Morris, G. L. Waldo, C. P. Downes, and T. K. Harden, J. Biol. Chem. 265, 13501 (1990).

13 j. Schacht, J. Lipid Res. 19, 1063 (1978). ~4 G. L. Waldo, J. L. Boyer, and T. K. Harden, this series, Vol. 237 [15]. 15 G. L. Waldo, J. L. Boyer, A. J. Morris, and T. K. Harden, J. Biol. Chem. 266, 14217 (1991). 16 D. H. Maurice, G. L. Waldo, A. J. Morris, R. A. Nicholas, and T. K. Harden, Bioehem.

J. 290, 765 (1993). ~7 j . L. Boyer, G. L. Waldo, T. Evans, J. K. Northup, C. P. Downes, and T. K. Harden,

J. Biol. Chem. 264, 13917 (1989). 18 A. J. Morris, G. L. Waldo, C. P. Downes, and T. K. Harden, J. Biol. Chem. 265,

13508 (1990), 19 G. L. Waldo, A. J. Morris, D. G. Klapper, and T. K. Harden, Mol. Pharmacol. 40,

480 (1991).


at room temperature for 1 hr. Wash the oxidized beads three times in a fritted disk funnel with 1 liter of distilled water. Add 200 ml of 60 mM neomycin sulfate, adjusted to pH 9 with NaOH, to the washed oxidized beads and incubate for 60 min at room temperature. Add 200 mg NaBH 4 to the reaction and incubate for 15 min. Both of these procedures are performed with stirring under vacuum. Wash the neomycin-linked beads three times with 1 liter of distilled water and once with 1 : 1 (v/v) CHC13/ CHaOH. The affinity resin may be stored at - 2 0 ° in 1 : 1 (v/v) CHC13/ CH3OH.

Collect approximately 20 ml of blood from the wing vein of a turkey with a sterile syringe fitted with a 19-gauge needle containing a small amount (I00/zl) of heparin solution (I000 units/ml). Centrifuge the blood at 300 g (Beckman J-6; 1200 rpm at 4 °) for 5 min. Discard the supernatant and buffy coat, and wash the erythrocyte pellet twice by resuspension in 5 volumes (40-50 ml) of cold Dulbecco's modified Eagle's medium (DMEM), followed by centrifugation at 300 g for 5 min at 4 ° and aspiration of the supernatant. Prepare four sterilized glass scintillation vials, each containing 1 ml washed, packed erythrocytes and 2.4 ml of inositol-free DMEM containing 2 mCi of 2-myo-[3H]inositol. Incubate the cells at 39 ° for 18-24 hr, stirring constantly, in a humidified atmosphere of 5% CO2.

Sediment the [3H]inositol-labeled erythrocytes by centrifugation at 300 g for 5 min. Hypotonically lyse the pelleted cells by vortexing for 20-30 sec in 15 volumes of ice-cold 5 mM NaH2PO4, pH 7.4, l mM [ethylene glycol bis(aminoethyl ether)]tetraacetic acid (EGTA), and 5 mM MgCI2 (phosphate lysis buffer). Centrifuge the lysate at 35,000 g (Beckman JA-17; 16,000 rpm) for 10 min at 4 °, discard the supernatant, and wash the pellet three times by resuspension in phosphate lysis buffer followed by centrifugation and aspiration of the supernatant. Resuspend the final pellet with 4 ml of 0.1 M ethylenediaminetetraacetic acid (EDTA) and 4 ml of 0. I M HCI (total volume -12 ml) and prepare lipid extracts of the radiolabeled membranes by adding 3.75 volumes (-45 ml) of 200 • 400 : 5 (v/v) CHCI3/CH3OH/H20, mixing, and incubating at room temperature for 30 min. Add 15 ml of CHC13 and 15 ml of 0.1 M HCI, mix, and centrifuge at 800 g (Beckman J-6; 2000 rpm) for 10 min at room temperature. Discard the aqueous upper phase and transfer the lower organic phase to a clean tube. Concentrate the organic phase to approximately 200/.d by evaporation under a stream of N2 gas while the tube is immersed in a beaker of room temperature water. Dilute the concentrated organic phase with 1 ml CHCIa and 1 ml CH3OH. The lipid extract is now ready for affinity purification of the 3H-labeled polyphosphoinositides.

Load the lipid extract by gravity on a 1-ml column of neomycin affinity resin equilibrated with 1 : 1 (v/v) CHCI3/CH3OH. Wash the column with 2 ml of 1 : 2 (v/v) CHC13/CH3OH followed by 2 ml of 5 : 10 : 2 (v/v) CHC13/


CHsOH/H20 and 20 ml of 0.2 M NH4COOH in 5 : 10 : 2 (v/v) CHCI3/ CHsOH/H20. Monitor the radioactivity eluting from the column and, if necessary, continue washing with the latter solvent until the radioactivity in the eluent decreases to less than 200 disintegrations per minute (dpm)/ /zl. If desired, Ptd[SH]Ins, which represents approximately 90% of total radioactivity applied to the column, can be recovered from this fraction by acetone precipitation and purified using additional steps, lz Elute Ptd [SH]Ins(4)P and Ptd[SH]Ins(4,5)P2 with five 2-ml washes of 0.6 M NH4COOH in 5 : 10 : 2 (v/v) CHC13/CH3OH/H20 and 1.2 M NH4COOH in 5 : 10 : 2 (v/v) CHCI3/CHsOH/H20, respectively. Add 588/~1 of CHC13 and 823/zl of 3 M HCI to each 2-ml peak fraction of 3H-labeled polyphosphoinositide and mix. Centrifuge the samples at 800 g for 10 rain at room temperature to separate the phases. Discard the upper phase and mix the lower organic phase with 2 ml of synthetic upper phase prepared by mixing CHCI3, CH3OH, and 3 M NaC1 in the ratio 1 : I : 0.9 (v/v). Centrifuge the samples, discard the upper phase, and transfer the lower phase containing Ptd[3H]Ins(4,5)P2 or Ptd[3H]Ins(4)P to a screw-capped vial and store at - 70 °. The radiochemical purity of each polyphosphoinositide is over 95%, with a yield of 100-200 p.Ci) 2

Preparation of Unlabeled Polyphosphoinositide Substrates

Neomycin affinity chromatography also can be used to purify microgram amounts of unlabeled polyphosphoinositides from a bovine brain lipid fraction (Folch fraction I) obtained from Sigma. Dissolve the lipid fraction (2 g) in 67 ml CHCI 3 and combine with 33 ml CH3OH. Apply the dissolved lipids at a flow rate of 3 ml/min to a 50-ml bed of neomycin affinity resin (2 × 16 cm) in a solvent-resistant column previously equilibrated with 2 : 1 (v/v) CHCIs/CHsOH. Wash the column with 50 ml of I : 1 (v/v) CHCI3/ CH3OH, followed by 50 ml of 1:2 (v/v) CHC13/CH3OH and 50 ml of 5 : 10 : 2 (v/v) CHCI3/CH3OH/H20.

Elute a fraction containing primarily Ptdlns and phosphatidylserine from the column with approximately 300 ml of 0.2 M NHaCOOH in 5 : 10 : 2 (v/v) CHCI3/CHsOH/H20. Determine the phosphate content of the eluate after evaporation to dryness and wet digestion in perchloric acid. We typically digest 50/zl of the eluate in 500/zl of 60% (w/w) perchloric acid for 30 min at approximately 200 ° in a fume hood. Measure the phosphate content in 10/xl of the digest using the microscale protocol of Itaya and Ui. z° When no phospholipid is detected in the eluate, Ptdlns(4)P is eluted with 240-300 ml of 0.6 M NHaCOOH in 5 : 10 : 2 (v/v) CHC13/CHsOH/ HzO, fractions (8 ml) are collected, the phosphate content determined,

2o K. Itaya and M. Ui, Clin. Chim. Acta 14, 361 (1966).


and the peak fractions pooled. Subsequently Ptdlns(4,5)P2 is eluted with 240-300 ml of 1.2 M NH4COOH in 5 : 10 : 2 (v/v) CHC13/CH3OH/H20 , fractions collected, analyzed, and pooled as described for Ptdlns(4)P. The pooled volume of polyphosphoinositide-containing fractions is typically 80-100 ml.

Extract the lipids from pooled fractions by adding 412/xl/ml of 3 M HCI and 267/A of CHCI 3 , mixing, and centrifuging at 800 g for 10 rain at room temperature to separate the two phases. Remove the upper phase and wash the lower phase once with synthetic upper phase prepared from CHC13, CH3OH, and 3 M NaCI (1 : 1 : 0.9, v/v). Recover the lower phase after mixing and centrifugation, remove the solvent by evaporation, and resuspend the lipids in 10 ml of 100:14:1 (v/v) CHCI3/CH3OH/H20. Determine the phosphate content of the purified lipids and calculate the mass knowing that Ptdlns(4)P and Ptdlns(4,5)P2 contain 2 and 3 mol of phosphate per mole of lipid, respectively. Typical yields are 20-40/xmol of each lipid from 2 g of Folch fraction I. Sodium salts of lipids are isolated by this procedure. The Na + can be replaced with an alternate countercation by evaporation of a portion of the lipid solution to dryness and resuspension in an appropriate volume of CHCI3/CH3OH/HEO (1 : 1 : 0.9, v/v) containing in the aqueous component the countercation of choice (e.g., NH4COOH or HC1 to prepare the NH4 ÷ or H ÷ salts). The lipid is recovered from the lower phase of the mixture. The purity of both labeled and unlabeled lipids can be assessed by thin-layer chromatography using potassium oxalate-impregnated Whatman (Clifton, NJ) PE SIL G (250/zm) plastic-backed silica plates. 2~


The assay is performed in 12 × 75 mm conical polypropylene tubes. Prepare substrate solution for 100 assays by mixing 1/zmol of PtdIns(4,5)P 2 with 2-3/~Ci of Ptd[3H]Ins(4,5)P2 and drying under nitrogen. Add 2.5 ml of 10 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), pH 7.4, to the dried lipids and disperse by briefly sonicating with a probe- type sonicator. Keep the substrate solution on ice.

Combine, on ice, 25/zl of a 4-fold concentrated assay buffer (10 mM HEPES, pH 7.4, 480 mM KC1, 40 mM NaC1, 8 mM EGTA, 23.2 mM MgSO4, and 8.4 mM CaC12), 25/zl of 2% sodium cholate in 10 mM HEPES, and 25/zl of 10 mM HEPES containing 1-5/zl of PLC sample. The reaction is started by rapidly adding 25/zl of substrate, mixing, and transferring the tubes to a 30 ° bath. The final assay mixture contains (final concentra-

21 j . Jolles, H. Zwiers, A. Dekker, K. W. A. Wirtz, and W. H. Gispen, Biochem. J. 194, 283 (1981).


tions) 10 mM HEPES, pH 7.4, 120 mM KC1, 10 mM NaC1, 2 mM EGTA, 5.8 mM MgSO 4 , 0.5% (w/v) sodium cholate, 2.1 mM CaCI2 to give approximately 100 tzM free Ca z+ , 100 tzM Ptdlns(4,5)P2, and 30,000-40,000 dpm Ptd[3H]Ins(4,5)P2 . Samples are incubated for 5-15 min and the reaction terminated by addition of 0.375 ml of 20 : 40 : 1 (v/v) CHCI3/CH3OH/HC! followed by 0.125 ml of CHC13 and 0.125 ml of 0. I M HC1. The samples are mixed and centrifuged at room temperature for 5 rain at 3250 g (Beck- man J-6; 4000 rpm). Four hundred microliters of the upper aqueous phase is removed and quantitated in a liquid scintillation counter.

Comments. Ptdlns(4)P may be substituted for Ptdlns(4,5)P 2 . Quantita- tion of PLC activity in the cytosol and resuspended (NH4)2SO 4 precipitate (see below) is problematic. This may follow from a low enzyme concentration in the samples, contaminating phospholipids that inhibit enzyme activity or decrease the specific activity of the substrate, Ca 2 ÷ binding proteins that alter the free Ca 2+ concentration in the assay, or other inhibitory factors present in the crude sample. Quantitation of activity improves following elution of the enzyme from Q-Sepharose.

The assays are carried out at subsaturating substrate concentrations and are not representative of Vmax values for the PLC determined by initial rates. Maximal PLC activity occurs in a range of cholate concentrations from 0.2 to 0.5% (w/v). The optimal cholate concentration is influenced by the total lipid concentration in the assay (lower substrate concentrations require slightly lower cholate concentrations for maximal activity).

Purification of Phospholipase C from Turkey Erythrocytes

Preparation of Turkey Erythrocytes

Collect approximately 20 liters of blood (requires 1-2 hr for one person) from freshly killed turkeys directly into an anticoagulant solution containing 95 mM EDTA, 5 mM EGTA, and 5 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.4 (100 ml of anticoagulant per 2 liters of blood). Filter the blood immediately through cheesecloth and store on ice for transporta- tion to the laboratory. Perform all subsequent operations at 4 ° . Sediment erythrocytes in I-liter bottles by centrifugation for 10 min at 525 g (Beck- man J-6, 1600 rpm) with the brake set at half-maximum. This brake setting reduces resuspension of the erythrocyte pellet caused by turbulence occurring at more rapid deceleration rates. Aspirate and discard the supernatant and lymphocyte-rich buffy coat. Wash the erythrocyte pellet twice by resuspension in 2-3 volumes of 2.5 mM HEPES, pH 7.4, 150 mM NaCI, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM benzamidine (whole cell wash buffer) followed by centrifugation and aspiration of the supernatant as described above. The sedimented, washed


erythrocytes (final volume 8-10 liters) may be stored overnight at 4 °. However, higher yields of PLC activity are normally obtained by immediately continuing with NE cavitation of the erythrocytes. The preparation of washed erythrocytes from 20 liters of whole blood typically requires 3-4 hr for one investigator using a single centrifuge.

Preparation of Turkey Erythrocyte Cytosolic Fraction

Nitrogen cavitation disrupts the erythrocyte plasma membrane relatively more selectively and more efficiently than does sonication, and it requires minimal dilution of the erythrocytes compared with hypotonic lysis protocols. Nuclei are stabilized during cavitation by the addition of Mg 2+ . Thus, add 50 ml of 200 mM MgCI2 in whole cell wash buffer to 1 liter of sedimented erythrocytes (10 mM MgCI2, final concentration) and transfer to the pressure chamber of a Parr cell disruption bomb (Model 4636, Parr Instrument Co., Moline, IL). Pressurize the chamber to 1300 psi with N 2 gas and allow the erythrocytes to equilibrate for 30 min while stirring constantly on a magnetic stir plate. Discharge the contents of the bomb into 1 liter of lysis buffer (5 mM Tris, pH 7.4, 5 mM MgCI z , 1 mM EGTA, 0.1 mM benzamidine, and 0.1 mM PMSF) while maintaining the pressure in the bomb.

Centrifuge the disrupted cells at 3250 g for 10 min with maximum brake (Beckman J-6; 4000 rpm). Collect the resulting dark red supernatant, containing cytosol and plasma membranes, and discard the pellet (predominately nuclei). Centrifuge the low-speed supernatant at 28,000 g for 20 min with the brake set at half-maximum (Beckman JA-14 rotor; 13,500 rpm). Carefully collect the upper 60-70% of the 28,000 g supernatant as the cytosolic fraction. The membrane pellet is difficult to identify at this stage owing to the dark red color of the supernatant. Proceed cautiously to avoid contaminating the cytosolic fraction with membranes. The easily resuspended pellet may be saved for plasma membrane purification. 14 Two investigators, one managing N2 cavitation and one managing centrifugation, can prepare the cytosolic fraction from the washed erythrocyte pellet in 7-8 hr using two cell disruption bombs, two Beckman J-21 centrifuges equipped with JA-14 rotors, and one J-6 centrifuge.

Ammonium Sulfate Precipitation

Phospholipase C can be easily concentrated from the cytosolic fraction (12-15 liters) and separated from hemoglobin by (NH4)2SO4 precipitation. Note that solid (NH4)eSO 4 salt added directly to cytosol frequently causes partial precipitation of the hemoglobin owing to localized high (NH4)2SO 4 concentrations. Therefore, dissolve 486 g of (NH4)2SO 4 in 1 liter of lysis


buffer, rapidly combine the (NH4)2SO 4 solution with 1 liter of cytosol, and allow precipitation to proceed for 10 min (alternatively, the precipitation may be left overnight at 4°). The final (NH4)2SO 4 concentration is 40% saturation (243 g added to 1 liter). Collect the precipitate by centrifugation at 28,000 g for 20 min (Beckman JA-14; 13,500 rpm) with maximum brake. Discard the supernatant and wash the precipitated protein pellet by resuspending with 6 liters of 40% (saturated) (NH4)2SO4 in 20 mM Tris, pH 7.4, 5 mM EDTA followed by homogenization in a plexiglass homogenizer (4.8 cm i.d. x 65 cm) of the type described by Stroobant and Scarborough) 2 Centrifuge the precipitate as described above, discard the supernatant, and resuspend the pellet in a small volume of 40% saturated (NH4)2SO4 buffer.

The washed (NH4)2SO 4 precipitate may be stored frozen at - 7 0 ° for 6 months with no detectable loss of PLC activity. Two researchers working continuously can collect and process 20 liters of blood and prepare a r e s u s p e n d e d (NH4)2SO 4 precipitate from the cytosolic fraction for loading on the first chromatography column within 12 hr. This approach normally results in the highest yields of PLC activity. Slightly lower yields are obtained when washed erythrocytes are stored overnight at 4 ° or when the (NH4)2SO 4 precipitate is stored frozen at -70 °.

Q-Sepharose Anion-Exchange Chromatography

All purification procedures are performed at 4 ° . Rapidly thaw the (NH4)2SO 4 precipitate or continue with freshly prepared (NH4)2SO 4 precipitate. Centrifuge the precipitate at 28,000 g for 20 rain and carefully discard all supernatant. Any residual (NH4)2504 solution associated with the pellet will require large volumes of resuspension buffer to dissolve the PLC. Resuspend the pellet by homogenization (plexiglass homogenizer) in 9 liters ef 20 mM Tris, pH 7.4, I mM EDTA, 1 mM dithiothreitol (DTT), 0.1 ~M benzamidine, and 0.6 mM PMSF (buffer A). Centrifuge the solution at 28,000 g for 30 min. Collect the supernatant and dilute with buffer A until the conductivity is less than that of a 100 mM solution of NaC1 [the final volume may range from 10 to 25 liters depending on the amount of (NH4)2SO 4 remaining in the pellet prior to resuspension].

Pour the resuspended protein into a 3-liter glass funnel with a 15 cm diameter coarse porosity fritted disk base containing a bed of 800 ml of Q-Sepharose fast flow resin (Pharmacia, Piscataway, N J) equilibrated with buffer A. Pass all of the protein solution (approximate flow rate 6 liters/ hr by gravity) through the funnel. Suspend the resin in buffer A, pour

22 p. Stroobant and G. A. Scarborough, Anal. Biochem. 95, 554 (1979).


into a 5 x 60 cm chromatography column, and pack at 6-7 ml/min with 1 liter of buffer A. Adjust the flow adapter to the surface of the resin and elute the column at 5 ml/min with a 2-liter linear gradient from 0 to 1 M NaCI in buffer A (Fig. 1A). Collect 14-ml fractions of the eluate. The PLC activity typically elutes from the Q-Sepharose column at 270 mM NaCI. Pool fractions containing PLC activity (pooled volume 150-175 ml) and desalt by gel-filtration chromatography at 7 ml/min on a 5 x 29 cm (570 ml) Sephadex G-25 column equilibrated with 20 mM Tris, pH 7.5, 1 mM DTT, 10 mM K2HPO4, 0. I mM benzamidine, and 0.6 mM PMSF (buffer B). Collect the void volume of the Sephadex G-25 column (activity measurements are not necessary).

Hydroxylapatite Chromatography

Apply the desalted Q-Sepharose pool at 4 ml/min to a 2.5 x 37 cm hydroxylapatite column (Bio-Rad HTP; Bio-Rad Laboratories, Richmond, CA) equilibrated with buffer B (Fig. IB). Wash the column with 100 ml of buffer B followed by 200 ml of buffer B containing 200 mM K2HPO 4 . Elute the PLC activity from the column with a l-liter linear gradient of 200-600 mM K2HPO4 in buffer B. Collect 11-ml fractions. The PLC activity elutes at approximately 335 mM K2HPO 4. Pool fractions containing PLC activity (pooled volume 90-100 ml) and desalt as described above on a Sephadex G-25 column (5 x 29 cm) equilibrated with buffer A containing 100 mM NaCI. Collect the void volume.

Heparin-Sepharose Chromatography

Apply the desalted hydroxylapatite pool at 1 ml/min to a 1 x 18 cm heparin-Sepharose 4B (Pharmacia, Piscataway, NJ) column (Fig. 1C) equilibrated with buffer A containing 100 mM NaCI, 2/zg/ml leupeptin, and 2/~g/ml aprotinin. Wash the column with 20 ml of equilibration buffer and elute with a 210-ml linear gradient from 100 to 900 mM NaCI in buffer A containing leupeptin and aprotinin. Collect 2-ml fractions. The PLC activity elutes at approximately 450 mM NaCI. Pool the fractions containing PLC activity (pooled volume 15-20 ml).

Sephacryl S-300 HR Gel-Filtration Chromatography

Apply the heparin-Sepharose pool at 2 ml/min directly to a 2.6 x 91 cm Sephacryl S-300 (Pharmacia, Piscataway, NJ) column (Fig. ID) equilibrated with buffer A containing 100 mM NaCI, 2/~g/ml leupeptin, and 2 ~g/ml aprotinin. Collect 5-ml fractions and pool the fractions containing PLC activity (pooled volume 30-35 ml). The elution volume (Ve) of the partially purified PLC is approximately 275 ml.


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FIG. 1. Chromatographic steps for the purification of G-protein-regulated PLC from turkey erythrocyte cytosol. Turkey erythrocytes were disrupted by N 2 cavitation, an (NH4)2SO 4 precipitate prepared from the cytosol as described in detail in the text, and PLC purified from the resuspended precipitate by chromatography through five sequential columns. The PLC activity in 2-p.l samples of fractions eluting from the chromatography columns was measured in the presence (O) or absence ((3) of 0.5% sodium cholate. The column type, fraction volume, and duration of assay were as follows: (A) Q-Sepharose, 14 ml/fraction, l0 min assay; (B) hydroxylapatite, l 1 ml/fraction, 10 min assay; (C) heparin-Sepharose, 2 ml/fraction, 5 min assay; (D) Sephacryl S-300 HR, 5 ml/fraction, 5 rain assay; (E) Mono Q FPLC, 0.3 ml/fraction, 2 min assay. Absorbance at 280 nm is illustrated by the dashed line.


TABLE I PROPERTIES OF TURKEY ERYTHROCYTE PHOSPHOLIPASE C a

Property Comments

Subcellular distribution >83% cytosolic SDS-PAGE Mr 150,000

Substrate preference (30 °) PtdIns(4,5)P2 Ptdlns(4)P PtdIns Other phospholipids

Ca2+-dependent enzyme pH optimum Products formed

pH 4.0; PtdIns(4,5)P2

pH7.5; Ptdlns(4,5)P 2

pH 4.0; PtdIns(4)P

pH 7.5; Ptdlns(4)P Immunoreactivity

Antisera (Western blot) X-region polyclonal Yes Y-region polyclonal Yes Turkey erythrocyte PLC Yes

polyclonal PLC-fl mono or polyclonal PLC-y mono or polyclonal

Immunoprecipitated Enzyme activity inhibited

G Protein activators Gqa/Glia Yes Gia/Goa No f17 subunits Yes

GTP-dependent receptor regulation P2v-purinergic receptor Yes

K m 5-40/~M; Vma x 10-80/zmol/min/mg K m 5-40/zM; Vma x 10-80/zmol/min/mg Poor substrate Not substrates Yes pH 4.0

68% lns(1,4,5)P 3 32% Ins(1 ;2 cyclic 4,5)P 3 98% lns(1,4,5)P 3 2% Ins(1 ;2 cyclic 4,5)P 3 47% Ins(1,4)P2 53% Ins(1 ;2 cyclic 4)P 2 99% Ins(1,4)P2

No No Yes (T.PLC polyclonal) Yes (T.PLC polyclonal)

a See Refs. 9, 12, 15, 18, and 19 for further details.

Fast Protein Liquid Chromatography Mono Q Anion Exchange

Apply the pool o f P L C act ivi ty eluting f rom the S-300 co lumn to a M o n o Q 5/5 F P L C (fast pro te in liquid c h rom a t og raphy ) co lumn (Pharmacia , P i sca taway , NJ) (Fig. 1E) equil ibrated with buffer A conta in ing 100 m M NaC1, 2 t~g/ml leupeptin, and 2 / z g / m l aprotinin. W a s h the co lumn with 5 ml o f equil ibrat ion buffer at 0.5 ml /min and elute with a 20-ml l inear gradient f rom 100 to 450 m M NaCI. Col lect 0.3-ml fract ions. The 150-kDa

[16] D E T E R M I N A T I O N O F P H O S P H O L I P A S E C A C T I V I T Y 207

PLC elutes at 270 mM NaCI in a volume of approximately 1.5 ml and is usually greater than 95% pure. Minor contaminants appearing between 97 and 116 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) are recognized by antisera raised against the 150-kDa PLC excised from a polyacrylamide gel and thus appear to be degradation products of the 150-kDa protein.

Conclusion

The purification procedure described in this chapter yields 200-500 /zg of PLC (from 17 separate purifications, the highest yield was about 1 mg and the lowest yield about 100/xg) from 20 liters of whole blood. A summary of properties of the purified PLC are presented in Table I. Dilute the purified PLC 1 : 1 (v/v) with buffer A containing 2/~g/ml leupeptin, 2/xg/ml aprotinin, and 40% glycerol. The enzyme may be stored in small aliquots at - 7 0 ° for at least 6 months without loss of activity. Activity diminishes with repeated freeze-thaw cycles or with prolonged storage at 4 °. Further properties of the enzyme have been described. 9'12'15'18'~9

[16] P h o s p h o l i p a s e C Ac t iv i t y in Dictyostelium discoideum Using E n d o g e n o u s N o n r a d i o a c t i v e Phospha t idy l inos i to l

4 , 5 - B i s p h o s p h a t e as S u b s t r a t e

By ANTHONY A. BOMINAAR and PETER J. M. VAN HAASTERT

I n t r o d u c t i o n

Inositol phosphates have received particular interest as second messengers in signal transduction. The commonly accepted inositol phosphate in signaling is inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] ,1,2 which is generated from phosphatidylinositol 4,5-bisphosphate [Ptdlns(4,5)P2] by the action of the enzyme phospholipase C. 3 In the same reaction the second messenger 1,2-diacyl-sn-glycerol is formed. Ins(1,4,5)P 3 has been shown to release Ca 2+ from internal stores. 4,5 For many higher eukaryotes the activity of

t M. J. Berridge and R. F. Irvine, Nature (London) 317, 315 (1984). 2 R. F. Irvine, in "Inositol Lipids in Cell Signalling" (R. H. Mitchell, A. H. Drummond,

and C. P. Downes, eds.), p. 135. Academic Press, London, 1989. 3 S. G. Rhee, P. G. Suh, S. H. Ryu, and S. Y. Lee, Science 244, 546 (1989). 4 M. J. Berridge and R. F. Irvine, Nature (London) 341, 197 (1989). 5 G. N. Europe-Finner and P. C. Newell, Biochim. Biophys. Acta 887, 335 (1986),

Copyright © 1994 by Academic Press, lnc. M ETHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved.


phospholipase C has been shown in vitro, and evidence has been obtained that in several systems the activity is regulated via G proteins (for review, see Meldrum et al.6). Assays for the determination of phospholipase C make use of either commercially available [3H]Ptdlns(4,5)P27 o r start with equilibrium labeling of cells to obtain radiolabeled substrate. 8 Subse- quently the production of radiolabeled inositol phosphates is determined.

In this chapter we describe a method for the analysis of phospholipase C activity in vitro, using endogenous unlabeled Ptdlns(4,5)P2. The Ins(1,4,5)P3 produced is detected using a specific Ins(1,4,5)P3-binding protein from bovine liver. Some characteristics of Dictyostelium phospholipase C and its regulation by the receptor and G-protein agonists cAMP and GTPyS are shown. This method complements other receptor/G protein/ effector assays in Dictyostelium described elsewhere in this s e r i e s . 8a


Principle

The phospholipase C assay described here is based on the fact that phospholipase C is inactive in the absence of Ca 2+. Cells are lysed in the presence of the Ca 2÷ chelator EGTA, and subsequently a fixed amount of Ca 2+ is added for a fixed period of time. On addition of Ca 2+ to the lysate Ins(l,4,5)P3 is produced from endogenous substrate. The Ins(1,4,5)P 3 produced is determined by an isotope dilution assay and is a measure for the activity of phospholipase C. To assay possible effects of G proteins, guanine nucleotide analogs can be included during lysis. To determine the effects of the cAMP surface receptor, cells are stimulated with cAMP prior to lysis.

The phospholipase C assay procedure is outlined in Fig. 1. The conditions and incubation times given here have been optimized for Dictyostel- ium discoideum and should be reconsidered if any other species is used.

Materials

Dictyostelium discoideum cells are starved for 4 hr by shaking at 150 rpm in 10 mM sodium/potassium phosphate buffer to acquire aggregation competence, washed, and resuspended in 40 mM HEPES-NaOH, pH 6.5, at 5 × 107 cell/ml

6 E. Meldrum, P. J. Parker, and A. Carozzi, Biochim. Biophys. Acta 1092, 49 (1991). 7 I. Litosch and J. N. Fain, J. Biol. Chem. 2611, 16052 (1985). 8 j . j . Baldassare and G. J. Fisher, J. Biol. Chem. 261, 11942 (1986). sa B. E. Snaar-Jagalska and P. J. M. Van Haastert, this series, Vol. 237 [30].

[16] DETERMINATION OF PHOSPHOLIPASE C ACTIVITY 209

Cells in appropriate buffer at 5x107 cells/ml

I 5 . 9 ~ EGTA

( + / - s t i m u l i )

I 20"

+PeA + 5.9 mM Ca 2÷

20"

+PCA Sample B Sample A

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A,B i

+ KHC% 15'

5' 850 g

20 ~i supernatant

20 ~i [~H]Ins(l,4,5)P3~ 20 ~i IAB

--Ins(l,4,5)P a BP

i0'

O 2' 14,000 g

l resuspend pelle~t in 100 ~i H20

l add scintillation cocktail

1 cpm A! cpm B

±

pmol A,~ pmol B

PLC = (B-A)/t

FIG. I. Schematic representation of the protocol for the phospholipase C assay.


Microtiter plates 96 wells, V-shaped) (Greiner B.V., Alphen a/d Rijn, The Netherlands)

CaC12, 59 mM EGTA, 118 mM Perchloric acid (PCA), 3.5% (v/v) Syringes, 1 ml (Terumo Europe, Leuven, Belgium) Hypodermic needles (Terumo Europe, Leuven, Belgium) Nuclepore polycarbonate filters (pore size 3 tzm) (Nuclepore, Roch-

ester, NY) Parafilm (American National Car, Greenwich, CT)

Standard Procedure

Preparations

1. Prepare the syringes by placing a piece of Nuclepore filter between the barrel and the needle and taping the needle to the barrel with Parafilm. Prepare a separate syringe for every sample. (Syringes and needles can be reused several times.)

2. To prepare the microtiter plates, the most convenient way is to work in rows. Place 5/zl of 59 mM CaC12 in every well and 50/zl of 3.5% PCA in the wells of every second row. Thus the plate will have Ca z+ in rows A, C, E, and G and both Ca z÷ and PCA in rows B, D, F, and H.

Assay

3. At t = 0 sec, add 7.5/zl of the 118 mM EGTA solution plus 7.5/xl of stimulus (buffer for control) to 135 tzl of cell suspension.

4. Transfer the mixture to the syringe and lyse, after 20 sec, by forcing the suspension through the needle (and hence through the membrane).

5. At 10 sec after lysis (t = 30 sec) transfer 50/zl of the lysate to the first well of the first row with only CaCI 2 and 50/zl to the first well of the second row.

6. Terminate the reaction in the first well after 20 sec (t = 50 sec) by addition of 50/zl of PCA.

Completion

7. Store the samples at - 20 ° or assay them directly for Ins(1,4,5)P 3

levels (see below).

Determination of Inositol 1,4,5-Trisphosphate Levels

The following is basically the standard procedure for the mass determination of Ins(1,4,5)P3 levels using an isotope dilution assay. The procedure


is derived from the assays as described by Palmer et al. 9 and Van Haastert. ~0

Materials

50% Saturated KHCO 3 (112 g/liter) Ins(1,4,5)P 3 assay buffer (IAB): 100 mM Tris-HC1, 4 mg/ml bovine

serum albumin (BSA), 4 mM EDTA, pH 9.0 Ins(1,4,5)P3-binding protein from bovine liver (see below) [3H]Ins(1,4,5)P 3 (20-60 Ci/mmol) diluted to 100,000 cpm/ml in water

(Amersham Radiochemical Centre, Amersham, UK) Ins(1,4,5)P3, 10 -5 M (Boehringer Mannheim, Mannheim, Germany)

Procedure

Preparations

1. Neutralize all samples by adding 30/A of a 50% saturated KHCO3 solution and allow to stand for 15 min to remove the CO2 produced.

2. Centrifuge the microtiter plates in a swing-out microtiter plate rotor for 5 min at 850 g at 4°C to precipitate the potassium perchlorate formed.

3. Randomly check the pH of a few samples using indicator paper. The pH should be between pH 7 and pH 9. If any sample is below pH 7 check all samples. If the pH is too low the binding capacity of the binding protein will be decreased, giving erroneous results. Store the samples on ice until used.

4. Prepare Eppendorf tubes containing 20/zl [3H]Ins(1,4,5)P3 (-2000 cpm) and 20/zl of IAB per sample.

5. Add 20/~1 of neutralized sample to the label/buffer mix. 6. To determine the maximal binding, 20 /zl of neutralized, PCA-

treated buffer without cells is used (this sample is called Co). To determine background (B/), 20/zl of 10 -5 M Ins(1,4,5)P3 is used instead of the sample.

Assay

7. Add 20/zl of binding protein and incubate for 10 min on ice. 8. Centrifuge the sample for 2 min at 14,000 g in a tabletop centrifuge;

aspirate the supernatant.

Completion

9. Resuspend the pellet in 100/zl water and either add 1.3 ml scintillation cocktail (if the scintillation counter can handle Eppendorf tubes) or

9 S. Palmer, K. T. Hughes, D. Y. Lee, and M. J. O. Wakelam, Cell. Signalling 1, 147 (1989). 10 p. j. M. Van Haastert, Anal. Biochem. 177, 115 (1989).


transfer 90/xl to a scintillation vial and add 1.5 ml scintillation cocktail. This will give the counts per minute (cpm) for each sample (Cx).

10. The amount of Ins(1,4,5)P 3 is determined using the following equation:

pmollns(1,4,5)P3(C-~xx -Bl ) = i x

X can be determined from a standard curve with known amounts of Ins(1,4,5)P3.

11. Phospholipase C activity (in pmol/min) is calculated from the difference between the sample incubated with Ca/+ (sample A) and the sample quenched directly (sample B) using the following equation:

PLC activity = (pmol A - pmol B)/t with t being the incubation time in the presence of Ca 2+ (in min).

Results In Table I some typical primary data are shown for an experiment

determining the basal and cAMP-stimulated activity of phospholipase C in Dictyostelium. In Fig. 2 the Ins(1,4,5)P3 production as a function of time and the stability of the enzyme in the absence of Ca 2+ are given. From Fig. 2A it is clear that the production of Ins(l,4,5)P 3 is linear with time up to 1 min of incubation in the presence of Ca a+ . Figure 2B shows that readdition of Ca 2÷ should occur within 20 sec. These data apply to

TABLE I DATA OBTAINED FROM STANDARD PHOSPHOLIPASE C ASSAY a

Basal activity Stimulated activity

Parameter 1 2 3 4 5 6

Primary data (cpm) A(t) 281.0 273.22 263.0 246.6 249.2 243.4 B(to) 574.3 518.8 624.2 629.2 422.6 544.0

Calculated Ins(l,4,5)P3 (pmol) A 5.68 6.35 7.46 10.18 9.64 10.93 B 0.51 0.77 0.33 0.32 1.51 0.64

Phospholipase C activity (pmol/20 sec) A - B 5.17 5.59 7.13 9.87 8.13 10.29 Average -+ SD 5.96 -+ 1.03 9.43 - 1.15

Cells were treated with buffer or stimulated with 10 -6 M cAMP prior to lysis and incubated under standard phospholipase C assay conditions. The value of Co for this experiment was 766.6 + 8.1, BI was 195.5 -+ 10.2, and the standard curve-derived factor X was 1.0.


Dictyostelium discoideum phospholipase C only, and it cannot be ruled out that in other organisms the situation is less critical. Figure 2C shows that following stimulation with the receptor agonist cAMP or lysis in the presence of GTPyS an increased activity of phospholipase C is observed.

The major source of variance in the data obtained is the determination of the Ins(1,4,5)P3 levels. First, the best results are obtained if the difference between specific binding (Co - B/) and nonspecific binding (BI) is as large as possible. A fair preparation of binding protein gives a Bl of approximately 200 cpm and a Co of 800 cpm. Second, the most accurate part of the displacement curve of [3H]Ins(1,4,5)P 3 by the Ins(1,4,5)P 3 from the sample is between 0.5 and 5 pmol Ins(1,4,5)P3/sample. If Cx gets close to either C o or Bl, little changes in counts per minute result in dramatic changes in the calculated amount of Ins(1,4,5)P 3 . A good preparation of Ins(1,4,5)P3-binding protein allows determination of changes of as little as 25% in phospholipase C activity, if cell density and/or sample size are chosen in such a way that the linear range between 0.5 and 10 pmol is used optimally.

Note. If the final free calcium concentration in the phospholipase C assay is in the millimolar range (e.g., in case of Ca 2+ dose-response experiments) it is advisable to include EGTA in the IAB to reduce the amount of Ca 2 + in the assay. The affinity of the Ins(1,4,5)P3-binding protein is modulated by Ca 2+, and high levels of Ca 2+ reduce the total binding.

Identification of Reaction Product as Inositol 1,4,5-Trisphosphate

When the assay is used for the first time for a certain cell type it is recommended to analyze the reaction product. In some systems Ins(1,4,5)P 3 is not the only substance cross-reacting with the binding protein (A. A. Bominaar and P. J. M. Van Haastert, unpublished results).

Two control experiments have been performed to demonstrate that in Dictyostelium the compound cross-reacting with the Ins(1,4,5)P3-binding protein is authentic Ins(1,4,5)P 3. In the first experiment a sample from the phospholipase C assay is mixed with authentic [3H]Ins(1,4,5)P3 and incubated with specific Ins(1,4,5)P3-degrading enzymes. If the cross-reactivity is not due to Ins(1,4,5)P3 it is very unlikely that it will be degraded to the same extent as Ins(1,4,5)P 3 by these enzymes. In the second experiment cochromatography on a high-performance liquid chromatography (HPLC) ion-pair system is used as a criterion for identity.

Materials Experiment 1

[3H]Ins(1,4,5)P 3 Dowex A-xl, formate form (Bio-Rad, Richmond, CA)

30 60

T ime . s

"T

E=

~: 75,

150"

150-

E

E

~ 75

, i

~ o c

6G 1io T ime . s

'T, ._= E

,T, 3 0 0 o~

E m o E Q.

150

cI ~ r

IT

+ * O.

k -


Ins(1,4,5)P 3 5-phosphate isolated from rat brain ~ Ins(1,4,5)P3 1-phosphatase isolated from Dictyostelium discoideum 12 MgClz, 20 mM Neutralized sample from the phospholipase C assay, the Ins(1,4,5)P 3

content of which has been determined using the isotope dilution assay

Procedure

1. Mix 10 /zl from the phospholipase C assay with 5 tzl of 20 mM MgC12 containing 1000 cpm Ins(1,4,5)P3 and a second sample of 10/xl with 5/zl of 20 mM MgC12.

2. Both samples are incubated with 5 tzl of either the 5- or l-phosphatase. The reaction is quenched with chloroform/methanol/HCl (20 : 40 : 1, v/v) for the samples with [3H]Ins(1,4,5)P 3 and by heating to 100 ° for 2 min for the other samples. The reaction time depends on the activity of the enzyme and is chosen in such a way that approximately 50% of the substrate is degraded.

3. The label-containing samples are analyzed for Ins(1,4,5)P 3 degradation on Dowex columns.13

4. The other samples are assayed for Ins(l,4,5)P3 content using the isotope dilution assay.

5. The extent of degradation of both [3H]Ins(1,4,5)P 3 and cross-reactivity are compared.

If the cross-reacting compound is Ins(1,4,5)P3 it should be degraded to the same extent as the [3H]Ins(1,4,5)P 3. The inset of Fig. 3 shows the results of such an experiment with Dictyostelium discoideum material.

I1 B. Verjans, R. Lecocq, C. Moreau, and C. Erneux, Eur. J. Biochem. 204, 1083 (1992). 12 A. A. Bominaar, P. Van Dijken, R. Draijer, and P. J. M. Van Haastert, Differentiation

46, 1 (1991). ~3 M. M. Van Lookeren Campagne, C. Erneux, R. Van Eijk, and P. J. M. Van Haastert,

Biochem. J. 254, 343 (1988).

FIG. 2. Characteristics of Dictyostelium discoideum phospholipase C. (A) Time course of incubation in the presence of Ca 2+. Cells were lysed and Ca -,+ was added at 10 sec after lysis (t = 0); samples were taken for Ins(1,4,5)Ps determination at the indicated time points. (B) Stability of phospholipase C in the absence of Ca -,+ . Cells were lysed and Ca 2+ was added back to the lysate at the indicated time points. Subsequently reactions were allowed to proceed for 20 sec before being quenched. (C) Effects of cAMP, GTP3,S, and GDP/3S on phospholipase C activity. Cells were stimulated for 20 sec with the indicated substances prior to lysis and incubated under standard phospholipase C conditions. Asterisks (*) mark activities significantly above the control (p < 0.05). Data shown are means (+-SEM) of three independent experiments performed in triplicate.

216 PHOSPHOLIPASES C [16J

'N ° ° g

c3

45 50 55

Fraction

FIG. 3. Identification of the cross*reactivity in the isotope dilution assay as Ins(],4,5)P~. HPLC profile of cross-reactivity (©, unstimulated cells; 0 , cells stimulated with cAMP prior to lysis) compared to authentic standards [A, Ins(1,4)P2 ; B, Ins(1,4,5)P3 ; C, Ins(1,3,4,5)P4]. Inset: Degradation of cross-reactivity (hatched bars) and [3H]Ins(1,4,5)p 3 (open bars) by rat brain 5-phosphatase (5) and Dictyostelium 1-phosphatase (1).

The degradations of cross-reactivity and radiolabeled Ins(1,4,5)P 3 are almost identical.

Materials Experiment 2

HPLC apparatus Lichrosorb RP-18 reversed phase column (10/.tm) Tributylarnmonium phosphate, 0.1 M, pH 6.5 (TBAP) Equilibration buffer: 10 mM TBAP, 25% methanol, pH 6.5 Radiolabeled inositol phosphate standards [for Ins(1,4,5)P 3 the 32p

form is recommended, but this is not readily available in some coun- tries]

Neutralized sample from the phospholipase C assay, the Ins(1,4,5)P 3 of which has been determined using the isotope dilution assay

Procedure

1. Equilibrate the column in equilibration buffer. 2. Mix approximately 20 pmol Ins(1,4,5)P 3 from a phospholipase C

experiment with the radiolabeled standards (1000 cpm each) and 500 tzl of 0.1 M TBAP.

3. Load the sample on the column and elute isocratically with equilibration buffer (1 ml/min).


4. Collect 0.5-ml fractions. 5. Mix 0.25 ml of each fraction with scintillation cocktail and quantify

the radioactivity in a scintillation counter. 6. Lyophilize the remainder of the fractions and resuspend them in

100 tzl of 100 mM Tris; the final pH of the sample will be approximately pH 9.0.

7. Determine the amount of cross-reactivity for each fraction using the isotope dilution assay.

If the observed cross-reactivity is due to Ins(1,4,5)P3, radiolabeled Ins(1,4,5)P 3 and cross-reactivity should elute with identical retention times. In Fig. 3 an example is given for Dictyostelium discoideum material before and after stimulation of phospholipase C.

In conclusion, the compound produced in the phospholipase C assay (i) cross-reacts with the highly specific Ins(1,4,5)P3-binding protein from bovine liver, (ii) is degraded to approximately the same extent as authentic Ins(1,4,5)P 3 by Ins(1,4,5)P3 5- and 1-phosphatases, and (iii) cochromato- graphs with authentic Ins(1,4,5)P 3 on a RP-18 HPLC system.

Isolation of Bovine Liver Inositol 1,4,5-Trisphosphate-Binding Protein

The procedure used here is a version of the isolation protocol described by Baukal et al. TM for preparing the Ins(1,4,5)P3-binding protein from bovine adrenal glands that has been adapted for bovine liver.15

Materials

Bovine liver, 500 g (fresh from the slaughterhouse) Tissue homogenizer NaHCO3, 20 mM, 2 liters Tris/HC1, 20 mM, pH 7.5 (at 4°), 0.5 liter Refrigerated centrifuge permitting 50-ml centrifuge tubes to be spun

at 20,000 g

Procedure

1. Keep everything on ice. 2. Cut the liver into small pieces and homogenize further in the homog-

enizer in NaHCO3. Use approximately 1 liter of buffer for every 500 g.

14 A. J. Baukal, G. Guillemette, R. Rubin, A. Spat, and K. J. Catt, Biochem. Biophys. Res. Cornmun. 133, 532 (1985).

15 G. Guillemette, T. Balla, and A. J. Baukal, A. Spat, and K. J. Catt, J. Biol. Chem. 262, 1010 (1987).


3. Centrifuge the homogenate for 10 rain at 500 g at 4°C to remove larger material.

4. Take the supernatant and centrifuge for 20 min at 20,000 g at 4°C. 5. Discard the supernatant and resuspend the pellet to half of the

original volume in NaHCO 3 buffer and centrifuge for 10 rain at 20,000 g. 6. Discard the supernatant and resuspend the pellet in 15 volumes of

Tris buffer. The protein concentration is now approximately 15 mg/ml. 7. Store the binding protein in portions of 20 ml at - 8 0 °. 8. When a 20-ml portion is thawed, divide into 1-ml portions and store

these at - 8 0 ° as well (1 ml is sufficient for 50 samples). Note. If following Step 5 the pellet is reddish-brown instead of yellow-

ish-brown, it is recommended to wash the pellet once more in NaHCO 3 . The reddish color is derived from blood components, and these are known to inhibit the binding protein. Processing of 500 g bovine liver yields approximately 400 ml binding protein (20,000 samples).

Comments and Discussion

The assay for phospholipase C described here has several advantages over the assays for phospholipase C more generally used (see Introduc- tion). In short the assay is relatively inexpensive, uses minimal amounts of radiolabeled products, allows processing of large numbers of samples, and uses the substrate endogenous to the organism, thus circumventing artifacts arising from differing compositions of the endogenous and applied substrate. There are no long prelabeling periods, and the rapidity of the assay allows unstable interactions between the enzyme and G proteins to be determined. As for any assay, the assay has drawbacks as well. The filter lysing method restricts the assay to cells which can be easily lysed in that way, although the idea behind the assay can probably be used in an adapted assay using other lysis methods. Another restriction, which is due to the nature of the assay, is that the substrate is not clearly defined and that there can be differences between batches of cells.

Acknowledgments

The authors thank Christophe Emeux for the gift of rat brain 5-phosphatase. Part of this work was supported by a grant from the NWO Council for Medical Research.

[17] PURIFICATION OF PLC-/33 FROM RAT BRAIN 219

[17] Purif icat ion o f P h o s p h o l i p a s e C-f i3 from Rat Brain

By DEOK-YOUNG JHON, DONGEUN PARK, and SUE G o o RHEE

Introduction

Phosphoinositide-specific phospholipase C (PLC) plays a central role in transmembrane signaling. The enzyme catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5@2) and thereby generates two second messenger molecules, inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol, in response to the binding of various ligands to cell surface receptors.1 Protein isolation and molecular cloning studies have revealed that PLC activities from a variety of species and cells belong to a family of isozymes. 2 A total of ten distinct PLC cDNAs (seven from mammalian species, 3-~2 two from Drosophila melanogaster, 13,14 and one from Dictyo- stelium discoideum 15) have been isolated. The proteins encoded by the

1 M. J. Berridge, Nature (London) 361, 315 (1993). 2 S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992). 3 D.-Y. Jhon, H.-H. Lee, D. Park, C.-W. Lee, K.-H. Lee, O. J. Yoo, and S. G. Rhee, J.

Biol. Chem. 268, 6654 (1993). 4 D. Park, D.-Y. Jhon, C.-W. Lee, K.-H. Lee, and S. G. Rhee, J. Biol. Chem. 268, 4573

(1993), 5 S. H. Ryu, K. S. Cho, K.-Y. Lee, P.-G. Sub, and S. G. Rhee, Biochem. Biophys. Res.

Commun. 141, 137 (1986). 6 S. H. Ryu, K. S. Cho, K.-Y. Lee, P.-G. Sub, and S. G. Rhee, J. Biol. Chem. 262,

12511 (1987). 7 S. H. Ryu, P.-G. Suh, K. S. Cho, K.-Y. Lee, and S. G. Rhee, Proc. Natl. Acad. Sci.

U.S.A. 84, 6649 (1987). 8 The molecules referred to as PLC-I, PLC-II, and PLC-III in Refs. 4-6 are now known

as PLC-fll, PLC-~/1, and PLC-S1, respectively. 9 S. G. Rhee, S. H. Ryu, K.-Y. Lee, and K. S. Cho, this series, Vol. 197, p. 502. The

molecules referred to as PLC-fl, PLC-y, and PLC-8 in this reference are, more specifically, now known as PLC-fll, PLC-3,1, and PLC-S1, respectively.

10 E. Meldrum, M. Katan, and P. Parker, Eur. J. Biochem. 182, 673 (1989). 11 y. Homma, Y. Emori, F. Shibasaki, K. Suzuki, and T. Takenawa, Biochem. J. 269,

13 (1990). 12 Z. Takenawa, Y. Homma, and Y. Emori, this series, Vol, 197, p. 511. 13 D. Park, D,-Y. Jhon, R. Kriz, J. Knopf, and S. G. Rhee, J. Biol. Chem. 267, 16048 (1992). t4 R. Kriz, L.-L. Lin, L. Sultzman, C. Ellis, C.-H. Heldin, T. Pawson, and J. Knopf, in

"Proto-Oncogenes in Cell Development" (Ciba Foundation Symposium 150), p. l l2. Wiley, Chichester, 1990.

15 A. Lyndsay Drayer and P. J. M. van Haastert, J. Biol. Chem. 267, 18387 (1992).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved


cDNAs vary markedly in size, with molecular masses ranging from 85 to 150 kDa, and contain two conserved X and Y domains, which appear to constitute a catalytic site.2 The PLC isozymes that have been characterized at the cDNA level can be divided into three structural types (fl, 3', and 8) on the basis of the relative locations of the X and Y domains in the primary structure. The/3 type includes three mammalian enzymes (PLC-/31, PLC-/32, and PLC-/33) and two Drosophila enzymes (PLC- norpA and PLC-p21); the 3' type includes two mammalian enzymes (PLC-yl and PLC-y2); and the 8 type includes two mammalian enzymes (PLC-81 and PLC-82) and one Dictyostelium enzyme. The distinct structural features of the different PLC types appear to be related to specific mechanisms underlying the receptor-mediated enzyme activation. PLC-3,1 and PLC-72 are specifically activated by receptor and nonrecep- tot protein tyrosine kinases.l Activation of/3-type isozymes is achieved by a completely different mechanism. The a subunit of the Gq class of G proteins have been shown to activate PLC-/3 isozymes in the order PLC-/31 -> PLC-/33 -> PLC-/32, 2 whereas the fly subunits of G proteins activate them in the order PLC-/33 > PLC-/32 > PLC-/31.3

A rich source of PLC isozymes is mammalian brain. Thus PLC-/31,4-8 PLC-71,4-8 PLC-81,4-8 and PLC-829 were purified from either rat or bovine brains. The PLC-y2 form was purified from bovine spleen. 10,11 The PLC-/32 was purified from extracts of HeLa cells that had been transfected with vaccinia virus containing PLC-/32 cDNA. 12 The distribution of PLC-/32 is not known except that cDNA corresponding to PLC-/32 was derived from HL-60 cells.13 PLC-83 is known only at the cDNA level. 13 We have purified two new/3-type enzymes, PLC-/333 and PLC-/34, from rat brain and bovine retina, respectively. We describe the procedures for the purification of PLC-/33 in this chapter and those PLC-/34 in [18].

Cloning and Sequencing of Phospholipase c-r3 cDNA

Phospholipase C-/33 was identified first at the cDNA level) With primers designed on the basis of the highly conserved amino acid sequences in the X and Y domains, we obtained a 580-bp polymerase chain reaction (PCR) product from a rat brain cDNA library that contained a PLC-like sequence distinct from those of the corresponding regions of known PLC isozymes. Screening of a rat FRTL cDNA library with the 580-bp PCR product followed by primer extension yielded four overlapping partial clones. Complete sequencing of the four cDNA inserts yielded a cumulative sequence of 3948 bp. Determination of the open reading flame was aided by comparison with known PLC sequences.


A comparison of the deduced amino acid sequence with known PLC sequences revealed the predicted protein to be similar in primary structure and overall structural organization to PLC-/31 and PLC-/32. Thus, the protein encoded by the isolated cDNA was termed PLC-fl3. The total number of amino acid residues identified in PLC-fl3 is 1234. Thus, the molecular mass of PLC-fl3 is expected to be slightly larger than that of the 1216-residue PLC-fll and substantially larger than that of the 1181- residue PLC-fl2. The amino acid sequence of PLC-fl3 is also more similar to that of PLCq31 than to that of PLC-fl2:PLC-fl3 is 56% identical to PLC-fll and 46% identical to PLC-fl2. The overall identity between PLC-/31 and PLC-fl2 is 48%. A distinctive feature of PLC-/33 is that the X and Y domains are separated by 121 residues compared to 70 and 76 residues in PLC-fll and PLC-/32, respectively. However, as in the case with PLC-/31 and PLC-/32, the region separating the X and Y domains in PLC-/33 is rich in acidic amino acids as well as serine and threonine: 20 of 70, 26 of 76, and 29 of 121 residues are acidic in PLC-fll, PLC-fl2, and PLC-/33, respectively; 8, 7, and 11 consecutive acidic amino acids are present in PLC-fll, PLC-fl2, and PLC-/33, respectively; and 19, 14, and 28 serine or threonine residues are present in PLC-fll, PLC-fl2, and PLC-/33, respectively. Another characteristic feature of fl-type enzymes is a high proportion of basic amino acids in the carboxyl-terminal region that follows the Y domain: 87, 76, and 72 arginine, lysine, or histidine residues are present in PLC-fll, PLC-/32, and PLC-/33, respectively.

Antibodies

Antisera to peptides corresponding to PLC-fl3 amino acid residues 75-84 (GRYARLPKDP) (antibody 1), residues 567-578 (TDPKKPTT- DEGT) (antibody 2), residues 1132-1145 (SVNSIRRLEEAQ) (antibody 3), and residues 1223-1234 (ADSESQEENTQL) (antibody 4) were generated in rabbits by injection of the synthetic peptides that had been conjugated to keyhole limpet hemocyanin with glutaraldehyde. Antibodies to PLC-/31 and PLC-/32 were described previously. 13

Distribution of/]-Type Phospholipase C Isoforms in Various Rat Tissues

With the use of rabbit antisera specific for PLC-/31, PLC-fl2, or PLC-fl3, we have investigated the distribution of the PLC isozymes in various rat tissues by immunoblotting. For the tissues studied, the relative amounts of PLC-fl3 were as follows: parotid gland > brain > liver, uterus, lung > heart, adrenal gland, ovary. The intensity of the signal for the remaining five tissue extracts studied was too weak to be classified


(Fig. I). On the basis of the results from several immunoblots, the amount of PLC-/33 in the parotid gland is estimated to be 2 to 3 times that in brain. From the results of the immunoblot shown in Fig. 1, which includes purified PLC-/33 as standard, the concentration of PLC-/33 was estimated to be approximately 5 ng per milligram of brain protein.

Antibodies to PLC-/31 recognized the 150kDa enzyme as well as a 140-kDa protein that is either a proteolytic fragment of 150-kDa PLC-fll or the product of an alternatively spliced mRNA. The PLC-/31 isozyme exists in much higher concentrations in the brain than other tissues. The estimated concentration of 150-kDa PLC-fll in brain is approximately 0.5 /zg per milligram of brain protein, which is about 100 times that of PLC-/33. The PLC-/32 form was not detectable in any of the 13 tissues.


Enzyme activity during purification is measured at 37 ° in a 200-/zl reaction mixture containing 20,000 cpm of [3H]phosphatidylinositol ([3H]PtdIns, Du Pont-NEN Research Products, Boston, MA), 150/zM soybean PtdIns, 0.1% (w/v) sodium deoxycholate, 3 mM CaCI 2, 2 mM EGTA, 50 mM HEPES (pH 7.0), and a source of enzyme.

The amount of PLC-fl3 is extremely low compared to other PLC isozymes, especially compared to PLC-fll in brain, which is one of the most abundant sources ofPLC-fl3. Thus, it is necessary to monitor the progress

o ._1 _. : 13. ~'~ co

- P L C ~3

- P L C [31

FIG. 1. Distribution of PLC-fl3 and PLC-fll in various rat tissues. Proteins were immunoprecipitated with an antibody (antibody 4) to PLC-fl3 from 1.9 mg of crude homogenates, separated on a 6% polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE), and immunoblotted with another antibody (antibody 1) (top gel). For the detection of PLC-fll, 100 /~g of each crude homogenate was directly analyzed on 6% SDS-PAGE and blotted with rabbit serum raised against PLC-fll (bottom gel). The left-hand lane of each panel received 5 ng of purified enzyme as standard.


of purification by immunoblotting with antibodies to PLC-/33. For this purpose, an antiserum (antibody 4) to a peptide corresponding to the carboxyl-terminal amino acid residues of PLC-/33 is used.

Phospholipase C-fl3 Purification Procedure

Frozen brains harvested in liquid nitrogen are obtained from Biopro- ducts for Science (Indianapolis, IN). All operations are carried out at 4 ° unless otherwise specified.

Step 1: Extraction of Phospholipase C-fl3 from Rat Brain Particulate Fraction. One thousand rat brains are washed twice with cold homogenization buffer [50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 p.g/ml of leupeptin, and 1 mM dithiothreitol (DTT)] and then homogenized with a Polytron (Brinkmann Instrument, Westbury, NY) (three times, each time for 10 sec) in 8 liters of homogenization buffer. The homogenate is centrifuged for 10 min at I000 g to remove debris. The supernatant is further centrifuged for 90 min at 23,000 g, and the resulting pellet is suspended in 4 liters of homogenization buffer with a Teflon pestle and then centrifuged for 60 rain at 23,000 g. The new pellet is washed and stored frozen at - 7 0 °. The final washed pellets from two identical preparations (corresponding to 2000 rat brains) are thawed and suspended in 6 liters of homogenization buffer containing 2 M KCI. The suspension is stirred for 2 hr at 4 ° and then centrifuged for 60 rain at 23,000 g. The supernatant is brought to 60% saturation with ammonium sulfate by adding solid salt. The suspension is stirred for 1 hr at 4 ° and then centrifuged for 20 min at 16,000 g. The resulting pellet is suspended in 1 liter of homogenization buffer and dialyzed overnight against homogenization buffer. The dialyzed solution is centrifuged for 30 rain at 13,000 g to remove insoluble particles, and the supernatant is diluted by adding homogenization buffer (final volume - 3 liters) to reduce the conductivity of the protein solution below 4 mho.

Step 2: Heparin-Sepharose CL-6B Column Chromatography. Three liters of dialyzed membrane extract (3.6 g of protein) is applied to a heparin-Sepharose CL-6B (Pharmacia, Piscataway, N J) column (5.9 x 11 cm) that has been equilibrated with 20 mM HEPES (pH 7.0), 1 mM EGTA. and 0.1 mM DTT. Unbound proteins are washed with 680 ml of equilibration buffer. Bound proteins are eluted from the column with a linear gradient from 0 to 1 M NaCI in 2 liters of equilibration buffer. Fractions (26 ml) are collected every 3 rain and assayed for PtdIns-hydrolyzing activity and PLC-fl3 protein (by SDS-PAGE followed by immunoblotting with antibodies to PLC-fl3). Peak fractions (75 to 93) of PLC-/33 (Fig. 2A) are pooled and concentrated to 32 ml on an Amicon (Danvers, MA) concentrator.

A i,~ ~ '/5 11 Jl 114 I'/ 90 9] ~

2'0 40 6'0 8.0 I10 110 1400 ~'~

B ~ "r" o

10 20 30 40 50 60 70

10 20 30 "40 50 60 70

D 4 2 ~ ~ ~ 46 47 4 49 50 5] S2

0,021

0.01

--I O.

20 30 40 50 60 70 - 1'0- 0'50 ~"~'~

E

Fraction Number

Z


Step 3: First Reversed-Phase Chromatography on TSK-Gel Phenyl- 5PW Column. Solid NaCI is added to the concentrated fractions from the heparin-Sepharose CL-6B column to give a concentration of 2.5 M, and the mixture is then centrifuged to remove insoluble particles. The supernatant is applied to a preparative high-performance (HPLC) TSK-Gel phenyl- 5PW column (21.5 x 150 ram) (TosoHaas, Montgomeryville, PA) equilibrated with 20 mM HEPES (pH 7.0), 1 mM EGTA, and 3 M NaCI. Proteins are eluted, at a flow rate of 5 ml/min, by successive application of (i) the equilibration buffer for 10 min, (ii) a decreasing NaCI gradient from 3 to 1.2 M for 10 min, and (iii) a decreasing NaCI gradient from 1.2 to 0 M for 25 min. Finally, the column is washed with NaCl-free buffer. Fractions (5 ml) are collected and assayed for Ptdlns-hydrolyzing activity and PLC-Il3 protein. Peak fractions (25 to 27) of PLC-Il3 (Fig. 2B) are pooled and adjusted to 2 M NaC1 by adding solid salt.

Step 4: Second Reversed-Phase Chromatography on TSK-Gel Phenyl- 5PW Column. The pool of active fractions (28 mg of protein in 15 ml) from the first TSK-Gel phenyl-5PW column is applied to an analytical HPLC TSK-Gel phenyl-5PW column (7.5 x 75 mm) (TosoHaas) equilibrated with the same buffer as that used for the preparative column. Proteins are eluted at a flow rate of 1 ml/min, by application of the same decreasing NaC1 gradients as for the preparative column. Peak fractions (23 to 25) (Fig. 2C) are pooled, concentrated to 1 ml, and then diluted to 3 ml by addition of 20 mM HEPES (pH 7.0) containing 1 mM EDTA.

Step 5: Absorption of Phospholipase C-ill on Monoclonal Antiphos- pholipase C-t1 Antibody Affinity Gel. An immunoaffinity gel is prepared by covalently attaching 3 mg of anti-PLC-ill monoclonal antibody (clone L54) per milliliter of Affi-Gel 10 (Bio-Rad, Richmond, CA) according to the manufacturer's instructions. The fraction (3 ml) from the second TSK- Gel phenyl-5PW column is incubated with 0.5 ml of immunoaffinity gel for 1 hr at 4 ° in a rotary mixer. Unbound proteins are removed by filtration through a sintered glass funnel and washing of the gel with 4 ml of 20 mM HEPES (pH 7.0) containing 1 mM EGTA. The filtrate is combined with the washing solution. The immunoabsorption procedure is repeated to ensure complete removal of PLC-Ill.

Step 6: Chromatography on TSK-Gel Heparin-5PW. The unbound proteins (2.2 mg) from the immunoaffinity gel are applied to an HPLC

FIG. 2. Purification of PLC-133 on a heparin-Sepharose CL-6B column (A), a preparative HPLC TSK-Gel phenyl-5PW column (B), an analytical HPLC TSK-Gel phenyl-5PW column (C), an HPLC TSK-Gel heparin-5PW column (D), and an HPLC Mono Q column (E). Detailed procedures are described in the text.


TABLE I PURIFICATION OF PHOSPHOLIPASE C-/33

Protein PLC-/33 a Yield Purification Step (mg) (rag) (%) (-fold)

KC1 extract of pellet 3600 b 0.62 100 1 Heparin-Sepharose 230 b 0.45 73 12 Phenyl, preparative 30 b 0.32 51 62 Phenyl, analytical 2.2 b 0.12 19 310 Heparin 0.32 b 0.05 8 850 Mono Q 0.04 C 0.04 6 5900

a Determined by immunoblot with the use of 125I-labeled protein A. b Determined by the method of Bradford. c Estimated using an average absorptivity (A°8~ %) of 1.14.

TSK-Gel heparin-5PW column (7.5 x 75 mm) (TosoHaas) equilibrated with 20 mM HEPES (pH 7.0) containing 1 mM EGTA and 0.1 mM DTT. Proteins are eluted, at a flow rate of 1.0 ml/min, by sequential application of equilibration buffer for 15 min and increasing linear NaCI gradients from 0 to 0.4 M for 25 min and from 0.4 to 0.9 Mfor 10 min. Peak fractions (46 to 48) (Fig. 2D) are identified by immunoblotting with antibodies to PLC-/33, pooled, concentrated to 0.5 ml in a Centricon microconcentrator, and diluted to 8 ml to reduce the salt concentration.

Step 7: Ion-Exchange Chromatography on M o n o Q Column. The diluted PLC-/33 sample (8 ml) from the TSK-Gel heparin-5PW column is applied to a Mono Q column (7 × 60 ram) (Pharmacia) equilibrated with

Antibodies PLC-[il PLC.[32

PLC 1 2 3 1 2 3

PLC-~3

1 2 3

~ .215 kDa

-105 kDa FIG. 3. Immunoblot analysis of PLC-/31, PLC-fl2, and PLC-fl3. Purified/3 isozymes (5-20

ng) were separated on a 6% SDS-PAGE, transferred to nitrocellulose, and incubated with rabbit antisera specific for PLC-/31, PLC-/32, or PLC-/33. Lanes 1, PLC-/31 ; lanes 2, PLC-/32; and lanes 3, PLC-/33. The positions of molecular size standards are shown on the right- hand side.

[ 1 8 ] P U R I F I C A T I O N OF PLC-fl4 F R O M BOVINE R E T I N A 2 2 7

20 mM Tris-HC1 (pH 7.4) containing 1 mM EGTA and 0.1 mM DTT. Proteins are eluted, at a flow rate of 1 ml/min, by a linear NaC1 gradient from 0 to 0.3 M for 25 min. Peak fractions (30 to 32) (Fig. 2E) are identified by immunoblotting, pooled, and concentrated to 0.4 ml. The final sample is divided into aliquots and stored at - 70 °. A summary of the purification steps is presented in Table I.

Immunoblot Analysis

Purified PLC-fl3 was recognized by any of the four antisera to peptides corresponding to PLC-fl3 amino acids but not by antisera to PLC-fll or PLC-fl2 (Fig. 3). Figure 3 also shows that, as predicted from the deduced amino acid sequences, the molecular size decreases in the order PLC-fl3 > PLC-fll > PLC-fl2.

[18] C h a r a c t e r i z a t i o n of P h o s p h o l i p a s e C I s o z y m e s in B o v i n e Re t i na : Pur i f i ca t ion of P h o s p h o l i p a s e C-fl4

By CHANG-WON LEE, K W E O N - H A E N G LEE, and SUE Goo RHEE


It has been proposed that signal transduction in photoreceptors involves the activation of phosphoinositide-specific phospholipase C (PLC), In retinas from a variety of invertebrates, the intracellular concentration of inositol 1,4,5-trisphosphate (IP3) is regulated by the light-induced breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2). Moreover, injection of IP 3 into invertebrate photoreceptor cells mimics the effect of light in inducing both excitation and adaptation. 1-4 Further evidence for the role of PLC was obtained from studies with a blind Drosophila mutant (norpA): PLC activity was found to be significantly reduced in the eyes of the norpA mutant compared to the wild type. 5 Subsequently, the norpA

t R. E. Anderson and J. E. Brown, Prog. Retinal Res. 8, 211 (1988). '~ A. Fein, R. Payne, D. W. Corson, M. J. Berridge, and R. F. Irvine, Nature (London)

311, 157 (1984). 3 j. E. Brown, L. J. Rubin, A. J. Ghalayini, A. P. Tarver, R. F. Irvine, M. J. Berridge,

and R. E. Anderson, Nature (London) 311, 160 (1984). 4 E. T. Szuts, S. F. Wood, M. S. Reid, and A. Fein, Biochem. J. 240, 929 (1986). 5 H. Inoue, T. Yoshioka, and Y. Hotta, Biochem. Biophys. Res. Commun. 135, 513 (1985).



gene was shown to encode a PLC that is expressed in the eye of Dro- sophila. 6

Despite some similarities, phototransduction in the vertebrate retina shows marked differences from that in the invertebrate retina. Whereas IP 3 functions as the primary second messenger in phototransduction in invertebrate photoreceptors, cGMP fulfills that role in vertebrate photoreceptors. 7 However, light has been shown to produce a transient increase in IP 3 in rod outer segments (ROS) of frog, chicken, and rat. 8-11 Evidence also indicates that Ca 2÷ mediates light adaptation in vertebrate ROS, suggesting an important role for PLC in ROS signaling. 12,~3 Bovine ROS were shown to contain at least two distinct PLC isoforms.14,~5 However, these isoforms were neither purified nor characterized in relation to previously identified isozymes.

As a step toward understanding the role of PLC in phototransduction, we have separated PLC isozymes from bovine retina by high-performance liquid chromatography (HPLC) and characterized them by immunoblot analysis with antibodies to known PLC isoforms. Retina was chosen as the source of enzymes because of the difficulty in obtaining sufficient quantities of ROS. The retinal homogenate contained six PLC isozymes, including a previously unidentified B-type enzyme, which we subsequently purified and designated PLC-fl4.

Materials

[3H]PIP2 and [3H]phosphatidylinositol ([3H]PI) are obtained from Du Pont-New England Nuclear (Boston, MA); PIP2 from Boehringer Mann- helm (Indianapolis, IN); PI from Sigma (St. Louis, MO); phosphatidylethanolamine and phosphatidylserine from Avanti Polar Lipids (Alabaster,

6 B. T. Bloomquist, R. D. Shortridge, S. Schneuwly, M. Perdew, C. Montell, H. Steller, G. Rubin, and W. L. Pak, Cell (Cambridge, Mass.) 54, 723 (1988).

7 L. C. Stryer, Annu. Rev. Neurosci. 9, 87 (1986). s j . E. Brown, C. Blazynski, and A. I. Cohen, Biochem. Biophys. Res. Commun. 146,

1392 (1987). 9 A. J. Ghalayini and R. E. Anderson, Biochem. Biophys. Res. Commun. 124, 503 (1984). to F. Hayashi and R. Amakawa, Biochem. Biophys. Res. Commun. 128, 954 (1985). 11 F. A. Millar, S. C. Fisher, C. A. Muir, E. Edwards, and J. N. Hawthorne, Biochim.

Biophys. Acta 970, 205 (1988). 12 H. R. Mathews, L. W. Murphy, G. L. Fain, and T. D. Lamb, Nature (London) 334,

67 (1988). 13 K. Nakatani and K. W. Yau, Nature (London) 334, 69 (1988). ~4 B. Gehm and D. G. McConnell, Biochemistry 29, 5447 (1990). 15 A. Ghalayini, A. P. Tarver, W. M. Mackin, C. A. Koutz, and R. E. Anderson, J. Neuro-

chem. 57, 1405 (1991).

[18] PURIFICATION OF PLC-fl4 FROM BOVINE RETINA 229

AL); and calpain inhibitors I and II from Calbiochem (San Diego, CA). Bovine retinas are either collected on dry ice from flesh eyes supplied by a local slaughterhouse or obtained from Pel-Freez Biologicals (Rogers, AR); they are stored at - 7 0 ° until use. Rabbit antisera specific to PLC- ill, 16 PLC-fl2,17 PLC-fl3, TM the sequence common to X regions, and the sequence common to Y regions 19 are as described. Monoclonal antibodies to PLC-71 and PLC-81 are as described. 19


Phospholipase C activity is assayed with either [3H]PIP2 or [3H]PI as substrate. The PIP2-hydrolyzing activity is measured with mixed phospholipid micelles containing phosphatidylethanolamine/phosphatidylserine/ [3H]PIP2 in a molar ratio of 1 : 1 : 1. The lipids in chloroform are dried under a stream of nitrogen gas, suspended in 50 mM HEPES, pH 7.0, 100 mM NaCI, and 1.6 mM sodium deoxycholate, and subjected to sonication. Assay incubations are performed for 20 min at 30 ° in a 100-~1 reaction mixture containing lipid micelles (15/~M [3H]PIP 2, 36,000 cpm), 50 mM HEPES (pH 7.0) 150 mM NaCI, 5 mM MgCI2, 2 mM EGTA, 0.8 mM sodium deoxycholate, and an enzyme source. CaCI2 is added to the assay mixture to give a final free Ca 2 + concentration of 1 ~M. The PI-hydrolyzing activity is assayed in a 200-/zl reaction mixture containing 150 ~M [3H]PI (20,000 cpm), 50 mM HEPES (pH 7.0), 3 mM CaC12, 2 mM EGTA, and 0.1% sodium deoxycholate, and enzyme incubations are performed at 37 ° for 3 to 10 min. For both assays, the reaction is stopped by adding 1 ml of chloroform/methanol/HC1 (100 : 100 : 0.6, v/v), followed by 0.3 ml of 1 M HC1 containing 5 mM EGTA. After brief centrifugation, 0.5 ml of the upper aqueous phase is assayed for 3H radioactivity by liquid scintillation spectroscopy.

Phospholipase C Isozymes in Bovine Retina

Ten frozen retinas are thawed in 10 ml of homogenization buffer [10 mM Tris (pH 7.4), 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), leupeptin (2 tzg/ml),

i6 S. H. Ryu, K. S. Cho, K.-Y. Lee, P.-G. Suh, and S. G. Rhee, J. Biol. Chem. 262, 12511 (1987).

17 D. Park, D.-Y. Jhon, R, Kriz, J. Knopf, and S. G. Rhee, J. Biol. Chem. 267, 16048 (1992). t8 D.-Y. Jhon, H.-H. Lee, D. Park, C.-W. Lee, K.-H. Lee, O. J. Yoo, and S. G. Rhee, J.

Biol. Chem. 268, 6654 (1993). ~9 P.-G. Sub, S. H. Ryu, W. C. Choi, K.-Y. Lee, and S. G. Rhee, J. Biol. Chem. 263,

14497 (1988).


and calpain inhibitors I and II (each at 4/xg/ml)] and homogenized in a glass homogenizer with a motor-driven Teflon pestle (I0 strokes). The homogenate is centrifuged at I000 g for 10 min. The supernatant (10 ml) is adjusted to 2 M KCI by adding solid KCI, stirred for 2 hr at 4 °, and then centrifuged at 23,000 g for 1 hr. The resulting supernatant is dialyzed overnight against 1 liter of homogenization buffer and centrifuged at 16,000 g for 20 min.

Proteins (20 mg) in the KCI extracts of the total homogenate are injected onto a TSK-Gel heparin-5PW HPLC column (7.5 × 75 mm) (TosoHaas, Montgomeryville, PA) that has been equilibrated with 20 mM HEPES (pH 7.0), 1 mM EGTA, and calpain inhibitors I and II (each at 4 p.g/ml). Proteins are eluted, at a flow rate of 1.0 ml/min, by applying successively the equilibration buffer for 15 min, a linear NaC1 gradient from 0 to 0.64 M for 40 min, a second linear NaCI gradient from 0.64 to 1.0 M, for 20 min, and equilibration buffer containing 1.0 M NaC1 for 10 min. Fractions (1 ml) are collected, and each fraction is assayed for PLC activity with either PI or PIPz as substrate (Fig. 1). Three prominent peaks of PI-hydrolyzing activity, centered at fractions 37, 44, and 65, are detected. Low but significant PI-hydrolyzing activity is also detected in fractions 47 to 60. With PIP 2 as substrate, three major peaks, centered at fractions 37, 54, and 65, and three partially resolved, minor peaks, between fractions 44 and 51, are observed. For immunoblot analysis, portions of each fraction are treated with 5 × electrophoresis sample buffer and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6% gel). Proteins are transferred to nitrocellulose paper, incubated with isoform-specific antibodies to PLC-/31, PLC-fl2, PLC-/33, PLC-yl, PLC-y2, or PLC-81, and visualized with alkaline phosphatase-conjugated secondary antibodies.

Immunoblotting results indicated that activity peaks centered at fractions 37 and 44 are mainly attributable to PLC-yl (145 kDa) and PLC-81 (85 kDa), respectively. As observed previously, antibodies to PLC-/31 recognized PLC-fll (150 kDa) as well as a 140-kDa product possibly arising from an alternatively spliced PLC-/31 mRNA; PLC-/31 immunoreactivity was centered at fraction 48. The intensity of the PLC-fl3 band (152 kDa) was maximal at fraction 50. Neither the 140-kDa protein recognized by antibodies to PLC-/32 nor the 142-kDa protein recognized by antibodies to PLC-72 were detected in the column fractions, suggesting that these enzymes are absent from, or present in low concentrations in, bovine retina.

None of the antibodies to known PLC isozymes 2° recognized PLC in

20 Al though PLC-82 has been purified and cloned, 21 antibodies to this enzyme were not available in our laboratory.

[18] PURIFICATION OF PLC-/34 FROM BOVINE RETINA 231

FRACTION NUMBER

3 2 3 4 3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0 5 2 5 4 5 6 5 8 6 0 62 64 66 68

iii ~ . . . . . . . . . . ~, ~ , ~ i ~ !i~i~!:! ii~;i~ : : . . . . . .

10000 3 0 3 2 3 4 3 6 3 8 4 0 4 2 4 4 4 6 4 8 5 0 5 2 5 4 5 6 5 8 6 0 6 2 6 4 6 6 6 8 7 0 i i i i e i i i i i e I i I i 1 i i i L

I~ 8000 6oool

! 20oo O'

3 0 3 2 3 4 3 6 3 0 4 0 4 2 4 4 4 6 4 8 5 0 5 2 5 4 5 6 5 8 6 0 6 2 6 4 6 6 6 8 7 0

FRACTION NUMBER

PLC.pl

PLC-~3

PLC-71

PLC-81

1.0

0.5 :~

o.o z

FIG. 1. Analysis of KC1 extract of total retinal homogenate for PLC isozymes. A KCI extract containing 20 mg of protein (equivalent to approximately four retinas) was resolved on a TSK-Gel heparin-5PW HPLC column as described in the text. Fractions (1 ml) were collected and assayed for PI- and PIP2-hydrolyzing activity. Alternate fractions were also subjected to immunoblot analysis with antibodies to PLC-/31, -/33, -yl, and -~51 (top, gels).

fractions 54 and 65. Under our assay conditions, the enzyme in fraction 54 showed higher activity with PIP2 than with PI, whereas the enzyme in fraction 65 exhibited similar activities with both substrates. These results suggested the possibility that two previously unidentified PLC isozymes are present in bovine retina. We tentatively designated the enzymes in fractions 54 and 65 PLC-retA and PLC-retB, respectively. On subsequent purification to homogeneity, PLC-retA and PLC-retB exhibited apparent molecular masses of 130 and 85 kDa, respectively, on SDS-PAGE. Puri- fied enzymes were digested with trypsin, and the resulting peptides purified and sequenced. Comparison with the sequences of known PLC isozymes reveals that all six tryptic sequences derived from PLC-retB were present in the sequence of PLC-82 that had been deduced from the nucleotide


80'

60' ,q-

, , o

2 0

1. LSI-IDR 2. AISQDK 3. YQQY 4. HISL 5. YGNELSADDLGHK 6. YVGATFNfl-IP 7. EWSDM]NT 8. AMGIETSDIAVXPDSTXK 9. LSTMINYAQP 6 10. NDEIEPATFTYEK l 1. AMQI1XMYXP 12. GLVTVEDEQAXMASY 5 /8'9~ 11,12

0 0 1 '0 2'0 3'0 4'0 5'0 6'0 7'0 8'0

Elution Time, min

FIG. 2. Isolation and sequences of tryptic peptides of PLC-retA (PLC-fl4). Purified PLC-retA (~150/~g) was digested with trypsin and subjected to chromatography on a C18 HPLC column. Some peptides were further purified by reversed-phase chromatography on a microbore C18 column (not shown). Peptides that were subjected to sequence analysis are indicated by numbers, and the sequences are listed. X indicates unidentified amino acids.

sequence of a bovine brain cDNA. 2~ Thus, we conclude that PLC-retB is PLC-82. Because PLC-82 had been purified from bovine brain, 22 which we believe is a better source for PLC-82 than bovine retina, we do not describe PLC-82 purification here.

The HPLC chromatogram of PLC-fl4 tryptic peptides and the sequences determined are listed in Fig. 2. The amino acid sequences of 12 tryptic peptides were compared with those of known PLCs (ill, f12,/33, norpA, p21, yl, y2, 81, and 82). Peptides I to 4 were too short to yield useful information. Peptide 6, YVGATFNIHP, and peptide 7, EWSDMINT, showed homology to the norpA sequences 539ySGSTTNVHP 548 and 943QWTDMIAR 95°, respectively, but no noticeable similarity to other PLC sequences. Peptide 9, LSTMINYAQP, was most similar to the PLC-norpA sequence 55°LSSMVNYAQp559 but also showed lesser similarity to sequences ofPLC-/3 1 (54°MSNLVNYIQp549), PLC-/32 (542MSSLVNYIQP551), and PLC-/33 (572MSTLVNYVEPS81). Peptide l l, AMQIIXMYXP, was similar to a region located immediately NH2-terminal to the X domain of /3-type and 8-type PLC isozymes, as exemplified by 253ARLLIEKYEpZ6z of PLC-/33 and 251ALSLIERYEp26° of PLC-81. No sequence homologous

21 E. Meldrum, R. W. Kriz, N. Totty, and P. J. Parker, Eur. J. Biochem. 196, 159 (1991). 22 E. Meldrum, M. Katan, and P. Parker, Eur. J. Biochem. 182, 673 (1989).

[ 18 ] PURIFICATION OF PLC-fl4 FROM BOVINE RETINA 233

to peptide 11 was found in PLC-yl and PLC-72. The other four sequences, YGNELSADDLGHK (peptide 5), AMGIETSDIAVXPDSTXK (peptide 8), NDEIEPATFTYEK (peptide 10), and GLVTVEDEQAXMASY (peptide 12), showed no homology to known PLC sequences. Taken together, these data indicate that PLC-retA is a new member of the PLC family that belongs, like Drosophila PLC-norpA, to the PLC-/3 subfamily. Therefore, PLC-retA was named PLC-/34. Thus, our study suggests that bovine retina contains at least six PLC isozymes: PLS-fll, PLC-/33, PLC-yl, PLC-81, PLC-82, and the newly discovered PLC-/34, all encoded by distinct genes. PLC-/32 and PLC-y2 were not detected. The six retinal enzymes hydrolyzed both PI and PIPz, but with different selectivities. The selectivity for PIP 2 over PI decreased in the order PLC-/34 > PLC-/33 -~ PLC-/31 > PLC-82 > PLC-61 > PLC-71, for enzymes partially purified from total retinal homogenate (Fig. 1).

Purification of Phospholipase C-fl4

All manipulations are performed at 4 ° to 6 ° in a cold room or on ice, unless otherwise indicated. During purification, PLC activity is monitored by measuring [3H]PI-hydrolyzing activity in 1 to 3 pA of column fractions, which are incubated with substrate for 3 to 10 min at 37 °. All chromatographic buffers used for the purification of PLC-fl4 contain calpain inhibi- tots I and II, each at a concentration of 2 p.g/ml.

Step 1: Preparation of Salt Extract of Retinal Particulate Fraction. Four thousand frozen retinas are thawed in 10 liters of homogenization buffer and homogenized with a Polytron (Brinkmann Instrument, West- bury, NY) and then in a glass homogenizer with a motor-driven Teflon pestle. The homogenate is centrifuged at 1000 g for 10 min. The supernatant is further centrifuged at 23,000 g for 1 hr. The resulting pellet is suspended with a glass homogenizer and Teflon pestle in 4 liters of homogenization buffer and centrifuged at 23,000 g for 1 hr. The pellet is suspended in 4 liters of homogenization buffer containing 2 M KC1, stirred for 2 hr, and centrifuged at 23,000 g for 1 hr. The supernatant, approximately 3.4 liters containing 6.8 g of protein, is dialyzed overnight against 40 liters of 20 mM HEPES (pH 7.0), 1 mM EGTA, 0.1 mM DTT, and calpain inhibitors. Insoluble materials are removed by centrifugation at 16,000 g for 20 min.

Step 2: Heparin-Sepharose CL-6B Column Chromatography. The slightly turbid supernatant, which contains 3.7 g of protein and approximately 0.2 M KC1 as estimated by conductivity, is applied at a flow rate of 10 ml/min to a heparin-Sepharose CL-6B column (5 × 13 cm) (Pharmacia LKB Biotechnology, Piscataway, N J) that has been equilibrated with 20 mM HEPES, pH 7.0, 0.2 M NaCI, 1 mM EGTA, and 0.1 mM DTT.

0001 A "3.0

4000

"2.0

2000 ~" "1.0

0 0.0 0 20 40 60 8 0

1 .5

'1.0

0 ,5

0 .0 oooli , 3ooo 1.o

2000

0.5

1000 0

° 20 40 0'0 ' 8'0 I00 °'° J

~,J 6000 C

20 40 60 8 0

• m

" 0 . 8

"1.0 ~

"0.6

- 0.4 0.5

"0.2

0.0

0.0

< ~ , 4000

2000

©

0

4°°°iD i f r "0.12 1.0

3000

"0.08 2000 1 0 .5

1000 0 . 0 4

0.0

0 ' ' ~ ' ' ' ' ' ' 0.00 0 20 4 0 60 8 0 1 0 0 120

F R A C T I O N N U M B E R

FIc. 3. Purification of PLC-retA (PLC-/34) from a bovine retinal particulate fraction. A KC1 extract of a bovine retinal particulate fraction was subjected to sequential chromatography on a heparin-Sepharose CL-6B column (A), a TSK-Gel phenyl-SPW column (B), a TSK-Gel heparin-SPW column (C), and a Mono Q FPLC column (D). The PLC activity of the column fractions was assayed with [3H]PI as substrate. Alternate fractions from the PLC activity peak (58 to 68) from the Mono Q column were subjected to SDS-PAGE on a 6% gel, and proteins were stained with Coomassie Brilliant Blue (E). The positions of molecular size standards (kDa) are shown at left.

[18] PURIFICATION OF PLC-/34 FROM BOVINE RETINA 235

E Mono Q HPLC

Fraction Number

58 60 62 64 66 68 | I I I I I

215 -

105 -

7 0 -

4 3 -

,i

FIG. 3. (continued)

The column is washed with 400 ml of equilibration buffer, at the end of which the absorbance of the effluent drops to near zero. Bound proteins are eluted at a flow rate of 6 ml/min with a linear gradient from 0.2 to 1.8 M NaCI in a total volume of 2 liters of equilibration buffer. Fractions (18 ml) are collected and assayed for PLC activity. Fractions 32 to 39, which contain PLC-/34, are pooled and concentrated to approximately 20 ml in a stirred ultrafiltration cell fitted with a YM100 membrane (Amicon, Danvers, MA).

Step 3: Reversed-Phase Chromatography on TSK-Gel Phenyl-5PW Column. Solid KCI is added to the concentrated solution of PLC-fl4 from the previous step to give a final salt concentration of approximately 3 M,


and any insoluble materials are removed by centrifugation. The clear supernatant (-270 mg of protein) is injected onto a preparative TSK-Gel phenyl-5PW HPLC column (21.5 x 150 mm) (TosoHaas) that has been equilibrated with 20 mM HEPES (pH 7.0), 3 M NaCI, and 1 mM EGTA. Proteins are eluted, at a flow rate of 5 ml/min, by successive application of the equilibration buffer for 12 min, a decreasing linear gradient from 3.0 to 1.2 M NaC1 for 10 min, and a second decreasing linear NaCI gradient from 1.2 to 0 M for 25 min. The column is then washed with NaCl-free buffer. Fractions (2.5 ml) are collected and assayed for PLC activity. Peak fractions (63 to 68) are pooled, then washed with 20 mM HEPES, pH 7.0, 1 mM EGTA in a Centriprep-100 (Amicon) to lower the salt concentration below 0.2 M.

Step 4: Chromatography on TSK-Gel Heparin-5PW Column. The pool of desalted fractions ( -8 mg of proteins) from the phenyl-5PW column is applied to a TSK-Gel heparin-5PW HPLC column (7.5 x 75 ram) (Toso- Haas) that has been equilibrated with 20 mM HEPES (pH 7.0) and I mM EGTA. Proteins are eluted at a flow rate of 1.0 ml/min by the application of equilibration buffer for 15 min, a linear gradient from 0 to 0.64 M NaC1 for 40 min, and a second linear NaCI gradient from 0.64 to 1.0 M for 10 min. Finally, the column is washed with equilibration buffer containing 1.0 M NaC1. Fractions (1 ml) are collected and assayed for PLC activity. Peak fractions (54 to 56) are concentrated and washed with 50 mM Tris- HC1 (pH 7.4) and 1 mM EGTA in a Centricon-100 (Amicon) to reduce the salt concentration below 50 mM.

Step 5: Ion-Exchange Chromatography on Mono Q Fast Protein Liquid Chromatography Column. The desalted PLC-fl4 sample ( - 1 mg of protein) from the heparin-5PW column is applied to a Mono Q FPLC (fast protein liquid chromatography) column (7 × 60 mm) (Pharmacia LKB Biotech- nology) that has been equilibrated with 50 mM Tris (pH 7.4) and 1 mM EGTA. Proteins are eluted at a flow rate of 1.0 ml/min by successive application of equilibration buffer for 10 rain, a linear NaCI gradient from 0 to 0.3 M for 25 min, and a second linear NaCI gradient from 0.3 to 0.9 M for 30 min. Fractions (0.5 ml) are collected and assayed for PLC activity. Peak fractions (60 to 64) are concentrated, and portions are stored at - 70 °.

Chromatograms obtained in the chromatographic steps described above are shown in Fig. 3A-D. Because of the presence of other isoforms of PLC in the initial samples, it was not possible to calculate the purification (-fold) and percent yield for PLC-retA. From 4000 bovine retinas, approximately 150/zg of PLC-retA was obtained. The purified PLC-/34 preparation exhibited other protein bands of similar size in addition to the 130-kDa major band on SDS-PAGE (Fig. 3E). All of these proteins were recognized by the X and Y antibodies, but none reacted with antibod-

[19] PURIFICATION OF ~T-STIMULATED PLC 237

ies to specific PLC isozymes. Furthermore, amino acid sequences of all 12 tryptic peptides derived from the PLC-/34 preparations exhibiting multiple bands could be found in the amino acid sequence deduced from the rat cDNA. These results suggest that PLC-/34 might exist in multiple forms derived from alternatively spliced mRNAs or from proteolysis; PLC-/31 exists in 150, 140, and 100-kDa forms.

[19] Pur i f ica t ion of 110 k D a Phospho inos i t i de Phospho l i pa se C Ac t iva t ed by G -P ro t e in fly Subun i t s

By J, L. BLANK and J. H. EXTON

Introduction

The/3 isoforms of phosphoinositide-specific phospholipase C (PLC) are activated by heterotrimeric G-protein a subunits of the Gq family in response to a variety of Ca 2 +-mobilizing agonists. 1-5 Subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) yields two intraceUular second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, which mobilize intracellular calcium and activate protein kinase C, respectively. This signaling pathway is unaffected by pertussis toxin (PTX) because a subunits of the Gq class lack the site for PTX-catalyzed ADP- ribosylation. 2

The PTX-sensitive G proteins are also implicated in transducing the signal from certain Ca 2 +-mObilizing agonists to PLC.5 Experiments suggest that/33' subunits derived from PTX-sensitive G proteins (e.g., Gi and Go) mediate this activation. 6-~2 Using a reconstitution assay that measures

I M. J. Berridge, Nature (London) 361, 315 (1993). z M. I. Simon, M. P. Strathmann, and N. Gautam, Science 252, 802 (1991). 3 S. G. Rhee and K. D. Choi, J. Biol. Chem. 267, 12393 (1992). 4 p. C. Sternweis and A. V. Smrcka, Trends Biochem. Sci. 17, 502 (1992). 5 j. H. Exton, Ann. Rev. Physiol. 56, 349 (1994). 6 M. Camps, C. Hou, D. Sidiropoulos, J. B. Stock, K. H. Jakobs, and P. Gierschik, Eur.

J. Biochem. 206, 821 (1992). 7 j. L. Blank, K. A. Brattain, and J. H. Exton, J. Biol. Chem. 267, 23069 (1992). 8 j. L. Boyer, G. L. Waldo, and T. K. Harden, J. Biol. Chem. 267, 25451 (1992). 9 M. Camps, A. Carozzi, P. Schnabel, A. Scheer, P. J. Parker, and P. Gierschik, Nature

(London) 360, 684 (1992). 10 A. Katz, D. Wu, and M. I. Simon, Nature (London) 360, 686 (1992). tt A. Carozzi, M. Camps, P. Gierschik, and P. J. Parker, FEBS Lett. 315, 340 (1993). 12 D. Park, D.-Y. Jhon, C.-W. Lee, K.-H. Lee, and S. G. Rhee, J. Biol. Chem. 268, 4573

(1993).



G-protein-stimulated PIPE hydrolysis, we describe the purification of a ll0-kDa PLC from bovine brain cytosol that is markedly activated by fly subunits. 13

Assay of Phospholipase C Activation

The PLC assay is based on that originally described by Taylor and Exton. 14 Phosphatidyl[3H]inositol 4,5-bisphosphate (100 ~M [3H]PIP 2) (900 cpm/nmol) from New England Nuclear (Boston, MA) is used as substrate and is incorporated into lipid vesicles containing phosphatidylethanolamine (PE) and phosphatidylserine (PS) in the molar ratio 1 : 4 : 1. The unlabeled PIP/ is commercially available (Sigma, St. Louis, MO; Boehringer-Mannheim, Indianapolis, IN), or may be prepared from mixed phosphoinositides (Sigma) on neomycin-linked glass beads (glyceryl-CPG- 240 ,~, 200-400 mesh, Fluka, Ronkonkoma, NY) as described by Schacht. 15 The lipids PE (bovine liver) and PS (bovine brain) are supplied by Avanti Polar Lipids (Birmingham, AL).

The assay is performed at 37 ° for 15 min or less in a final volume of 200 izl. The phospholipids are dried under a stream of N2 and vesicles prepared at twice the final desired concentration by sonication into assay buffer containing 75 mM HEPES, pH 7.0, 150 mM NaCl, 4 mM EGTA, and 1 mg/ml bovine serum albumin (BSA). The CaCI2/EGTA buffer system maintains the free Ca 2 + concentration at 220 nM, as calculated from the COMICS program. 16 To monitor the enzyme during purification, fractions from each column step are assayed for PLC activity in the absence and presence of purified bovine brain fly subunits. The purification of bovine brain fly subunits is described elsewhere. 7 Routinely, fly subunits are added to the assay mixture in l0/zl or less to give a final concentration of 60 nM. Reactions are initiated by addition of 15/A or less of column fraction such that the total volume of fly plus PLC is 20 tA. The reaction is terminated by adding 200 tzl of 10% (w/v) trichloroacetic acid, followed by 100 tzl of 1% (w/v) BSA. After 5 rain on ice, the mixture is centrifuged (900 g for 4 rain), and 400 tzl of supernatant is counted.

Purification of fly-Stimulated Phospholipase C

Preparation of Bovine Brain Cytosol. All steps in the preparation of the phospholipase C are performed at 4 °. Cerebra ( - 1 kg) from four bovine

13 j. L. Blank, K. Shaw, A. H. Ross, and J. H. Exton, J. Biol. Chem. 268, 25184 0993). 14 S. J. Taylor and J. H. Exton, Biochem. J. 248, 791 0987). 15 j. Schacht, J. Lipid Res. 19, 1063 0978). 16 O. D. Perrin and I. G. Sayce, Talanta 14, 833 (1967).

[19] PURIFICATION OF fly-STIMULATED PLC 239

brains are homogenized using a Waring blender in 2 liters of 20 mM PIPES (1,4-piperazinediethanesulfonic acid), pH 6.8, containing 1 mM EDTA, I mM EGTA, 1 mM dithiothreitol (DTT), 2.7 mM KC1, I0 ~g/ml leupeptin, 10/xg/ml antipain, 5 /xg/ml aprotinin, and 5 ~g/ml APMSF (4-amidino- phenylmethanesulfonyl fluoride). The homogenate is centrifuged at 13,700 g for 30 min and the supernatant recentrifuged at 100,000 g for 60 min.

Heparin-Sepharose Chromatography. The 100,000 g supernatant (-1.5 liters; 10 g total protein) is loaded at 5 ml/min onto a 300-ml heparin-Sepharose CL-6B (Pharmacia-LKB, Piscataway, N J) column (2.6 × 57 cm) equilibrated with 20 mM PIPES, pH 6.8, 1 mM EDTA, I mM DTT (buffer A), containing 2.7 mM KC1, 10 /zg/ml leupeptin, 10 p.g/ml antipain, 5/zg/ml aprotinin, and 5/~g/ml APMSF. After washing overnight with 3 liters of buffer A, the column is developed with a linear gradient from 0 to 300 mM NaCI in 1140 ml of this buffer, and fractions of 12 ml are collected at a flow rate of 3 ml/min. Fractions eluting from heparin-Sepharose column are assayed for PLC activity in the absence and presence of purified bovine brain fly subunits. Fractions are pooled on the basis of fl~/-stimulated PLC activity, which elutes as a single peak at approximately 200-250 mM NaC1. Heparin-Sepharose removes PLC-fll, PLC-yl, and PLC-81 from the principal fly-stimulated PLC activity, as assessed by Western blotting with corresponding monoclonal antisera (Upstate Biotechnology Inc., Lake Placid, NY). The procedure for immunoblot analysis of PLC isozymes has been described elsewhere. 7 The enzymes remain bound to heparin-Sepharose under the conditions described, and they elute in order PLC-yl, PLC-fll, then PLC-81 when the column is developed with a second gradient of NaC1, between 400 and 700 mM.

Phenyl-Sepharose Chromatography. The pool from the heparin- Sepharose step (-168 ml) is brought to 35% saturation with (NH4)2SO 4 (20.9 g/100 ml) and stirred for 30 min at 4°C; insoluble proteins are removed by centrifugation at 10,800 g for 20 min. The supernatant is loaded at 3 ml/min onto a 150-ml phenyl-Sepharose 6 Fast Flow-Low Sub (Phar- macia-LKB) column (2.6 × 28 cm) equilibrated in buffer A containing 1.2 M (NH4)2SO 4. The column is washed with 300 ml of column equilibration buffer at the same flow rate and eluted at 2 ml/min with a reverse gradient of (NH4)2SO 4 from 1.2 to 0 M in 800 ml of buffer A. Elution is completed with a further 150 ml of buffer A, and fractions of 10 ml are collected. The fly-stimulated PLC activity elutes as a single peak at approximately 0.4 M (NH4)2SO 4. Peak fractions ( -90 ml) are pooled and dialyzed overnight against two changes of 2 liters of buffer A containing 2.7 mM KC1.

Q-Sepharose Chromatography. The dialyzed proteins are applied at 4 ml/min to a 100-ml Q-Sepharose Fast Flow (Pharmacia-LKB) column


(2.6 x 19 cm), equilibrated with buffer A containing 2.7 mM KCI. After washing with 200 ml of equilibration buffer containing 50 mM NaC1, the column is eluted at 2 ml/min with an 800-ml gradient from 50 to 300 mM NaC1, followed by a 150-ml gradient to 1 M NaCI in this buffer. The fraction size is I0 ml. A peak of basal (fly-independent) PLC activity is detected in fractions containing the trailing edge of the fly-stimulated activity which, on some occasions, appears as a partially resolved double peak. Therefore, fractions containing the peak and leading edge of the fly-stimulated PLC activity, which display little or no basal PLC activity, are pooled (-150 ml) to partly avoid this second activity.

Hydroxylapatite Chromatography. The Q-Sepharose pool is concentrated to approximately 20 ml using an Amicon (Danvers, MA) filtration cell fitted with a YM30 membrane and loaded onto a 24-ml column of high-performance liquid chromatography (HPLC)-grade hydroxylapatite (Calbiochem, La Jolla, CA) (1.0 x 30 cm) equilibrated in 20 mM PIPES, pH 6.8, containing 0.1 mM EDTA, 1 mM DTT, 2.7 mM KCI, and 100 mM NaCI (buffer B). The column is washed with 50 ml of this buffer and eluted at 2 ml/min with a linear gradient of K2HPO4 from 0 to 250 mM in 320 ml of buffer B, collecting 4-ml fractions. Hydroxylapatite chromatography resolves the fly-dependent PLC activity into three distinct peaks. The second and third minor peaks elute later in the gradient and are associated with a significantly higher basal activity than that detected in the first, major fly-stimulated peak. Fractions containing the principal fly-stimulated PLC activity eluting at approximately 1 I0 mM KzHPO4 are pooled (-28 ml) and dialyzed overnight against 2 liters of buffer A.

Blue-Sepharose Chromatography. The dialyzed protein solution is loaded at 1 ml/min onto an 8-ml column of blue-Sepharose Fast Flow (Pharmacia-LKB) (1.0 x 10 cm) that has been equilibrated in buffer A. After washing, the column is eluted at 2 ml/min with a linear gradient from 0 to 1.2 M NaCI in 240 ml of buffer A, and 4-ml fractions are collected. The fly-stimulated activity elutes as a broad peak between approximately 340 and 600 mM NaCI; active fractions are pooled (-50 ml) and concentrated to approximately 2-3 ml using a Centriprep 30 concentrator (Amicon).

Sephacryl S-300 Gel Filtration. The concentrate is applied to a 320-ml Sephacryl S-300 HR HiLoad 26/60 column (Pharmacia-LKB) that has been equilibrated in 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM DTT (buffer C) containing 150 mM NaCI. The column is eluted at 0.5 ml/min, and 3-ml fractions are collected. The fly-stimulated PLC activity elutes as a symmetrical peak centered at fractions 60-61 (-180 ml elution volume).

Mono Q Chromatography. Active fractions are pooled (12 ml), diluted with 2 volumes of buffer C, and applied to a l-ml Mono Q HR5/5 column

[19] PURIFICATION OF By-STIMULATED P L C 241

TABLE I PURIFICATION OF PHOSPHOLIPASE C

Total activity ~ Total protein b Specific activity Purification Yield Column (/zmol/min) (rag) 0xmol/min/mg) (-fold) (%)

Hepar in-Sepharose 2.82 663 0.0043 l 100 Phenyl-Sepharose 2.41 71.8 0.0336 8 86 Q-Sepharose 1.33 1.11 1.20 279 47 Hydroxylapat i te 0.62 0.095 6.53 1520 22 Blue-Sepharose 0.51 N.D. - - - - 18 Sephacryl S-300 0.19 N.D. - - - - 6.7 Mono Q 0.12 0.010 c 12.0 2790 4.3

Determined in the presence of 63 nM/33,. b Determined by AMIDO black staining. N.D. indicates that protein concentrations were not

detectable above blank values. ¢ Estimated by silver staining using PLC-/31 of known concentration to construct a standard curve. ~3

(Pharmacia-LKB) equilibrated with this buffer. The column is washed with 10 ml of buffer C containing 100 mM NaC1, then eluted with a linear gradient from 100 to 350 mM NaCI in 25 ml of buffer C, followed by a 5-ml gradient to 1 M NaC1. Fractions of 1 ml are collected at a flow rate of 1 ml/min. Fractions containing the peak of PLC activity, which elutes at approximately 240-280 mM NaCI, are supplemented with 20% (v/v) glycerol, divided into aliquots, frozen in liquid N2, and stored at - 70 °. Omission of glycerol or repeated freezing and thawing results in a complete loss of enzyme activity after purification.

As summarized in Table I, this procedure results in an approximately 2000-fold purification of the enzyme from the pool obtained by heparin- Sepharose chromatography of bovine brain cytosol, with a final yield of approximately 4%. Approximately 10/zg of the purified enzyme is obtained from four bovine brains. Because the enzyme has a negligible basal activity in the absence of fly, purification is assessed using a fixed concentration of fl~/in the reconstitution assay, routinely 60 nM. Values are calculated based on the pool of activity obtained by heparin-Sepharose chromatography of bovine brain cytosol, from which the basal (fly-independent) PLCs have been largely removed.

Properties of fl*/-Stimulated Phospholipase C

The enzyme migrates on 10% polyacrylamide-sodium dodecyl sulfate (SDS) gels with an apparent molecular mass of 1 l0 kDa and is essentially pure as judged by silver stain analysis (Fig. 1). The enzyme, designated


200 kDa-

116 kDa- 97 kDa-

66 kDa-

55 kDa- ~ i ~

J

~ 1 1 0 kDa PLC

37 kDa-

FIG. 1. Silver stain analysis of purified fly-stimulated PLC-110. Purified fly-stimulated PLC from Mono Q chromatography (36 ng PLC-110, right-hand lane) was subjected to 10% polyacrylamide-SDS gel electrophoresis and stained with silver according to previously published procedures. 7 The left-hand lane shows the marker proteins myosin (200 kDa), fl- galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), glutamate dehydrogenase (55 kDa), and lactate dehydrogenase (37 kDa).

16

E

• ~ 12 o E

• -~ 8

2 R "l-

f f 4 a .

f I I

/ /

/ /

~ 0

J

0 100 200 300 400 500 600

[ ~ y ] ( n i )

FIG. 2. Stimulation of purified PLC-110 by bovine brain fly subunits. The hydrolysis of PIPz by 12 ng PLC-110 was determined in the presence of varying amounts of purified brain fly subunits as described in the text. The basal (fly-independent) specific activity of PLC- 110 in the experiment shown was 0.1 /zmol/min/mg, and maximal activation by fly was approximately 150-fold.

[19] PURIFICATION OF fly-STIMULATED PLC 243

F=

E e'}

£

0., .

/ /

/ ; q / , ~ ? o - o - o - o - - o - o - - o ~ o

i I , I , i i l i I

0 0.01 0.1 1 10 100 1000

[ Ca 2+ ] ( I.tM )

FIo. 3. Calcium dependence of/3y-stimulated PLC-110. The hydrolysis of PIP2 by 19 ng PLC-I10 was measured in the absence (O) and presence (0) of 63 nM/33, as described in the text, except that CaZ+/EGTA buffers were used to vary the free calcium concentration, as indicated. Basal PLC-110 activity was in the range of 0-0.16/zmol/min/mg.

PLC-110, is not recognized by monoclonal antisera raised to bovine brain PLC-/31, PLC-yl, and PLC-81, which have apparent molecular masses of 150, 145, and 85 kDa, respectively. 3 An antiserum to a synthetic peptide corresponding to a region of the Y domain conserved among mammalian PLC isozymes L7 cross-reacts with PLC-110, confirming its relatedness to these enzymes. PLC-110 is also recognized by two antisera selective for N-terminal amino acid residues 58-67 (GRYARLPKDP) and for residues 550-561 (TDPKKPTTDEGT) in PLC-/33, ~8 indicating that PLC-110 is related to PLC-/33. However, PLC-110 is not recognized by an antiserum raised to a sequence corresponding to amino acid residues 1206-1217 (ADSESQEENTQL) at the C-terminus of PLC-/33, TM indicating that PLC-110 is a C-terminal truncated form of PLC-/33. Further proof that PLC-110 is derived from PLC-/33 is provided by the sequences of 15 tryptic peptides obtained from the 110-kDa protein (J. L. Blank, S. Afendis, C. Moomaw, C. A. Slaughter, and J. H. Exton, unpublished findings). These show the presence of the PLC-/33 sequence up to residue 856,

17 D. Park, D.-Y. Jhon, R. Kriz, J. Knopf, and S. G. Rhee, J. Biol. Chem. 267, 16048 (1992). L8 D.-Y. Jhon, H.-H. Lee, D. Park, C.-W. Lee, K.-H. Lee, O. J. Yoo, and S. G. Rhee, J.

Biol. Chem. 268, 6654 (1993).


consistent with PLC-110 being a C-terminally truncated form of the enzyme.

Activation of PLC-110 by/3y is direct and produces a greater than 100-fold stimulation of PIP2 hydrolysis over basal. Half-maximal activation requires approximately 60 nM fly, and full activation is observed at about 500 nM f17 (Fig. 2). Purified liver and brain/3y are equipotent. PLC-110 has no activity with phosphatidylinositol as substrate, and it hydrolyzes phosphatidylinositol 4-phosphate and PIPE maximally at 1-I00/zM Ca 2÷ . The calcium dependence of PLC-110 with PIP2 as substrate is shown in Fig. 3. Whereas/33/subunits produce a dramatic activation of PLC-110, GTPyS-liganded Ogq, which is a potent activator ofPLC-/313-5 and PLC-/33,18 has no effect. This observation indicates that the site at which/3Y interacts with PLC-110 is distinct from that at which O~q regulates the activity of PLC-/3 isozymes. As experiments have demonstrated that the O~q interaction site is located in the C terminus of PLC-fll,19,2o our findings support the conclusion that PLC-I I0 is a PLC-/3 isozyme that has lost this domain but has retained that involved in fly interaction.

19 D. Park, D.-Y. Jhon, C.-W. Lee, S. H. Ryu, and S. G. Rhee, J. Biol. Chem. 268, 3710 (1993).

2o D. Wu, H. Jiang, A. Katz, and M. I. Simon, J. Biol. Chem. 268, 3704 (1993).

[20] Ampl i f ica t ion of Phospha t idy l inos i to l -Spec i f ic P h o s p h o l i p a s e C-fl I soforms Us ing D e g e n e r a t e P r i m e r s

By HAI-WEN MA, RAVI IYENGAR, and RICHARD T. PREMONT


Hydrolysis of phosphatidylinositol (PI) on the inner leaflet of the cell membrane to liberate phosphorylated inositols and diacylglycerol is catalyzed by a family of calcium-dependent, phosphatidylinositol-specific phospholipase C (PLC) enzymes. Three distinct classes of PI-specific phospholipase C enzymes, called/3, 3~, and 8, have been characterized by both protein purification and cDNA cloning and expression. 1 Multiple members of each class have been identified. Hormonal stimulation of PI

I S. G. Rhee, Trends Biochem. Sci. 16, 297 (1991).


[201 DEGENERATE PRIMERS FOR PLC-fl ISOFORMS 245

hydrolysis mediated by G proteins is due to activation of members of the PLC-/3 class, 2 whereas hormones which activate tyrosine kinases can stimulate PI hydrolysis through tyrosine phosphorylation and activation of members of the PLC-y class. 1 Physiological regulation of the PLC-8 class remains unclear.

The PLC-/3 enzymes have been shown to be stimulated by a subunits of the Gq family of heterotrimeric G proteins,2 although the three expressed PLC-/3 subtypes that have been tested differ as to which members of the Gqa family best stimulate PI hydrolysis. 3-5 The PLC-/31 and PLC-/33 isozymes are stimulated best by Gq~ and GHa, whereas PLC-/32 is better stimulated by Gl6o~. 3-5 Additionally, activation of PLC-/3 enzymes by the /33 ̀subunits of heterotrimeric G proteins, in the absence of ~ subunits, has also been demonstrated. 4'6-9 The potency of/33` subunits to stimulate PLC activity varies among the PLC-fl subtypes. 9 Hence, regulation of known PLC-/3 enzymes by G-protein a and/33, subunits is very complex, and additional PLC-/3 subtypes may exhibit additional regulatory specificities.

Several distinct PLC-fl types have been cloned from mammals, Xenopus, and Drosophila. Comparison of the deduced amino acid sequences with one another and with cloned PLC-3` and PLC-6 sequences has allowed the identification of two regions of high similarity (called the X and Y boxes), which are thought to be involved in substrate binding and catalysis. ~ Analysis of the aligned protein sequences has allowed the identification of conserved regions which may be useful for preparing degenerate oligonucleotide primers for the polymerase chain reaction (PCR) amplification of additional PLC subtypes or identifying the presence of known subtypes in tissues or cultured cell lines. Sequences common to known PLC-/3, -3`, and -8 sequences, as well as to each individual class of PLC enzyme, have been identified. The PLC-/3-specific sequences are presented here. For a discussion of general

2 A. V. Smrcka and P. C. Sternweis, Trends Biochem. Sci. 17, 502 (1992). 3 C. H. Lee, D. Park, D. Wu, S. G. Rhee, and M. I. Simon, J. Biol. Chem. 267, 16044

(1992). 4 D.-Y. Jhon, H.-H. Lee, D. Park, C.-W. Lee, K.-H. Lee, O. J. Yoo, and S. G. Rhee, J.

Biol. Chem. 268, 6654 (1993). J. R. Hepler, T. Kozasa, A. V. Smrcka, M. I. Simon, S. G. Rhee, P. C. Sternweis, and A. G. Gilman, J. Biol. Chem. 268, 14367 (1993).

6 M. Camps, A. Carozzi, P. Schnabel, A. Scheer, P. J. Parker, and P. Gierschik, Nature (London) 3611, 684 (1992).

7 A. Katz, D. Wu, and M. I. Simon, Nature (London) 360, 686 (1992). 8 A. J. Carozzi, M. Camps, P. Gierschik, and P. J. Parker, FEBS Lett. 315, 340 (1993). 9 A. V. Smrcka and P. C. Sternweis, J. Biol. Chem 268, 9667 (1993).


methods for amplification from degenerate oligonucleotide primers, see Refs. 10-12.

Subtype-Selective Primers for Phospholipase C-fl Subtypes

Four vertebrate PLC-fl subtypes (bovine 13 and rat 14 PLC-fl l , human PLC-fl2,15 human 16 and rat 4 PLC-fl3, and Xenopus PLC-f117) and two Dro- sophila PLC-fl sequences (norpA TM and plc2119) were aligned to identify regions of high sequence conservation. Each region was then compared to the corresponding regions in PLC-T and -8 sequences to identify the residues conserved among all PLC subtypes. Several regions longer than six amino acids and containing one or more PLC-fi-specific residue were identified in the X and Y domains. In many instances, distinct differences between the four vertebrate and the two Drosophila PLC-fl sequences were present which would require the synthesis of different primer sequences for each class. Portions of the alignment are shown in Fig. 1.

Taking into account amino acid degeneracies and codon usage, primer sequences corresponding to HGFTMTT(E/D) , L S F E N H V D , and QQAK- MAEYC in the X box and NYMPQ(L/M)FWN and VEV(D/E)(M/L)FG in the Y box appear appropriate for preparing oligonucleotide primers for amplifying vertebratelike PLC-fl sequences. For primers biased toward the Drosophila PLC-fl sequences, the X box sequence QQAK(M/I/L)A- (E/N/K)YC and the Y box sequence VEV(D/E)(M/L)(F/Y)G can be used to prepare more highly degenerate primers, or less degenerate primers based solely on the Drosophila sequences [i.e., L S F E N H C in X box or FWN(A/S)GCQ in Y box] can be prepared.

Because the X and Y boxes are 170 and 250 amino acids in length, the conserved PLC-fl sequences within the X and Y boxes are necessarily

l0 R. T. Premont, this volume [9]. IIT. W. Wilkie, A. M. Aragay, A. J. Watson, and M. I. Simon, this series, Vol. 237 [26]. 12 C. Gallagher and N. Gautam, this series, Vol. 237 [37]. 13 M. Katan, R. W. Kriz, N. Totty, R. Philp, E. Meldrum, R. A. Aldape, J. L. Knopf, and

P. J. Parker, Cell (Cambridge, Mass.) 54, 171 (1988). 14 P.-G. Suh, S. H. Ryu, K. H. Moon, H. W. Suh, and S. G, Rhee, Cell (Cambridge, Mass.)

54, 161 (1988). 15 D. Park, D.-Y. Jhon, R. Kriz, J. Knopf, and S. G. Rhee, J. Biol. Chem. 267, 16048 (1992). 16 A. J. Carozzi, R. W. Kriz, C. Webster, and P. J. Parker, Eur. J. Biochem. 210, 521 (1993). 17 H.-W. Ma, R. D. Blitzer, E. C. Healy, R. T. Premont, E. M. Landau, and R. lyengar,

J. Biol. Chem. 268, 19915 (1993). 18 B. T. Bloomquist, R. D. Shortridge, S. Schneuwly, M. Perdrew, C. Montell, H. Steller,

G. Rubin, and W. L. Pak, Cell (Cambridge, Mass.) 54, 723 (1988). ~9 R. D. Shortridge, J. Yoon, C. R. Lending, B. T. Bloomquist, M. H. Perdew, and W. L.

Pak, J. Biol. Chem 266, 12474 (1991).

[]20] DEGENERATE PRIMERS FOR PLC-/3 ISOFORMS 247

X BOX

cow I~1 rat [31 hum 1~2 hum [33 rat 1~3 cow 134 rat 1~4 Xen 13 Dro norpA Dro plc21 rat plc y1 rat plc 81

PVITHGFTMTTEISFKEVIEAIAECAFKTSPFPILLSFENHVDSPKQQAKMAEYCRLIFG PVITHGFTMTTEISFKEVIEAIAECAFKTSPFPILLSFENHVDSPKQQAKMAEYCRLIFG PIITHGFTMTTDIFFKEAIEAIAESAFKTSPYPIILSFENHVDSPRQQAKMAEYCRTIFG PFITHGFTMTTEVPLRDVLEAIAETAFKTSPYPVILSFENHVDSAKQQAKMAEYCRSIFG PFITHGFTMTTEVPLRDVLEAIAETAFKTSPYPVILSFENHVDSAKQQAKMAEYCRSIFG PIITHGKAMCTDILFKDVIQAIKETAFVTSEYPVILSFENHCS-KYQQYKMSKYCEDLFG PIITHGKAMCTDILFKDVIQAIKETAFVTSEYPVILSFENHCS-KYQEYQMSKYCEDLFG PFITHGFTMTTEIPFKEVIEAIAESAFKTSPFPVILSFENHVDSSKQQAKMAEYCRNIFG PIVTHGHAYCTEILFKDCIQAIADCAFVSSEYPVILSFENHCN-RAQQYKLAKYCDDFFG PVIVHGYTFVPEIFAKDVLEAIAESAFKTSEYPVILSFENHCN-PRQQAKIANYCREIFG PVIYHGHTLTTKIKFSDVLHTIKEHAFVASEYPVILSIEDHCS-IAQQRNMAQHFRKVLG PIIYHGYTFTSKILFCDVLRAIRDYAFKASPYPVILSLENHCS-LEQQRVMARHLRAILG

Y BOX

cow 131 rat 151 hum 132 hum 133 rat ~3 cow 134 rat !34 Xen 13 Dro norpA Dro plc21 rat plc y1 rat plc 81

QLSRIYPKGTRVDSSNYMPQLFWNAGCQMVALNFQTVDLAMQINMGMYEYNGKSGYRLKP QLSRIYPKGTRVDSSNYMPQLFWNAGCQMVALNFQTVDLAMQINMGMYEYNGKSGYRLKP QMSRIYPKGTRMDSSNYMPQMFWNAGCQMVALNFQTMDLPMQQNMAVFEFNGQSGYLLKH QLSRIYPKGTRVDSSNYMPQLFWNVGCQLVALNFQTLDVAMQLNAGVFEYNGRSGYLLKP QLSRIYPKGTRVDSSNYMPQLFWNVGCQLVALNFQTLDLPMQLNAGVFEYNGRSGYLLKP QMSRIYPKGGRVDSSNYMPQIFWNSGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKP QMSRIYPKGGRVDSSNYMPQIFWNAGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKP QLSRIYPKGTRVDSSNYMPQLFWNAGCQMVALNFQTLDLPMQLNVGIFEYNRRSGYLLKP QMSRIYPKGTRADSSNYMPQVFWNAGCQMVSLNFQSSDLPMQLNQGKFEYNGGCGYLLKP QLSRVYPAGTRFDSSNFMPQLFWNAGCQLVALNFQTLDLAMQLNLGIFEYNARSGYLLKP QLSRIYPKGQRLDSSNYDPLPMWICGSQLVALNFQTPDKPMQMNQALFMAGGHCGYVLQP CLSRIYPAGWRTDSSNYSPVEMWNGGCQIVALNFQTPGPEMDVYLGCFQDNGGCGYVLKP

Ft6. 1. Partial alignment of PLC-/3 X and Y box regions. Deduced amino acid sequences of the PLC-fl enzymes were aligned using the GeneWorks software (Intelligenetics, Mountain View, CA).

quite close together. Primers entirely within the X box [HGFTMTT- (D/E) to QQAKMAEYC] would amplify only 34 internal codons, whereas primers in the Y box [NYMPQ(L/M)FWN to VEV(D/E)(M/L)FG] would amplify 76 internal codons. The close proximity of the X and Y boxes to one another in the PLC-/3 class (from 71 to 130 amino acids apart) allows the efficient amplification from X to Y box sequences. Thus, the primers given below are oriented for amplification from X (sense) to Y (antisense) box sequences, and the predicted size of amplified products for each pair is given in Table I. All primers contain a site for Xhol and a six-base pair (bp) enzyme "clamp" at the 5' end.

Primer pair for amplifying vertebratelike PLC-fl sequences: HGFTM- TT(E/D) sense


TABLE I PREDICTED POLYMERASE CHAIN REACTION PRODUCT SIZES (bp) FOR VARIOUS

PHOSPHOLIPASE C-fl FORMS a

Primer pair /31 /32 r3 /34 Xen/3 plc21 norpA

HGFTMTT(ED) 741 759 891 - 846 - - NYMPQ(ILM)FWN

LSFENHC - - - 744 - 837 681 FWN(AS)GCQ

QQAK(IML)(AS)(ENK)YC 873 891 1023 963 978 1053 897 VEV(ED)M(FY)G

a Fragments which are not expected to amplify with a given primer pair are indicated b y " - "

5'-cccgtcctcgagca(c/t)gg(i)tt(c/t)ac(i)atgac(i)ac(i)ga NYMPQ(L/M/I)- FWNantisense

5'-tggcacctcgagttcca(a/g)aa(i)a(a/g/t)(c/t)tg(i)ggcat(a/g)ta(a/g)tt

Note. i, Inosine. Bovine 2°'2~ and rat 22 retinal PLC-fl4 cDNAs share highest similarity with the Drosophila norpA sequence, and have distinctly "Drosophila"-like sequences quite different from the "mammalian" X box sequence primer above (HGKAMCTD). However, the Y box primer shown should amplify PLC-fl4, as should both "Drosophila"-biased primers and both "general" primers given below.

Primer pair for amplifying Drosophila-like PLC-fl sequences: LSFENHC sense

5'-gtgatcctcgagct(i)tc(i)tt(c/t)ga(a/g)aa(c/t)ca(c/t)tg FWN(A/S)GCQ antisense

5'-gagtgcctcgagctg(a/g) ca(i)cc(i)g(a/c)(a/g)ttcca(a/g)aa

Primer pair for amplifying general PLC-fl-like sequences: QQAK- (I/M/L)(A/S)(E/N/K)YC sense

5'-gactccctcgagca(a/g)ca(a/g)gc(i)aa(a/g)(a/c)t(i)(g/t)c(i)(a/g)a(i)ta- (c/t)tg VEV(E/D)M(F/Y)G antisense

5'-ccgagtctcgagccc(a/g)(a/t)acat(i)tc(i)ac(c/t)tc(i)ac

20 p. A. Ferreira, R. D. Shortridge, and W. L. Pak, Proc. Natl. Acad. Sci. U.S.A. 90, 6042 (1993).

21 C.-W. Lee, D. J. Park, K.-H. Lee, C. G. Kim, and S. G. Rhee, J. Biol. Chem. 268, 21318 (1993).

22 M. J. Kim, Y. Y. Bahk, D. S. Min, S.-J. Lee, S. H. Ryu, and P.-G. Suh, Biochem. Biophys. Res. Commun. 194, 706 (1993).

[20] DEGENERATE PRIMERS FOR PLC-/~ ISOFORMS 249

Polymerase Chain Reaction Methods

RNA Isolation. Total RNA is isolated by the guanidiniurrdCsCl gradient method 23 and poly(A) + RNA selected on oligo(dT) spin columns (Phar- macia/LKB, Piscataway, N J) according to the manufacturer's instructions. Alternatively, poly(A) ÷ RNA may be isolated directly from cell lysates by oligo(dT) spun column chromatography using the FastTrack system (Invitrogen, San Diego, CA).

cDNA Synthesis. First-strand cDNA is prepared using total or poly(A)- RNA as a template for reverse transcriptase. The reverse transcription reaction is performed in an RNase-free tube using 1 ~g of poly(A) ÷ RNA or 5 tzg of total RNA and 1 p.l of 100/zM oligo(dT)]s primer brought to a volume of 11 tzl with diethyl pyrocarbonate (DEPC)-treated water. The RNA and primer are heated for 5 min at 75 ° and cooled on ice. Two microliters of 100 k~M dithiothreitol (DTT), 1 ~l (40 U) of RNasin placental RNase inhibitor (Promega, Madison, WI), 1 izl of 10 mM mixed deoxynucleoside triphosphates (dNTPs) (Boehringer Mannheim, Indianapolis, IN), 4 Izl of 5 x SuperScript buffer, and 1 tzl (200 U) of SuperScript reverse transcriptase (GIBCO-BRL, Gaithersburg, MD) are added to the tube on ice. The 20-~1 reaction is incubated for 1 hr at 42 °, diluted to 100 Izl with DEPC-treated water, and stored at -20 ° until use. One microliter contains the cDNA prepared from 10 ng of poly(A) RNA or 50 ng total RNA. The synthesis can be monitored by adding 1 tzCi of [a-a2p]dCTP or other dNTP tracer to the reaction.

Polymerase Chain Reaction Amplification. Amplification of DNA sequences is performed in a programmable thermal cycler using the heat- stable Taq DNA polymerase. 24 Standard reactions are performed in 100-1A volumes and contain 1 × Taq buffer with 1.5 mM MgCI 2 , 200 tzM each dNTP, 500 nM each of the sense and antisense primers, and l0 ng of first-strand cDNA template. Reactions are covered with 100 tzl of light mineral oil (Sigma, St. Louis, MO) and heated for 5 min at 95 ° to denature the templates initially. While the samples are held above the chosen annealing temperature, 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT) is added to the individual tubes through the mineral oil layer, and cycling is begun immediately. For the primers given here, the standard reaction conditions are 35 cycles of 95 ° for 1 min denaturation, 55 ° for 1 min annealing, and 72 ° for 3 min extension, followed by a final 10-min extension at 72 °.

23 j. Sambrook, E. F. Fritsch, and T. Maniatis (eds.), "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.

24 M. A. Innes, D. H. Gelfand, J. J. Sninsky, and T. J. White (eds.), "PCR Protocols: A Guide to Methods and Applications." Academic Press, San Diego, 1990.


Probe: PLC-I] I

Probe:PLC-~2

Probe: PLC-I~3

PLC-~I PLC-p2 PLC-p3

i i

FIG. 2. Specificity of three PLC-/3 DNA probes. One hundred nanograms of full-length bovine PLC-/31 and human-/32 cDNAs and 30 ng of a 750-bp fragment of PLC-fl3 cDNA (VPLRDVL to LTKSPMI) were dot blotted in triplicate on nitrocellulose. Strips containing all three PLC-fl cDNAs were then hybridized to random-prime labeled PLC-fll, -/32, or -/33 cDNA probes. Hybridization was performed in 6× NET, 5x Denhardt's, 0.1% SDS, and 100/~g/ml salmon sperm DNA at 65 ° overnight. Blots were washed and exposed to X-ray film to determine probe cross-reactivity with distinct PLC-/3 cDNA sequences.

Characterization of Polymerase Chain Reaction Products. The completed reaction is extracted with 175 ~1 of chloroform to remove the mineral oil, and the upper aqueous phase is transferred to a clean tube and dried in a SpeedVac (Savant, Farmingdale, NY) concentrator. The PCR reactions separated on 1% agarose gels are transferred by capillary action to nitrocellulose membranes and probed by hybridization at high stringency to individual random-prime labeled DNA probes 23 that are specific for the PLC-/3 subtypes. The probes were a 560-pb DNA fragment from PLC-fll (FENHVDS to MQLSRIY), a 560-bp DNA fragment from PLC-fl2 (FENHVDS to FVDYNKR), and a 750-bp DNA fragment from PLC-/33 (VPLRDVL to LTKSPMI). These fragments were obtained by PCR amplification using primers corresponding to the following amino acids: for PLC-/31, FENHVDS (in box X) and MQLSRIY (in box Y); for PLC-/32, FENHVDS (in box X) and FVDYNKR (in box Y); and for PLC-/33, VPLRDVL (in box X) and LTKSPMI (in box Y). Probe DNAs were labeled to greater than 108 cpm//xg, and were present at 10 6 cpm/ml. Hybridization is performed in 6x NET (Ix NET is 150 mM NaCI, 1 mM EDTA, 15 mM Tris-HCl, pH 7.5), 5x Denhardt's [1 x Denhardt's is 0.2 mg/ml Ficoll, 0.2 mg/ml bovine serum

[20] DEGENERATE PRIMERS FOR PLC-/3 ISOFORMS 251

A

1 2 3 4 5 6 7 8 9 1 0 1 1 12

1 3 5 3 . . . . . . . . . . . . 1 0 7 8 - -

8 7 2 - - 6 0 3 - -

3 1 0 - -

- 1 3 5 3

- 1 0 7 8

- 8 7 2

- 6 0 3

1 3 5 3 - 1 0 7 8 -

8 7 2 -

6 0 3 -

1 2 3 4 5 6 7 8 9 10 11 12

C

1 3 5 3 - - 1 0 7 8 - -

8 7 2 - -

6 0 3 - -

3 1 0 - -

1 2 3 4 5 6 7 8 9 10 11 12

- - 1 3 5 3

- - 1 0 7 8

- - 8 7 2

- - 6 0 3

FIG. 3. Identification of PLC-fl-like sequences in PCR mixtures (using "mammalian" primers) from various tissues. Ten nanograms of first-strand cDNA was amplified for 35 cycles as described using the "mammalian"-biased PLC-/3 primer pair [HGFTMTT(ED) sense and NYMPQ(LMI)FWN antisense]. Thirty microliters of the reaction was separated in 1% agarose gels, which were all transferred to nitrocellulose. Each blot was hybridized with an individual PLC-/3 probe under conditions as described for Fig. 2. Autoradiograms are shown for PLC-/31 (A), PLC-/32 (B), and PLC-/33 probes (C). Expected products are 741 bp (ill), 759 bp (02), and 891 bp (03). Lane 1, bovine brain; lane 2, bovine kidney; lane 3, bovine lung; lane 4, bovine heart, lane 5, bovine liver; lane 6, bovine spleen; lane 7, rat brain; lane 8, rat liver; lane 9, human HEK-293 cells; lane 10, rabbit liver; lane 1 l, chicken liver; lane 12, rat skeletal muscle.

a lbumin (BSA), 0.2 mg/ml poly-v inylpyr ro l idone] , 0.1% sodium dodecy l sulfate (SDS), and 100 ~g /ml sonica ted sa lmon sperm D N A at 65 ° overnight , fo l lowed by washing for 15 rain at 65 ° in 2x SSC ( l x SSC is 150 m M NaCI , 15 m M sodium citrate, p H 7.5) and 0.1% SDS. 23 The c ross - reac t iv i ty o f the PLC-f l p robes , assessed under these condi t ions by hybr id iz ing each p robe with dot blots conta in ing each PLC-f l c D N A , was negligible (Fig. 2).


Amplification of Phospholipase-fl Sequences

The X box sense HGFTMTT(E/D) and Y box antisense NYMPQ- (L/M)FWN primers were synthesized and used to amplify PLC-fl-like sequences from various tissues and cell lines. Under cycling conditions as described (35 cycles of 95 ° for 1 min, 55 ° for 1 min, 72 ° for 3 min), product bands have been obtained which are of appropriate size to represent PLC- fll or PLC-fl2 (741-759 bp) as well as PLC-fl3 (891 bp) in several tissues. To determine whether the PCR products represent PLC-fl-like sequences, the PCR mixtures were separated on agarose gels and hybridized at high stringency with PLC-fll, -f12, and -f13 probes. As seen in Fig. 3, PCR product bands of the size expected for PLC-fll, -f12, and -f13 are detected in various tissues. In addition, bands of distinct sizes, which may represent novel PLC forms or alternatively spliced forms of known PLCs, are also observed in several tissues. Further analysis of these unexpected PCR products may reveal even greater diversity in the PLC-fl family.

[2 II GUANINE NUCLEOTIDES ASSOCIATED WITH Ras 255

[21] Analysis of Guan ine Nucleot ides Associated with

Pro tooncogene Ras

By RXCHARD R. VAILLANCOURT, ANNE E. HARWOOD, and SIM WINITZ


The 21-kDa protooncogene Ras is a member of the family of low molecular weight guanine nucleotide-binding proteins whose biological activity is pivotal for cellular growth and differentiation. Ras binds GTP on stimulation of quiescent cells and is active in the GTP-bound form. Growth factors such as thrombin which binds to a G-protein-coupled seven-transmembrane receptor and the classic tyrosine kinase receptors which bind epidermal growth factor (EGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF) are examples of receptor-mediated pathways which, on stimulation, lead to Ras activation. Tyrosine phosphorylated receptors recruit specific adaptor proteins, such as Grb2 (growth factor receptor-bound protein),l which are characterized by having both SH2 and SH3 (Src homology) domains. The Grb2 protein, through the SH2 domain, recognizes specific phosphotyrosine domains within a receptor and the Ras guanine nucleotide exchange factor, Sos (the homo- log of Drosophila Son of Sevenless), through the SH3 domain, 2-8 The complex of receptor, Grb2, and Sos converts Ras from a GDP-bound, inactive state, to a GTP-bound, active state. The GTPase-activating protein (GAP) tightly regulates Ras activity by stimulating the intrinsically low GTPase activity of Ras. The effector(s) which GTP-bound Ras activates has not been identified although it has been shown that activated

t E. J. Lowenstein, R. J. Daly, A. G. Batzer, W. Li, B. Margolis, R. Lammers, A. Ullrich, E. Y. Skolnik, D. Bar-Sagi, and J. Schlessinger, Cell (Cambridge, Mass.) 70, 431 (1992).

2 M. A. Simon, G. S. Dodson, and G. M. Rubin, Cell (Cambridge, Mass.) 73, 169 (1993). 3 S. E. Egan, B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, and R. A.

Weinberg, Nature (London) 363, 45 (1993). 4 M. Rozakis-Adcock, R. Fernley, J. Wade, T. Pawson, and D. Bowtell, Nature (London)

363, 83 (1993). s N. Li, A. Batzer, R. Daly, V. Yajnik, E. Skolnik, P. Chardin, D. Bar-Sagi, B. Margolis,

and J. Schlessinger, Nature (London) 363, 85 (1993). 6 N. W. Gale, S. Kaplan, E. J. Lowenstein, J. Schlessinger, and D. Bar-Sagi, Nature

(London) 363, 88 (1993). 7 L. Buday and J. Downward, Cell (Cambridge, Mass.) 73, 611 (1993). 8 K. Matuoka, F. Shibasaki, M. Shibata, and T, Takenawa, EMBO J. 12, 3467 (1993).


256 GROWTH AND TRANSFORMATION [21]

Ras binds another protooncogene, Raf, in oi t ro . 9-12 Thus, activated Ras triggers a series of serine/threonine kinase reactions including the mitogen- activated protein kinase (MAPK) cascade.

The activation of Ras in response to cell growth and differentiation stimuli can be assessed by measuring the level of guanine nucleotides bound to the protein. The activity of Ras is measured by first radiolabeling the phosphate pool of quiescent cells with ortho[aEp]phosphate. The cells are then stimulated with either hormones or growth factors. Following cell lysis Ras is immunoprecipitated, and the guanine nucleotides are eluted from the protein and then separated by thin-layer chromatography. The levels of GTP and GDP can be quantitated and the data expressed as the percentage of the ratio of GTP relative to the amount of total guanine nucleotides [GTP/(GDP + GTP) × 100].

Methods

The following protocol can be used with many different cell types such as rat pheochromocytoma PC-12 cells 13 and fibroblasts. 14 Cells which are 80% confluent (10-cm dish) are washed once with Tris-buffered salien (25 mM Tris, pH 7.5, 150 mM NaC1) and serum-starved to induce quiescence with 10 ml phosphate-free Dulbecco's modified Eagle's medium (DMEM) (GIBCO-BRL, Gaithersburg, MD) supplemented with 1 mM sodium pyruvate, 100 units/ml penicillin, 100/zg/ml streptomycin, and 0.1% bovine serum albumin (BSA). After 12-18 hr, 30/zl of 32po 4 (10 mCi/ml water, NEN, Boston, MA) is added to the medium, and the cells are maintained in an incubator for up to 6 h.

The cells are stimulated with 3/zl of 100 ~g/ml human recombinant EGF (Upstate Biotechnology, Inc., Lake Placid, NY) at ambient temperature. After 5 min, the cells are washed twice with ice-cold PBS (10 mM Na2HPO 4, pH 7.4, 1.7 mM KHEPO 4, 136 mM NaCI, and 2.6 mM KCI) and then lysed with buffer containing 25 mM Tris, pH 7.5, 150 mM NaCI, 16 mM MgCl2, 1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% (v/v) aprotonin (Sigma, St. Louis, MO), and 0.1 mg/ml

9 S. A. Moodie, B. M. Willumsen, M. J. Weber, and A. Wolfman, Science 260, 1658 (1993). 10 X.-F. Zhang, J. Settleman, J. M. Kyriakis, E. Takeuchi-Suzuki, S. J. Elledge, M. S.

Marshall, J. T. Bruder, U. R. Rapp, and J. Avrueh, Nature (London) 364, 308 (1993). IIp. H. Warne, P. R. Viciana, and J. Downward, Nature (London) 364, 352 (1993). 12 L. Van Aelst, M. Barr, S. Marcus, A. Polverino, and M. Wigler, Proc. Natl. Acad. Sci.

U.S.A. 90, 6213 (1993). 13 B.-Q. Li, D. Kaplan, H.-F. Kung, and T. Kamata, Science 256, 1456 (1992). 14 T. Satoh, M. Endo, M. Nakafuku, T. Akiyama, T. Yamamoto, and Y. Kaziro, Proc.

Natl. Acad. Sci. U.S.A. 87, 7926 (1990).

[21] GUANINE NUCLEOTIDES ASSOCIATED WITH Ras 257

A

GDP

G T P

Origin

B

60-

o 50-

x 40. n I--

+ 3 0 - o.. D

~,~ 20 - I.- (.9 10-

0 a b Basal EGF

FIG. 1. Analysis of guanine nucleotides associated with immunoprecipitated Ras. Rat l a cells were labeled with [32p]p i and left untreated (a) or stimulated with 30 ng/ml EGF for 5 min (b). Ras was immunoprecipitated with monoclonal antibody Y13-259, and the guanine nucleotides were analyzed by thin-layer chromatography on polyethyleneimine cellulose using 0.75 M KH2PO 4 , pH 3.5, as solvent. The autoradiogram is shown in (A). Nonradioactive GDP and GTP were used as standards and migrated with relative mobilities of 0.36 and 0.18, respectively. Radioactive GDP and GTP were quantitated using a Molecular Dynamics Phosphorlmager (B).

leupeptin, which is supplemented with Ras monoclonal antibody YI3- 25915 at a final concentration of 20/xg/ml (Santa Cruz Biotechnology). The cells are immediately removed from the dish with a disposable cell scraper (Fisher Scientific), and the lysate is placed in a 1.5-ml screw- capped microcentrifuge tube.

Nuclei and cell debris are removed from the lysate by centrifugation at 14,000 g for 10 min. The supernatant is incubated at 4 ° for 1 hr. Mean- while, 3/zl of the supernatant is quantitated by liquid scintillation counting. An equal amount of radioactivity is added to 30/zl of goat anti-rat immunoglobulin coupled to agarose (Sigma) which is prepared by diluting 30/zl of the suspension into 30/zl oflysis buffer. The mixture is centrifuged in a microcentrifuge at 1000 rpm for 1 min followed by removal of the supernatant. The lysate and agarose beads are rotated for 1 hr at 4 °. The immune complexes are collected by centrifugation at 1000 rpm for 1 rain and washed with 1 ml of buffer consisting of 25 mM Tris, pH 7.5, 150 mM NaCI, 16 mM MgC12, and 1% (v/v) Nonidet P-40 followed by gentle agitation.

After 5 washes the immune complexes are resuspended with 20/xl of elution buffer consisting of 2 mM EDTA, 0.2% sodium dodecyl sulfate

~5 M. E. Furth, L. J. Davis, B. Fleurdelys, and E. M. Skolnick, J. Virol. 43, 294 (1982).

258 GROWTH AND TRANSFORMATION 122l

(SDS), and 2 mM dithiothreitol (DTT), then heated at 100 ° for 3 min. The supernatants are collected by centrifugation at 14,000 g for 5 min and are stable to freezing at - 8 0 °. Nucleotides are analyzed by thin-layer chromatography by applying 5-7/xl to a polyethyleneimine cellulose plate coated with fluorescent indicator (J. T. Baker Chemical Co., Phillipsburg, NJ). GDP and GTP are resolved by chromatography in 0.75 M KH2PO4, pH 3.5. Nonradioactive GDP and GTP are used as standards. In addition to autoradiography, the radiolabeled nucleotides are imaged and quantitated using a PhosphorImager (Molecular Dynamics) (Fig. 1).

The assay can be used to measure the change in GTP/GDP ratio of guanine nucleotides bound to Ras. A time course of exchange reactions should be performed for each stimulus and cell type.

Acknowledgments

This work was supported by National Institutes of Health Grant DK 08897 and the Cancer League of Colorado.

[22] M e a s u r i n g Ac t iva t i on o f K inases in M i t o g e n - A c t i v a t e d P r o t e i n K i n a s e R e g u l a t o r y N e t w o r k

By ANNE M. GARDNER, CAROL A. LANGE-CARTER, RICHARD R. VAILLANCOURT, and GARY L . JOHNSON

Introduction

Mitogen-activated protein kinases (MAPKs) are serine threonine kinases that are rapidly activated in response to a variety of growth factors in many cell types (reviewed in Refs. 1-3). Phosphorylation of both tyrosine and threonine residues is required for activation. 4 This family of kinases is proposed to play a key role in the conversion of growth factor receptor tyrosine kinase [i.e., epidermal growth factor (EGF) receptor and platelet-derived growth factor (PDGF) receptor] signaling to SER/

1 S. L. Pelech and J. S. Sanghera, Science 257, 1355 (1992). 2 Z. G. Boulton, S. H. Nye, D. J. Robbins, N. Y. Ip, E. Radziejewska, S. D. Morgenbesser,

R. A. DePhinho, N. Panayotatos, M. H. Cobb, and G. D. Yancopoulos, Cell (Cambridge, Mass.) 65, 663 (1991).

3 G. L'Allemain, J. Pouyssegar, and M. J. Weber, Cell Regul. 2, 675 (1991). 4 N. G. Anderson, J. L. Mailer, N. K. Tonks, and T. W. Sturgill, Nature (London) 343,

651 (1990).


[22] MAP KINASE REGULATORY NETWORK 259

Tyrosine Kinases Serpentine Receptors

Ras / G Proteins / / \X

/ \

I I Raf MEKK

MEK

MAPK

R k 90 EGFR) ( c-Myc, c-Jun, cPLA 2 , s ,

FIG. 1. Mitogen-activated protein kinase network. Arrows indicate regulation by phosphorylation between kinases acting in the pathway(s) which lead to the activation of MAPK. Known substrates for MAPK are also listed.

THR kinase activation. However, pertussis toxin-sensitive Gi2-coupled receptors (i.e., thrombin receptor) also induce the rapid activation of MAPKs. 3

Regulation of MAPK activation by different receptor types is poorly defined but involves a series of upstream phosphorylation events within parallel but integrated "kinase networks" (Fig. 1). Thus, certain cytoplasmic SER/THR protein kinases within the networks are likely to behave as a convergence point(s) for diverse membrane receptor-initiated signaling events. Of great interest is the identification and biochemical characterization of unique effectors in the receptor-tyrosine kinase versus G-protein-mediated pathways leading to the activation of MAPK. A direct upstream activator of vertebrate/mammalian MAPK (MEK- 1; MAPK kinase) has been cloned and characterized by several laboratories. ~-7 The MEK-1 protein activates MAPK by specific phosphorylation of both re-

5 C. M. Crews, A. Alessandrini, and R. L. Erickson, Science 258, 478 (1992). 6 R. Seger, D. Seger, F. J. Lozeman, N. Ahn, L. Graves, J. S. Campbell, L. Ericsson, M.

Harrylock, A. M. Jensen, and E. G. Krebs, J. Biol. Chem. 267, 25628 (1992). 7 j. Wu, J. K. Harrison, L, A. Vincent, C. Haystead, T. A. J. Haystead, H, Michel,

D. F. Hunt, K. R, Lynch, andT. W. Sturgill, Proc. Natl. Acad. Sci. U.S.A. 90, 173 (1993).


quired tyrosine and threonine residues 8,9 and is itself regulated by phosphorylation. ~0 Activation of MEK-1 appears to be tightly linked to MAPK activation. I~ However, cell-specific and receptor-specific differences in the regulation of MEK-I suggest that it is regulated by multiple upstream activators which are also kinases. Raf-1 is a SER/THR protein kinase which is an immediate upstream activator of MEK-1. ~2 A second SER/ THR protein kinase, MEK kinase (MEKK), also phosphorylates and activates MEK-1 independent of Raf. 13 Thus, MEKK and Raf define a diver- gence point upstream of MEK (Fig. 1). In this chapter, assays for measuring the activity of these key kinases (MAPK, MEK, Raf, and MEKK) acting in the MAPK regulatory network are described.

Mitogen-Activated Protein Kinase Assay

The elution profile for pp42 and pp44 MAPK from anion-exchange chromatography is illustrated in Fig. 2. Anion-exchange chromatography of PC-12 cell extracts is performed at 4 ° with a Pharmacia LKB Biotechnology Inc. (Piscataway, NJ) FPLC (fast protein liquid chromatography) system. The PC-12 cells are cultured as described TM and are serum-starved in Dulbecco's modified Eagle's medium (DME) supplemented with 0.1% bovine serum albumin (BSA) for 12-18 hr at 37 °, and stimulation with 30 ng/ml PDGF-BB (Upstate Biotechnology, Inc., Lake Placid, NY) is performed for 10 rain at 37 °. After stimulation the cells are washed twice with ice-cold phosphate-buffered saline (PBS) followed by the addition of 650/xl of lysis buffer (70 mM fl-glycerophosphate, pH 7.2, I00/zM sodium vanadate, 2 mM MgCI2, 1 mM EGTA, 0.5% Triton X-100, 5 /zg/ml leupeptin, 2 /.Lg/ml aprotinin, and 1 mM dithiothreitol). The attached cells are scraped from the dish, collected into a microcentrifuge tube, and spun for 15 min at 15,000 rpm. Soluble extracts (500 tzl, 0.5-1 mg protein) are applied to a Mono Q HR 5/5 FPLC column equilibrated in buffer (70 mM/3-glycerophosphate, pH 7.2, 100/zM sodium vanadate, 1 mM EGTA, and 1 mM dithiothreitol),

8 M. H. Cobb, T. G. Boulton, and D. T. Robbins, Cell Regul. 2, 965 (1991). 9 L. B. Ray and T. W. Sturgill, Proc. Natl. Acad. Sci. U.S.A. 85, 3753 (1988). x0 S. Matsuda, H. Kosako, K. Takenaka, K. Moriyama, H. Sakai, T. Akiyama, Y. Gotoh,

and E. Nishida, EMBO J. 11, 973 (1992). 11 A. M. Gardner, R. R. Vaillancourt, and G. L. Johnson, J. Biol. Chem. 268, 17896 (1993). 12 j. M. Kyriakis, H. App, X.-F. Zhang, P. Banerjee, D. L. Brautigan, U. R. Rapp, and J.

Aruch, Nature (London) 358, 417 (1992). 13 C. A. Lange-Carter, C. M. Pleiman, A. M. Gardner, K. J. Blumer, and G. L. Johnson,

Sc&nce 260, 315 (1993). 14 L. E. Heasley and G. L. Johnson, Mol. Biol. Cell 3, 545 (1992).


t - -

o m~--

#_o ~ ' ~

n~ ,,,-~

W

100-

75-

50-

25-

- 250

- 20O

- 150

L - 100

i ~ m ~ , . ~ m m m , ~ ~ , m ~ 5 0

10 12 14 16 18 20 22

t¢ Z

E

i

Fraction, ml

FIG. 2, Analysis of growth factor-stimulated PC-12 cells containing fl-PDGF receptor. The PC-12 cells were stimulated (Q) with 30 ng/ml PDGF-BB (Upstate Biotechnology, Inc.) for 10 rain at 37 ° or were unstimulated (A). Soluble PC-12 cell lysates (1.4 mg/ml) were prepared and fractionated on Mono Q FPLC. Phosphorylated pp42 and pp44 were eluted with 150 and 175 mM NaCI, respectively. The activated enzyme was measured by quantitating phosphorylated EGFR (662-681) peptide which contains the consensus MAPK phosphorylation site PXTP [A. K. Erickson, D. M. Payne, P. A. Martino, A. J. Rossomando, J. Shabanowitz, M. J. Weber, D. F. Hunt, and T. W. Sturgill, J. Biol. Chem. 265, 19728 (1990)].

The column is washed with 2 ml of buffer and the bound proteins eluted with a 28-ml linear 0-350 mM NaC1 gradient in the equilibration buffer.

Aliquots (20/~1) of 1-ml fractions are mixed with 20/xl of 50 mM/3- glycerophosphate, pH 7.2, 100/zM sodium vanadate, 20 mM MgCI z , 200 /zM [y-32p]ATP (2000-5000 dpm/pmol), 50 ~g/ml IP20 (inhibitor peptide, TTYADFIASGRTGRRNAIHD, cAMP-dependent protein kinase inhibitor), ~5 1 mM EGTA, and 400/~M EGFR 662-681 peptide (RRELVEPLTP- SGEAPNQALLR). 16 After 15 min at 30 °, 10/zl of 25% trichloroacetic acid (TCA) is added, and 45/xl of the reaction mix is spotted onto 2-cm squares of P-81 phosphocellulose paper (Whatman, Chifton, N J). The papers are washed three times (5 min each) in 400 ml of 75 mM phosphoric acid and dehydrated once by washing with acetone. The phosphocellulose papers are allowed to dry and are placed into 7-ml vials with 4 ml scintillation fluid and counted by liquid scintillation counting.

t5 H.-C. Cheng, B. E. Kemp, R. B. Pearson, A. J. Smith, L. Misconi, S. M. Van Patten, and D. A. Walsh, J. Biol. Chem. 261, 989 (1986).

~6 K. Takishima, I. Griswold-Prenner, T. lngebritsen, and M. R. Rosner, Proc. Natl. Acad. Sci. U.S.A. 88, 2520 (1991).


Myelin basic protein (MBP) can be used as an alternative substrate to assay MAPK activity. 17 The assay is terminated by spotting an aliquot of the reaction mixture on P-81 phosphocellulose paper, rather than by TCA precipitation, and the washing procedure is the same as described above.

The fractions obtained by chromatography can be concentrated for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting by TCA precipitation. ~8 The sample (9 volumes) is precipitated with 1 volume of 72% (w/v) TCA, 0.15% sodium deoxycholate in a microcentrifuge tube for 2 hr at 4 °. The precipitate is collected by centrifugation for 10 min at 15,000 rpm and then washed twice with 1 ml ice- cold acetone. The precipitated pellet is allowed to dry and then resuspended in 100 t~l SDS-PAGE sample buffer (1 × SDS buffer contains 2% sodium dodecyl sulfate, 5% glycerol, 62.5 mMTris-Cl, pH 6.8, 5% (v/v) 2-mercaptoethanol, and 0.001% bromphenol blue) and boiled for 3 rain.

MEK Activity Assay

Activation of MAPK by tyrosine kinase-encoded receptors is dependent on Ras, 19 whereas G-protein-mediated activation of MAPK appears to occur predominately through Ras-independent pathways. 1 To examine the role of MEK in transducing receptor-mediated signals, we developed an in vitro assay using recombinant MAPK to measure MEK activity in growth factor-stimulated cell lysates. H Rat-la fibroblasts are placed in serum-free medium containing 0.1% BSA for 16-18 hr to induce quiescence. Cells are treated with growth factors [30 ng/ml recombinant human EGF (Upstate Biotechnology, Inc.) for 5 rain, 0.2 U/ml thrombin for 3 rain] or are untreated, washed twice in I0 ml ice-cold PBS, and lysed in 650 /.d of cell lysis buffer [70 mM fl-glycerophosphate, 20 mM 2-(N- morpholino)ethanesulfonic acid (MES), pH 6.0, 100/xM sodium vanadate, 2 mM MgCl 2 , 1 mM EGTA, 0.5% Triton X-100, 2/.tg/ml leupeptin, 2/xg/ ml aprotinin, and 1 mM dithiothreitol]. After centrifugation for 10 min at maximum speed in a microcentrifuge, cell lysates containing 1-2 mg soluble protein are applied to a Mono S HR 5/5 FPLC column equilibrated in loading buffer (70 mM fl-glycerophosphate, 20 mM MES, pH 6.0, 100 /zM sodium vanadate, 1 mM EGTA, and 1 mM dithiothreitol). The column is washed with 2 ml of buffer and the bound proteins eluted with a 28-ml linear 0-350 mM NaCl gradient in loading buffer.

17 A. K. Erickson, D. M. Payne, P. A. Martino, A. J. Rossomando, J. Shabanowitz, M. J. Weber, D. F. Hunt, and T. W. Sturgill, J. Biol. Chem. 265, 19728 (1990).

18 A. Bensadoun and D. Weinstein, Anal. Biochem. 70, 241 (1976). 19 G. Thomas, Cell (Cambridge, Mass.) 68, 3 (1992).


Thirty microliters of each 1-ml column fraction is assayed for the ability to phosphorylate recombinant kinase-inactive MAPK by mixing with 10 /zl kinase buffer {100 mM/3-glycerophosphate, 160 mM HEPES, pH 7.2, 200/xM sodium vanadate, 40 mM MgC12 , 100 IzM [y-3Zp]ATP (2000-5000 dpm/pmol), I00/~g/ml IPz0, 2.0 mM EGTA, 30/xg/ml recombinant kinase- inactive MAPK}. After a 30-min incubation at 30 °, the reaction is terminated by the addition of 10/xl of 5X SDS sample buffer, boiled for 3 min, and subjected to SDS-PAGE and autoradiography. Figure 3A shows a typical elution profile for MEK-1 activity from the Mono S cation-exchange column. A prominent peak of MAPK phosphorylating activity was found in fractions 10 to 14 (100-150 mM NaC1) of EGF- or thrombin- stimulated cell lysates.

Coupled MEK Activity Assay

To demonstrate that the peak of MAPK phosphorylation activity represents a MAPK activator, we have used a coupled assay in which the fractions are assayed for the ability to activate wild-type recombinant MAPK which, in turn, phosphorylates the EGFR (662-681) peptide substrate. In this assay, 30 ~1 of each column fraction is assayed for the ability to activate recombinant wild-type MAPK by mixing with 10 /xl kinase buffer {100 mM/3-glycerophosphate, 160 mM HEPES, pH 7.2,200 /xM sodium vanadate, 40 mM MgC12, 400 /zM [7-32p]ATP (3000-4000 dpm/pmol), 100/xg/ml IP20,2.0 mM EGTA, 30/zg/ml recombinant MAPK, and 800 IzM EGFR (662-681) peptide}. After a 20-min incubation at 30 °, the incorporation of 32p into EGFR (662-681) peptide is measured by binding to P-81 paper, as described for the MAPK assay.

Two major peaks of activity were detected by this assay (Fig. 3B). In reactions that lack recombinant MAPK, the early eluting peak of activity was still present (Fig. 3D), indicating that it most likely represents endogenous activated MAPK, which directly phosphorylates the EGFR peptide substrate. Indeed, p42 MAPK was detected in these fractions by immunoblotting (Fig. 3E). The major form of MAPK found in these cells is p42 MAPK; p44 MAPK is undetectable in the immunoblot. We consistently found more MAPK activity in fraction 2, whereas the majority of the protein was found in fraction 3. Apparently, active MAPK binds Mono S less tightly than inactive nonphosphorylated MAPK. The second peak corresponds to the elution position of the MEK activity detected by phosphorylation of kinase-inactive MAPK. This peak (fractions 11-13) contained a protein recognized by an affinity-purified rabbit antiserum raised against a peptide encoding a COOH-terminal region of MEK- 1 (STIGLN- QPSTPTH) (Fig. 3C). A minor peak of activity was often found in fraction


B 40

C

N

lO

~ o

A + EGF

Control ~ Kinase"

MAPK

2 4 6 8 10 12 14 16

2 4 6 8 10 12 14 16 Fraction, ml

D 30

" i 20

lO

u 0

MEK-1 -'~

8 10 12 14 16

2 4 6 8 10 12 14 16 Fraction, ml

E MAPK

2 4 6 8 10 12 14 16

FIG. 3. Activity of MEK in EGF-stimulated Rat-1 a cells. (A) Phosphorylation of kinase- inactive MAPK was measured after Mono S FPLC fractionation of lysates from Rat-1 a cells challenged for 5 rain with EGF (30 ng/ml) or without (control). (B) Activation of recombinant wild-type MAPK. Column fractions of EGF-stimulated and unstimulated cell lysates were analyzed for the ability to activate recombinant wild-type MAPK, which then phosphorylates the EGFR (662-681) peptide substrate. The later eluting peak of EGFR peptide kinase activity corresponds to the peak of EGFR peptide kinase activity detected by phosphorylation of kirtase-inactive MAPK in (A). (C) Immunoblotting detects MEK-1 in the fractions from


7 (Fig. 3B,D). This peak may correspond to an uncharacterized EGFR peptide kinase activity that is distinct from MAPK and MEK. ~

Expression and Purification of Recombinant Kinases for Use as $ubstrates

Expression Plasmids

A construct designed to express MAPK with a polyhistidine sequence at the NHz terminus is prepared by ligating NspBII-digested Xenopus oocyte p42 MAPK cDNA with PvuII-digested bacterial expression vector pRSETB, u A kinase-inactive MAPK fusion protein is created by site- directed mutagenesis in which the active site lysine-57 is converted to methionine using the oligonucleotide 5-TGCATATCAT*GAAAATCAG- 3'. A MEK-1 cDNA is obtained by polymerase chain reaction (PCR) cloning of cDNA templates from mouse B cell poly(A) + RNA based on the sequence reported by Crews et al. 5. The sense primer is 5'-TTCCCG- GATCCAAGATGCCCAAGAAGAAGCCGAC-Y, which overlaps the initiation methionine and incorporates a 5' BamHI site. The antisense primer 5'-CTTTGAAGCTTCCTAAAGGCTCAGATGCTGGC-3' corresponds to a region 3' to the termination codon and incorporates a 3' HindlII site. The PCR product is subcloned into the pRSETA expression vector. The kinase-inactive MEK-1 is generated by site-directed mutagenesis of lysine-97 to methionine using the oligonucleotide 5'-GTGGATCAG- CA*TTCTAGCCAT-Y.U

Growth and Induction of Recombinant Proteins

One difficulty in expressing recombinant MAPK and MEK-1 for use as substrates is that the proteins are predominantly insoluble. However, the following protocols produce enough soluble recombinant MAPK and MEK-1 for many assays. We use the BL21(DE3)LysS strain of Esche-

the peak of MAPK activator (fractions 11-13). No MEK-1 immunoreactive band was detected in fractions 2 and 3 (data not shown). (D) The early eluting EGFR peptide kinase peak contains endogenous MAPK. Fractions were analyzed as in (B), except reactions lacked recombinant MAPK and were incubated for 15 rain. The early eluting peak is not dependent on addition of recombinant MAPK. (E) Immunoblotting detects p42 MAPK in the early eluting EGFR peptide kinase peak. Fractions were probed with monoclonal anti- MAPK antibody, revealing MAPK to be present in fractions 2 and 3. In some cell types, unactivated MAPK is found in fractions 15 and 16.


richia coli (reviewed by Studier et al. 2°) because it is deficient in the Ion protease and lacks the ompT outer membrane protease that can degrade proteins during purification. 21 Bacteriophage DE3 is a h derivative that carries a DNA fragment containing the lac! gene, the LacUV5 promoter, the beginning of the lacZ gene, and the gene for T7 RNA polymerase. 22 The LacUV5 promoter, which is inducible by isopropyl-/3-D-thiogalactopyranoside (IPTG), directs transcription of the T7 RNA polymerase gene. The presence of IPTG induces T7 RNA polymerase expression which in turn transcribes the MAPK or MEK-1 DNA in the plasmid. The basal level of T7 RNA polymerase activity results in some transcription of the kinases in uninduced cells, and because MAPK and MEK-1 are insoluble proteins they tend to be toxic. To reduce the basal T7 RNA polymerase activity, the LysS strain of BL21 (DE3) is used. The LysS strain expresses a low level of T7 lysozyme, which binds to T7 RNA polymerase and inhibits transcription. 23 This strain has the further advantage that freezing and thawing allows the endogenous lysozyme to lyse the cell efficiently. The LysS plasmid is maintained by 25/xg/ml chloramphenicol.

The E. coli BL21(DE3) LysS cultures containing kinase constructs are grown in Luria broth [10 g tryptone, 5 g yeast extract, 10 g NaC1 per liter water (pH 7.2-7.4)] with 50 tz/ml ampicillin and 25/zg/ml chloramphenicol to an OD600 of approximately 0.5. Glycerol stocks are prepared 24 and stored at -70 °. To start cultures for induction, 50 t-d of the glycerol stocks is inoculated into 5 ml of SOB [20 g tryptone, 5 g yeast extract, 0.58 g NaC1, 0.186 g KCI per liter water (pH 6.8-7.0)] with 0.4% glucose, 50 /~g/ml ampicillin, and 25/~g/ml chloramphenicol. The constructs do not induce well if started from diluted overnight cultures or from plates more than 1 day old. The cultures are incubated at 37 ° with shaking until slightly turbid ( - 3 - 4 hr). The 5-ml culture is then used to inoculate 500 ml of Luria broth containing 50 ~g/ml ampicillin and 25/.Lg/ml chloramphenicol in 2-liter flask. The incubation is continued at 37 ° with shaking until an OD600 of 0.5 to 0.6 is attained ( -4 -5 hr). Freshly made IPTG in water (0.5 mM final) is added to induce fusion protein synthesis, and the cultures are incubated for an additional 2 to 3 hr. The cultures are harvested by centrifugation in GSA bottles at 8000 g for 15 min at 4 °. The pellets can be stored at -20 ° indefinitely. To analyze induction, 1-ml samples of bacteria are removed 0, 1, 2, and 3 hr after induction. The bacteria are

20 F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, this series, Vol. 185, p. 60.

21 j . Grodberg and J. J. Dunn, J. BacterioL 170, 1245 (1988). 22 F. W. Studier and B. A. Moffat, J. Mol. Biol. 189, 113 (1986). 23 B. A. Moffat and F. W. Studier, Cell (Cambridge, Mass.) 49, 221 (1987). z4 T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual."

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982.


microcentrifuged at 14,000 rpm for 30 sec, and the pellets are resuspended in 200 tA of lysis buffer (3% SDS, 3% (w/v) 2-mercaptoethanol, and 0.5% (w/v) bromphenol blue) and boiled for 7 min. Then 10 to 20 t~l of each lysate is fractionated by SDS-PAGE and stained with Coomassie blue.

Purification of Recombinant Histidine-Tagged Kinases

For each 500 ml of bacterial culture, the pellet is resuspended in 50 ml of ice-cold lysis buffer (50 mM sodium phosphate, pH 8.0, 100 mM KC1, 0.1% Tween 20, 10 mM (v/v) 2-mercaptoethanol, 5 tzg/ml leupeptin, 2.1 p~g/ml aprotinin). The cells are lysed by freezing in liquid N z or dry ice/methanol and by thawing quickly at 37 °. The lysate is put on ice, and the DNA is sheared by sonication. The lysate is clarified by centrifugation at 10,000 g for 15 min at 4 °. The inclusion bodies, which contain the insoluble recombinant protein, are found in the pellet. The clear supernatant, containing the soluble MAPK or MEK-1 proteins, is mixed with 0.5 ml of NiZ+-nitrilotriacetic acid (NTA)-agarose (Qiagen, Chatsworth, CA) on an end-over-end mixer for 1 hr at 4 °. The beads are washed at 4 ° 3 times with 10 ml of lysis buffer and 3 times with 10 ml of lysis buffer, pH 6.3, and are eluted 3 times with 1 ml of lysis buffer, pH 4.5. The eluate is dialyzed first against 10 mM HEPES, pH 7.2, 1 mM EDTA, 0.1% 2- mercaptoethanol, 0.025% Triton X-100, 2.1 t~g/ml aprotinin for 1 hr and then against 50% glycerol, 10 mM HEPES, pH 7.2, 1 mM EDTA, 0.1% 2-mercaptoethanol, 0.025% Triton X-100, 2.1 /zg/ml aprotinin for 2 to 3 hr and stored at -20 °. Approximately 2 mg of purified wild-type or kinase- inactive MAPK and 1 mg of purified wild-type or kinase-inactive MEK- 1 is routinely obtained per 500 ml of bacteria. As judged by Coomassie blue staining, the purity of the recombinant kinases is between 50 and 80%. The purity can vary widely depending on the batch of Ni2+-NTA - agarose used.

Immunoprecipitation of Raf-1 and Kinase Assay

Figure 4 illustrates Raf-1 immunoprecipitation and kinase activity assayed from lysates of cultured cells. Cells are cultured in 10-cm dishes until they reach 70-80% confluence (I-2 dishes per experimental condition) and deprived of serum for 18-20 hr prior to immunoprecipitation. To immunoprecipitate c-Raf-1, cells in 10-cm dishes are placed on ice, washed twice with ice-cold PBS, and lysed by scraping in 0.5 to 1 ml of RIPA [10 mM sodium phosphate, pH 7.0, 150 mM NaCI, 2 mM EDTA, 1% sodium deoxycholate, 1% Nonidet P-40 (NP-40), 0.1% SDS, 1% aprotinin, 50 mM NaF, 200 p.M Na3VO4,0.1% (v/v) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF). The insoluble material is pelleted by centrifugation at 10,000 g for 10 min at 4 ° and discarded. To reduce


K inase " MEK-1 - - ~

m D

B. 2 - ,.- (.9

o ILl o LU C) + 0 +

- 4 - Raf

FIG. 4. Immunoprecipitation of Raf and kinase assay. Raf was immunoprecipitated as described in the text from quiescent (Control) COS cells or COS cells treated with human EGF (30 ng/ml) for 10 min (+EGF). (A) Raf kinase assay using purified recombinant catalytically inactive (kinase-) MEK-1 as substrate. (B) Immunoblot of immunoprecipitated Raf protein.

background arising from nonspecific binding sites, soluble cell lysates may be precleared by preincubation with 25 tzl of a suspension of formalin- fixed Staphylococcus aureus (protein A; Sigma No. P-7155) in RIPA for 30 min at 4°; this step is optional and will depend on the particular cell type used. The formalin-fxed Staphylococcus aureus (protein A) is prepared by washing with RIPA (1 : 2, v/v), spinning at 4000 rpm for 10 min, and then resuspending with the original volume of RIPA. If preclearing, the protein A may be removed by centrifugation at 10,000 g for 5 rain.

The soluble cell lysate (or precleared supernatant) is incubated with a rabbit antibody (I/I00 dilution) to the C terminus ofRaf-125 (No. SC133, Santa Cruz Biotechnology) for 90 min at 4 °. The immune complexes are then incubated with 25 Izl of washed protein A for 30 min at 4 ° and spun through a 600-/zl cushion of RIPA/10% sucrose in 10 × 75 mm polystyrene tubes for 20 min at 2500 rpm. The immune complexes are washed twice with 1 ml ice-cold RIPA, twice with 1 ml ice-cold PAN buffer (10 mM PIPES, pH 7.0, 100 mM NaCI, 20/zg/ml aprotinin) containing 0.5% NP-40, and once with 1 ml ice-cold PAN. For each wash the immune complexes are pelleted by centrifugation at 2500 rpm for 7 min and resuspended by gentle vortexing. The pellet is resuspended in 0.5 ml PAN buffer, transferred to a 0.5-ml Eppendorf tube, and repelleted by centrifugation for I min at 5000 rpm. This final pellet is resuspended in 10-20/~1 PAN buffer.

For an in vitro kinase assay, 5-10 tzl of the PAN suspension is incubated with catalytically inactive MEK- 1 (50-100 ng) and 10-30/zCi of [~/-32p]ATP (2000-5000 dpm/pmol) in buffer (20 mM PIPES, pH 7.0, 10 mM MnCI2, and 20/zg/ml aprotinin) in a final volume of 20-50/zl for 15-30 min at

25 A. M. Schultz, T. O. Copeland, G. E. Mark, U. R. Rapp, and S. Orosylan, Virology 146, 78 (1985).

t22] MAP KINASE REGULATORY NETWORK 269

+ + - M E K K

+ - + w t M E K - 1

-41-wt MEK-1 "4-Kinase- MAPK

FIG. 5. Coupled MEKK activity assay. The MEKK protein was partially purified from COS cells transiently overexpressing MEKK by FPLC Mono Q column chromatography as described in the text. The MEKK (contained in column fraction 22) was incubated in the presence (+) or absence (-) of purified recombinant wild-type (wt) MEK-1 and in the presence of catalytically inactive MAPK (Kinase- MAPK). The MEKK phosphorylated and activated wild-type MEK-1, leading to MAPK phosphorylation. Autophosphorylated MEKK is also visible in the +MEKK lanes.

30 ° with frequent gentle mixing. Reactions are stopped by the addition of an appropriate volume of 5X SDS sample buffer. The samples are boiled for 3 min and subjected to SDS-PAGE and autoradiography. Figure 4A illustrates a Raf kinase assay in which Raf was immunoprecipitated from COS cells treated with or without EGF. As measured by MEK- 1 phosphorylation, Raf was robustly activated in the EGF-stimulated cells compared to unstimulated control cells. Figure 4B shows an immunoblot of the immunoprecipitated Raf contained in 5-10/zl of the same PAN suspension used in the kinase assay. Equal amounts of Raf were immunoprecipitated for each experimental condition.

Some purchased preparations of Staphylococcus aureus-derived protein A may contain contaminating phosphatase which can affect the kinase assay results. Protein A-Sepharose CL-4B (10-20/zl of a 1 : 1 slurry in RIPA) may be substituted. To prepare protein A-Sepharose CL-4B, pre- swell the beads by incubation in an excess volume of PBS for 1 hr, wash twice in fresh PBS, and wash three times in RIPA. The resin may be prepared in advance and stored as a 1 : 1 slurry in RIPA at 4 °. Raf-1 is more efficiently immunoprecipitated from some cell types if lysis is done with extraction buffer (EB) (1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaC1, 50 mM NaF, 0.1% w/v bovine serum albumin, 20/zg/ml aprotinin, 1 mm PMSF, 2 mm Na3VO4). EB is then used in place of RIPA to prepare formalin-fixed staphylococcus aureus (protein A).


Purification of MEK Kinase and Coupled Activity Assay

Figure 5 illustrates a coupled assay system in which purified recombinant MAPK and MEK-1 were used to assay the activity of MEKK, a protein kinase which phosphorylates and activates MEK independently of Raf.~3 This assay system demonstrates the great utility of purified recombinant proteins in the biochemical characterization of kinases acting upstream of MEK in the MAPK network. The MEKK protein is transiently overexpressed in COS-1 cells as described, ~3 and active MEKK contained in cell lysates is partially purified by fractionation on a FPLC Mono Q column using the same column conditions as described above for the MAP kinase assay. A portion (20/~1) of the peak fraction containing MEKK (fraction 22 as determined by immunoblotting) is mixed with buffer (50 mM fl-glycerophosphate, pH 7.2, 100/zM sodium vanadate, 20 mM MgCI 2 , 1 mM EGTA, 50 ~M ATP, 50/zg/ml IP20, and 10/zCi [3' -32p]ATP) in a final reaction volume of 40 tzl and incubated for 30 rain in the presence or absence of purified recombinant wild-type MEK-I (150 ng) and in the presence of purified recombinant catalytically inactive MAPK (300 ng). Reactions are stopped by the addition of 5X SDS sample buffer (10 ~1), and samples are boiled for 3 min and subjected to SDS-PAGE and autoradiography. Phosphorylation of recombinant wild-type MEK-1 by MEKK enhanced the phosphorylation of catalytically inactive MAPK (Fig. 5). However, MEKK does not significantly phosphorylate MAPK in the absence of added recombinant MEK-1.

Conclusions

We have reviewed methods for assaying kinases acting in the MAPK regulatory network based on phosphorylation of recombinant protein and synthetic peptide substrates. These methods have been successfully used in our laboratory for defining the relative contributions of these key enzymes in the transduction of signals mediated by multiple growth factor receptors. The major advantages of these assays are the use of purified preparations of highly specific peptide and/or protein substrates, allowing for a high degree of sensitivity and selectivity.

Acknowledgments

This work was supported by National Institutes of Health Grants DK 37871, GM 30324, CA 58187, GM 15843 to C.A.L.C., DK 08897 to R.R.V., and the American Heart Association. A.M.G. and R.R.V. are fellows of the Cancer League of Colorado.

1 2 3 ] T R A N S C R I P T I O N A L A C T I V A T I O N A N A L Y S I S 271

[23] T r a n s c r i p t i o n a l Ac t iva t ion Analys i s of O n c o g e n e F u n c t i o n

By CRAIG A. HAUSER, CHANNING J. DER, and ADRIENNE D. Cox


Many classes of oncogenes activate the transcription of a set of cellular and viral genes, including collagenase, stromelysin, c-los, and polyoma virus T antigen. Analysis of the promoter DNA sequences of oncogene- activated genes has revealed that a number of oncogene-responsive promoter elements (originally called ras-responsive elements) consist of closely linked binding sites for AP-1 and an Ets-related protein (reviewed in Refs. 1 and 2). The presence of oncogene-responsive elements (OREs) in a promoter leads to a 5- to 40-fold increase in transcription by a variety of oncogenes, including oncogenic ras, v-src, v-mos, v-raf, v-fms, polyoma middle T antigen, and gip2. Thus, oncogenes with a wide range of functions, including receptor and nonreceptor tyrosine kinases, serine/threonine kinases, small GTP-binding proteins, and heterotrimeric GTP-binding proteins, can all activate transcription from ORE-containing promoters. In contrast, nuclear oncogenes such as v-myc, adenovirus Ela, and polyoma or simian virus 40 (SV40) large T antigens do not activate transcription from these promoter elements. 3-6 The activated oncogenes c-jun, c-los, c-ets-1, and c-ets-2 also activate promoters containing OREs, but unlike the case for nonnuclear oncogenes, which require both the Ets and AP-! sites, activation by each of these transcription factors requires only their own binding site .7,8 The mechanism of transcriptional activation of OREs by nonnuclear oncogenes is not totally defined but includes phosphorylation of c-Jun and a yet unidentified 120-kDa Ets-related protein. 6,9

D. M. Bortner, S. J. Langer, and M. C. Ostrowski, Crit. Rev. Oncogen. 4, 137 (1993). 2 A. Aoyama and R. Klemenz, Crit. Rev. Oncogen. 4, 53 (1993). 3 C. Wasylyk, J. L. Imler, and B. Wasylyk, EMBO J. 7, 2475 (1988). 4 C. Wasylyk, P. Flores, A. Gutman, and B. Wasylyk, EMBO J. 8, 3371 (1989). 5 C. Gallego, S. K. Gupta, L. E. Heasley, N,-X. Qian, and G. L. Johnson, Proc. Natl.

Acad. Sci. U.S.A. 89, 7355 (1992). 6 M. A. Reddy, S. J. Langer, M. S. Colman, and M. C. Ostrowski, Mol. Endoerinol. 6,

1051 (1992). 7 R. D. Owen and M. C. Ostrowski, Proc. Natl. Acad. Sci. U.S.A. 87, 3866 (1990). 8 C. Wasylyk, A. Gutman, R. Nicholson, and B. Wasylyk, EMBO J. 10, 1127 (1991). 9 B. Binetruy, T. Smeal, and M. Karin, Nature (London) 351, 122 (1991).



Because the transcriptional activation of ORE-containing genes is a feature of many oncogenes, transactivation assays provide another test for oncogene function that complement the biological assays described in [24] in this volume. The ability of an oncogene to activate an ORE-containing reporter gene transcriptionally can be determined by cotransfection analysis in about 3 days. Where it has been examined, there is a good correlation between the ability of nonnuclear oncogenes to activate transcription from OREs and to transform cells. Analysis of Ha-Ras mutant proteins revealed that mutants with reduced transformation activity also show a corresponding reduction in transactivation. ~°,N It has also been found that the gip2 oncogene, which encodes a mutant form of the heterotrimeric Gi2cz protein, has the same cell type specificity for transformation and ORE transcriptional activation. 5 The correlation between transformation and transactivation indicates that, during mutational analysis of oncogenes, a rapid initial screening of mutant proteins can be accomplished by assaying for the ability to activate transcription from an ORE. After the identification of the oncogene sequences required for transactivation, the more interesting mutants can then be examined for full transforming activity. One must be cautious when interpreting negative results of transactivation assays, however, because, as in biological assays of oncogene function, not all transforming oncogenes will give a positive result in every transactivation assay.

Approaches

A simple assay for transactivation of OREs can be performed by cotransfection of a plasmid expressing the oncogene to be tested with a reporter gene containing an ORE upstream of a minimal promoter. The level of reporter gene expression is then compared to that seen when the reporter gene is cotransfected with the expression vector lacking the oncogene sequences. If there appears to be oncogene activation of the reporter construct, then further experiments can be performed to demonstrate the specificity of activation. This can be accomplished by comparing the expression of the cotransfected ORE-containing reporter gene to a similar cotransfected reporter construct lacking functional OREs. The use of a transformable cell type in the transactivation assay can be crucial, as the results obtained with the gip2 oncogene suggest that a cell type that is refractory to transformation by gip2 (NIH 3T3 cells) also shows virtually no transactivation. 5

i0 p. Sassone-Corsi, C. J. Der, and I. M. Verma, Mol. Cell. Biol. 9, 3174 (1989). ~1 j . K. Westwick, A. D. Cox, C. J. Der, M. H. Cobb, M. Hibi, M. Karin, and D. A.

Brenner, Proc. Natl. Acad. Sci. U.S.A. (1994). In press.

[23] TRANSCRIPTIONAL ACTIVATION ANALYSIS 273

An alternative approach to the cotransfection assay for transactivation is to compare the expression of ORE-containing reporter constructs after transient transfection into either untransformed cells or the same cell line transformed by the oncogene to be analyzed. A drawback to this method is that it is more difficult to demonstrate the specificity of observed effects, owing to the wide variety of metabolic changes in stably transformed cells. Examples of the approaches for assaying transactivation by oncogenes, and the variety of ORE-containing reporter constructs that have been used, can be found elsewhere. 3-6,9,1°,12

When comparing the expression of reporter genes in untransformed cells to expression in cells that have been transiently transfected with an oncogene, or to expression in stably transformed cell lines, there may be significant differences in DNA uptake and in overall transcription rates, owing to altered cell growth. There may also be variations in the transfection efficiency of different DNA samples. An approach to control for these variables is to cotransfect simultaneously an internal control plasmid, such as an expression construct for fl-galactosidase. Transcriptional levels of the reporter genes can then be normalized to the fl-galactosidase expression in each transfection. ~3 The drawback to this approach is that expression of the internal control plasmid may also be influenced by oncogene expression, and normalization to this altered expression could obscure significant changes in reporter gene expression. A method to control for variations in transfection, that does not use normalization to an internal control plasmid, is to repeat each transfection several times, using at least two different preparations of each DNA, and to normalize to the amount of protein in each cellular extract. This approach, when combined with a comparison of mutant and wild-type ORE reporter constructs, should yield an accurate assay of transcriptional activation. Of course, for many investigators, the absolute amount of transactivation is not an important quantity. If this is the case, a few simple transfection experiments, such as the one shown below, will indicate whether an oncogene can transactivate gene expression, and also indicate the qualitative effect that mutations in the oncogene have on this transactivation.

Example

An example of an initial screen for transactivation of a reporter gene by various oncogenes is shown in Fig. I. Detailed descriptions of the use

12 j. Bruder, G. Heidecker, and U. R. Rapp, Genes Dev. 6, 545 (1992). 13 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual,"

2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989.


of expression constructs, transfection by calcium phosphate precipitation, and chloramphenicol acetyltransferase (CAT) assays, can be found in Sambrook et al.~3 The reporter construct used for the experiment shown in Fig. 1 is pB-4x-CAT, which contains four copies of the ORE for the polyoma virus enhancer inserted upstream of the/3-globin promoter fused to the CAT gene.12 The NIH 3T3 cells used in the assay are split the day before transfection to a density of 5 × 105 cells per 60-mm dish. Each 60-ram dish receives 1 /xg of reporter gene cotransfected with 0.5/xg of the indicated oncogene expression construct, along with 20/zg calf thymus carrier DNA. The amount of oncogene expression construct used in transactivation assays (0.1-5.0/zg) needs to be optimized to provide measurable transactivation in the linear range of the CAT assay, and it depends on the expression vector used and the activation potential of the oncogene. To enhance the consistency of transfections, all of the plasmid DNAs are prepared by cesium chloride-ethidium bromide density gradient centrifugation and quantitated by OD260 measurements, and the quantity and quality of the DNA are checked by ethidium bromide staining following agarose gel electrophoresis.13

Transfection is carried out by calcium phosphate precipitation in a total volume of 0.55 ml, and each duplicate is precipitated separately. The cells are glycerol shocked for 3 hr after transfection and then harvested after 48 hr. The final volume of the freeze-thaw lysate (in 0.25 M Tris- HC1, pH 7.8) from each dish is 0.1 ml. For the initial screening, it is assumed that there is the same amount of protein in each cellular extract,

(vector) ras fms src raf myc E la SV40 Ig T

I . f i i t i i J i J ~ i i J

2 83 29 39 30 <1 <1 <1

FIG. 1. Screen for transcriptional activation by various oncogenes. Expression constructs of different oncogenes (0.5/zg) were transfected into NIH 3T3 cells along with the pB-4x- CAT reporter construct (1/zg), and the CAT activity resulting from transcriptional activation of the reporter was assayed as described in the text. Expression of CAT activity causes the conversion of chloramphenicol from the unacetylated (lower spots) to acetylated forms (upper spots). This representative experiment, performed in duplicate, shows the average percent conversion for each duplicate pair, indicating the amount of acetylated [14C]chloramphenicol as a percentage of total [Inc]chloramphenicol present.

[23] TRANSCRIPTIONAL ACTIVATION ANALYSIS 275

but to measure protein concentration for more quantitative analysis, 25 /zl of cell extract gives a good reading in a l-ml Bradford protein assay.~4

The CAT assay is performed in a total volume of 140/zl, with 20/zl of cellular extract, 70/.~g acetyl-CoA, and 0.05/zCi [~4C]chloramphenicol. The assay mixture is incubated for 45 rain at 37 °. The acetylated and nonacetylated forms of [~4C]chloramphenicol are separated by thin-layer chromatography and quantitated after a 30-min scan on an AMBIS (San Diego, CA) Radioanalytic Imaging System scanner. The entire assay, from the start of cell harvest to quantitative results from the scanned TLC plate, takes about 7 hr.

The results in Fig. 1 show the large magnitude of transcriptional activation by nonnuclear oncogenes. In fact, the 40-fold transcriptional activation seen with an oncogenic mutant of human H-ras(61-Leu) is a minimum estimate of activation, because at 83% conversion the CAT assay is beyond the linear range. For more precision, the assay would need to be repeated using less cell extract. To summarize the results shown in Fig. 1, transcription of the pBX4-CAT reporter gene is strongly activated by cotransfection with nonnuclear oncogenes H-ras(61-Leu), v-fms, v-src, or activated c-raf, but not with the nuclear oncogenes v-myc, adenovirus Ela, or SV40 large T antigen.

Transactivation Assays in G-Protein Analysis

A constitutively activated mutant a subunit of a heterotrimeric Gi2

protein, the gip2 oncogene, acts not only to transform cells as determined by the biological assays (see [24] in this volume), but also to transactivate an ORE-containing reporter gene? Thus, it is reasonable to anticipate that other activated heterotrimeric G proteins, such as the mutant a subunit (Q205L) of Go, which has all the features of a transforming oncogene in biological assays, ~5 will also transactivate expression of ORE-containing genes. Similarly, a mutant a subunit (Q209L) of GQ also causes focus formation and anchorage-independent growth of NIH 3T3 ceUs, 16 and it is a good candidate for a transactivator of ORE-containing genes. Both mutant G-protein oncogenes show cell type specificity for transformation and, like gip2, may also show cell type specificity for transactivation. In the proper pituitary cell type, an activated mutant G~a subunit, the gsp

14 M. M. Bradford, Anal. Biochem. 72, 248 (1976). 15 S. D. Kroll, C. Chen, M. DeVivo, D. J. Carry, A. Buku, R. T. Premont, and R. lyengar,

J. Biol. Chem. 267, 23183 (1992). 16 M. DeVivo, J. Chert, J. Codina, and R. [yengar, J. Biol. Chem. 267, 18263 (1992).


oncogene, ~7 may also activate ORE-containing genes. Experiments to assess the function of G-protein oncogenes as transcriptional activators could lead to important insights into how they alter regulation of cell growth.

Summary

Because no single assay provides a complete analysis of the transformed phenotype, transactivation assays complement the cell growth and tumorigenicity analyses of oncogene function. Transactivation of ORE- containing genes is such a common feature of a diverse variety of viral and cellular oncogenes that it can be considered one aspect of the oncogene- induced phenotype. After the initial identification of oncogenes that activate transcription, studies of the mechanisms of activation and the identification of the downstream target genes should lead to a better understanding of the events leading to cellular transformation. The fact that cell type specificity of transactivation and transformation can be similar means that the transactivation assay may be a useful tool in dissecting cell type- specific transformation. The transactivation assay of oncogene function also has the advantage that it is easy to perform and significantly more rapid than assays based on altered cell growth. This is of particular advantage when one wishes to examine the function of a large number of oncogene mutants generated in vitro. Overall, transactivation assays provide another tool for examining transforming potential and a starting point for the analysis of the downstream targets of oncogenes.

Acknowledgments

Our research is supported by grants from the National Institutes of Health to C.A.H. (HD28525), to C.J.D. (CA42978, CA52072, CA 55008, and to A.D.C. (CA61951). C.J.D. is the recipient of an American Cancer Society Faculty Research Award.

17 C. A. Landis, S. B. Masters, S. B. Spada, A. M. Pace, H. R. Bourne, and L. Vallar, Nature (London) 340, 692 (1989).

[ 24 ] BIOLOGICAL ASSAYS FOR CELLULAR TRANSFORMATION 277

[24] Biological Assays for Ce l lu l a r T r a n s f o r m a t i o n

By ADRIENNE D. Cox and CHANNING J. DER

Introduction

In vitro and in vivo growth properties that distinguish malignant cells from normal counterparts include alterations in cellular morphology, decreased dependence on serum growth factors, loss of density-dependent growth inhibition, the ability to proliferate in suspension or in semisolid medium, and the ability to form tumors when introduced into the appropriate animal hosts.LZ These components of the transformed phenotype constitute the basis for the majority of currently used transformation assays. In this chapter we describe some of the most commonly applied in vitro and in vivo assays, emphasizing those that use the NIH 3T3 mouse fibroblast cell line, to determine whether a particular gene displays growth- promoting and growth-deregulating activities characteristic of viral and cellular oncogenes.

Focus-Formation Assays

Background

The most widely used and best characterized assays for determining the transforming potential of a particular gene are gene transfer assays using rodent fibroblast cell lines such as NIH 3T3 or Rat-1 fibroblasts. 3-6 Because these established cell lines are considered to be preneoplastic, rather than fully normal, and can undergo single-step malignant transformation following the introduction of a single oncogene, they provide very sensitive recipient hosts for transformation assays. Furthermore, DNA can be readily introduced into the cells by calcium phosphate transfection techniques. The basic assay, generally referred to as a focus-formation assay, measures the ability of an exogenously introduced gene to induce morphological transformation and/or loss of density-dependent growth regulation. These alterations result in the appearance of clusters of mor-

i V. H. F reedman and S. Shin, Cell (Cambridge, Mass.) 3, 335 (1974). 2 E. J. Stanbridge and J. Wilkinson, Proc. Natl. Acad. Sci. U.S.A. 75, 1466 (1978). 3 R. A. Weinberg, Biochim. Biophys. Acta 651, 25 (1981). 4 G. M. Cooper , Science 217, 801 (1982). 5 H. Land, L. F. Parada, and R. A. Weinberg, Science 222, 771 (1983). 6 j . M. Bishop, Science 235, 305 (1987).



FIc. 1. Morphology of normal and transformed NIH 3T3 cells. Cells were transfected with G418-resistant mammalian expression constructs (see Transfection of NIH 3T3 Cells), and drug-resistant transfectants were selected in G418 (see Establishment of Stable Cell Lines). (a) Empty vector [pZIP-neoSV(X)1]; (b) H-ras (61 L) [pZIP-rasH(61L)]; (c) activated c-raf(p22W-raf); (d) SV40 large T antigen (pSV-TAg). The untransformed cells (a) possess a fiat morphology and exhibit a regular, orderly pattern of growth, whereas the cells transformed by the different oncogenes (b-d) have rounded or spindle-shaped, refractile cell bodies with long cytoplasmic extensions and exhibit a disorganized pattern of growth.

phologically transformed cells (rounded or spindle-shaped cells that appear highly refractile when viewed under phase-contrast microscopy, see Fig. 1) growing in a disorganized pattern that is readily detectable against the background of a confluent monolayer of untransformed cells (Fig. 2).

In addition to relative ease of performance, an important attribute of the focus-formation assay is the vast wealth of literature documenting the biological consequences of the expression of a wide variety of cellular and viral oncogenes in rodent fibroblast cell lines.7 The transforming poten-

7 j. M. Bishop, Cell (Cambridge, Mass.) 64, 235 (1991).

[24] BIOLOGICAL ASSAYS FOR CELLULAR TRANSFORMATION 279

FIG. 2. Focus formation on NIH 3T3 cells. NIH 3T3 cells were transfected with 50 ng of plasmid DNA encoding either activated c-raf(22W) (a) or H-ras (12V) (b) (see Transfection Assay), maintained in growth medium, and observed for focus formation at 14 days. Both oncogenes produce foci of morphologically transformed cells that can be seen under the light microscope (4 x ) as heaped masses of darker cells that grow in a swirling pattern over the very fiat background of normal, untransformed cells (see top).

tial of newly discovered oncogenes can therefore be compared and con- trasted with the activities associated with previously characterized oncogenes. However, it is also important to mention several cautions which preclude this assay, like all others, from being used as the sole indicator of transforming potential of a given gene. One major disadvantage of focus-formation assays lies in their dependence on morphological transformation. Some cellular oncogenes may induce alterations in growth properties without alterations in cellular morphology, and would therefore score negative in this assay. A second potential disadvantage is that the recipient cells are of fibroblast origin and so may not be susceptible to transformation by all the oncogenes that are active in other cell types, particularly in epithelial or hematopoietic cells, from which the majority of human cancers are derived. Third, since these established cell lines contain a number of genetic mutations, only a subset of oncogenes which promote specific steps in malignant progression may transform the cells. Finally, certain oncogenes actively promote morphological changes in only a subset of the commonly used rodent fibroblast lines. Nevertheless, despite these cautions, the focus-formation assay can be a clean, straightforward


assay capable of giving a very clear indication of transforming potential, and it remains one of the most important biological assays of cellular transformation.

Although the performance of focus-forming assays can be straightforward, several important conditions must first be met. Because cells such as NIH 3T3 cells 8 or Rat-1 cells 9 are preneoplastic, they have a strong tendency to transform spontaneously. 1° Therefore, it is critical to use a subclone of the recipient cell line which does not contain a subpopulation of spontaneously transformed variants. Thus, it is strongly advised that cells be acquired from investigators who use the cells specifically for focus-formation assays. To minimize spontaneous transformation of these cell lines, they should be subcultured at regular intervals to maintain the cells in subconfluent cultures in logarithmic growth phase. Procedures for basic cell line maintenance that minimize spontaneous transformation of NIH 3T3 cells and methods for establishing inventories of frozen cell stocks to increase assay reproducibility over time are described below.

A number of similar protocols for the use of NIH 3T3 cells and other rodent fibroblast cell lines for focus-formation assays are in common use. H-13 Generally, small amounts of the plasmid DNA containing the gene to be tested are mixed with larger amounts of carrier DNA. Calcium phosphate-based protocols are the standard techniques used for stable transfer of the cloned gene into the monolayer cultures. 14 The density of the cultures and the time of exposure of the cells to the precipitated DNA may vary. Additionally, some protocols suggest a subsequent glycerol shock step to increase DNA uptake, whereas others suggest subculturing the cells after exposure to DNA. 15,~6 As cells vary in culture, even though derived from the same parental line, it is generally best to apply the protocol used by the investigator from whom the cells were received. Alternatively, an investigator may determine the conditions that optimize the efficiency of focus-forming activity and minimize the background from

8 j. L. Jainchill, S. A. Aaronson, and G. J. Todaro, J. Virol. 4, 549 (1969). 9 M. Wigler, A. Pellicer, S. Silverstein, and R. Axel, Cell (Cambridge, Mass.) 14, 725 (1978). l0 H. Rubin and K. Xu, Proc. Natl. Acad. Sci. U.S.A. 86, 1860 (1989). 11 C. Chen and H. Okayama, Mol. Cell. Biol. 7, 2745 (1987). 12 j. Ray and F. H. Gage, BioTechniques 13, 598 (1992). 13 j. Carcagne, K. T. Ha, and J. Armand, Biologicals 19, 317 (1991). 14 F. L. Graham and A. J. van der Eb, Virology 54, 536 (1973). ~5 J. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual,"

2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. t6 F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K.

Struhl, eds. "Current Protocols in Molecular Biology." Wiley, New York, 1991.

[ 2 4 ] BIOLOGICAL ASSAYS FOR CELLULAR TRANSFORMATION 281

spontaneous transformation using a known oncogene. Sample procedures for NIH 3T3 and Rat-1 transfections are described below, z7'~8

Maintenance of Stock NIH 3T3 Cells

The NIH 3T3 cells are propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) calf serum (Colorado Serum Co.), 100 U/ml penicillin, and 100 t~g/ml (w/v) streptomycin, at 37 ° in a humidified 10% CO 2 atmosphere. To establish frozen stocks, subconfluent NIH 3T3 cultures are trypsinized and seeded at 103 cells per 100-ram dish. To establish a large inventory of cells for focus-formation assays, 10-40 dishes are plated. The culture.s are fed with fresh growth medium every 3 days until they reach approximately 70% confluency (13-15 days). Following trypsinization, the cells from each plate are washed once and resuspended in 1 ml freezing medium (DMEM supplemented with 20% calf serum and 10% (v/v) dimethyl sulfoxide) before transfer to freezing ampules (one per 100-mm dish) for storage in liquid N2. The use of cells from a common frozen stock, together with using the cells for a limited number of passages after they are reconstituted in culture, optimizes the reproducibility of results from independent assays done over a long period of time.

A weekly schedule for maintaining sufficient NIH 3T3 cells in culture to provide cells for two assays per week (Tuesdays and Fridays) is described below. This schedule is initiated from cells freshly reconstituted from frozen cell stocks; the cells can be used for a total of four successive focus-formation assays before they are discarded. This short-term use of cells minimizes the frequency of spontaneous tranformation.

Monday. Thaw one frozen ampule, resuspend the cells in 10 ml growth medium, transfer the cell suspension to a 15-ml sterile centrifuge tube, and centrifuge to pellet the cells. Resuspend the cell pellet and transfer to three to six 100-ram dishes. The exact number of dishes to be plated will depend on how many cells are available to achieve a density of approximately 5-6 × 106 per dish after 3 days.

Tuesday. Feed cultures with fresh growth medium. Thursday. Trypsinize and resuspend the cells in growth medium. Deter-

mine cell numbers and plate some cells at 5 × 105 cells per 60-mm dish for focus-formation assays and some cells at 1 × 105 cells per 100-ram dish for the short-term stock cells that will be used for plating the next transfection assay (the following Monday). Four dishes should be used for each DNA, such that a reasonable-sized assay may range from 12 to

iv C, J. Der, B. Weissman, and M. J. MacDonald, Oncogene 3, 105 (1988). 18 M. H. Ricketts and A. D. Levinson, Mol. Cell. Biol. 8, 1460 (1988).


60 dishes. When determining the number of dishes to be used, include both a positive control DNA, such as an oncogenic ras expression construct, and a negative control DNA, such as an empty vector construct, to determine the background of spontaneous transforming activity in the cells used for that particular assay.

Friday. Conduct the transfection assay (described below); the cells should be subconfluent and well separated, Feed stock plates with fresh growth medium.

Monday. Use stock 100-mm cultures that were plated the previous Thursday for both focus-forming assays (Tuesday) and for stock cultures for the next transfection (Friday). Each stock plate should contain 4-6 × 10 6 cells and should provide enough cells to plate 40-60 dishes for transfection.

Transfection of NIH 3T3 Cells

Reagents. Reagents required for the transfection assay include 1.25 M CaC12 dihydride in water (autoclave sterilized); HEPES-buffered saline, pH 7.05 (HBS); 15% glycerol/HBS. The HBS is prepared by adding the following to 900 ml distilled water: 8.00 g NaCI, 0.37 g KC1, 0.19 g Naz HPO4" 7H20), 1.0 g glucose, and 5.0 g HEPES. Adjust to exactly pH 7.05 with 5 M NaOH. The correct pH is critical for good DNA precipitation. 14 Adjust the volume to 1 liter, autoclave to sterilize, and store at room temperature. The 15% glycerol/HBS solution is prepared by simply mixing glycerol (glycerin) with HBS (15 : 85, v/v). Both the HBS and the CaCI2 solutions are stable for several months at room temperature; however, both can deteriorate with prolonged storage.

A good source of high molecular weight DNA is needed as carrier DNA for generating the calcium phosphate DNA precipitant. This can be isolated either from normal cell lines (e.g., NIH 3T3 cells) or from animal tissues. Two commercial sources of high molecular weight calf thymus DNA are Sigma Chemical Company (St. Louis, MO) and Boehringer Mannheim Biochemicals (BMB, Indianapolis, IN). To obtain a sufficiently clean preparation of DNA, the Sigma DNA requires phenol-chloroform extraction prior to use, whereas the BMB DNA can be used directly; we have obtained the best results in focus-formation assays using the DNA from Boehringer Mannheim. Because the success of a transfection is critically dependent on generating a good calcium phosphate precipitate of the DNA, a trial precipitation should be done to determine that all the reagents are working properly (see below). The most critical reagent is the HBS, whose most critical variable is pH (pH 7.05-7.12). 14 Although the generation of satisfactory DNA precipitate (see below) is a good indica-


tion of the quality of the HBS reagent, the testing of new reagents in an actual transfection assay is strongly recommended. Additionally, the focus-forming activity can vary significantly with different lots of calf serum. We have found that calf serum from Colorado Serum Co. gives consistently good results. Different lots of calf serum from other vendors should be tested using both positive and negative control DNAs to determine which give the cleanest focus formation.

Transfection Assay . Focus-formation assays should be performed in quadruplicate for each DNA to be tested. As described above, 60-mm dishes are seeded with 5 x 105 cells 16 to 24 hr prior to the transfection assay. To prepare the DNAs for precipitation, first determine the total number of dishes that will be transfected. Each dish will require 20 tzg carrier DNA in 0.5 ml HBS. Then add the carrier DNA stock (! mg/ml) to enough HBS for the entire assay to make a final concentration of 40 /xg/ml DNA. Place a 2-ml aliquot of the HBS/carrier DNA mixture in a separate 16 x 100 mm clear polystyrene tube for each plasmid being transfected. Add plasmid DNA, 40 ng to 4/zg, to each tube and vortex to mix thoroughly. Add 200/~1 of 1.25 M CaC12 to each tube, mix with gentle pipetting to initiate precipitation, and complete the precipitation by vortexing for 10-15 sec. Precipitation should be visible immediately. Allow precipitates to form for at least 20 min at room temperature. Ideally, the precipitated DNA should appear as fine dust rather than as long white strings. The generation of a good precipitate is critical for high transfection efficiencies. Gently pipette the precipitates dropwise (0.55 ml per dish) directly onto the growth medium of the appropriate recipient cultures, swirl the dishes gently to distribute the DNA, and then incubate at 37 ° for 3-4 hr.

For the initial transfection analysis of an uncharacterized oncogene, a range of DNA concentations, from 10 to 1000 ng per dish, should be used. Once the transforming potency is established, then lower concentrations can be used to allow accurate determination of the focus-forming activity of a particular gene. As a guide, oncogenic ras in the pZIP-NeoSV(x)I retrovirus vector, 19 from which a high level of constitu- tive expression is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), produces 4-6 x 104 foci per microgram (focus- forming units, flu) of transfected DNA and therefore requires only 10 ng to achieve 40-60 foci per 60-mm dish. 2° In contrast, normal ras transforms NIH 3T3 cells only when overexpressed (30-fold) and therefore no foci will be seen when 10 ng DNA is transfected, whereas 20-40 foci per dish

~9 C. L. Cepko, B. Roberts, and R. C. Mulligan, Cell (Cambridge, Mass.) 37, 1053 (1984). :0 C. J. Der, T. Finkel, and G. M. Cooper, Cell (Cambridge, Mass.) 44, 167 (1986).


will be seen when it is transfected at 1 ~g per dish. When in doubt, 1/zg per dish is a good starting point. For focus-formation assays, it is necessary to use a high-quality preparation of plasmid DNA (e.g., cesium chloride purified). Impurities in the DNA will reduce precipitation efficiency, and hence transfection efficiency. However, for establishing cell lines that stably express the gene of interest (see below), even minipreparation DNA can be used, at approximately 5/zl per dish.

To increase the efficiency of DNA uptake, glycerol-shock the transfected cultures following incubation with the precipitated DNA. Aspirate the growth medium containing the precipitated DNA, rinse once with fresh growth medium, and add 1 ml of 15% glycerol/HBS to each 60-ram dish. Aspirate the glycerol in sufficient time to rinse each dish with growth medium at 4-5 min following the initial glycerol shock. Because the glycerol treatment is rather toxic to the cells, the timing of this step is critical. Following the rinse step, feed each dish with growth medium and return to the 37 ° incubator. Feed the dishes three times a week with fresh growth medium until the transformed foci are well established but not too crowded to count.

Although foci induced by oncogenic ras can usually be visualized as early as 8 days after transfection, they are usually counted after 14 days. The time of appearance of transformed foci will vary with different oncogenes and with the same oncogene when expressed from different promoters. The most accurate method to quantitate the transformed foci is to count them while scanning the plates under 4 x magnification using an inverted phase-contrast microscope. The frequency of spontaneously transformed foci is usually less than one per four dishes. However, the appearance of foci of densely growing cells that mimic the appearance of transformed foci may be quite high (up to 20-30 per dish) when the assays are done under suboptimal conditions. Because the bogus "foci" can usually be distinguished from authentic transformed foci by comparing the appearance of cells growing on the negative control dishes with those on the positive control dishes, the inclusion of both negative and positive control dishes in each assay is crucial.

Maintenance o f Stock Rat-1 Cells

Rat-I cells are propagated in Ham's F12/DMEM (1 : 1) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100/zg/ml streptomycin, at 37 ° in only 5% CO2. The cells are very flat and very adherent, but quite tolerant of trypsinization. Using 0.25% (5 x) trypsin/EDTA, split cultures 1 : 10 every 3-5 days. Although Rat-1 cells are less susceptible


to spontaneous transformation than are NIH 3T3 cells, care should be taken to maintain stock cultures at subconfluent cell densities.

Transfection of Rat-1 Cells

For the most part Rat-1 cells are transfected similarly 21'22 to NIH 3T3 cells as just described. The differences largely reflect the slower growth rate and lower transfection efficiency of Rat-1 cells. Seed cells at 1 × 105 per 60-mm dish 16-24 hr prior to the transfection, when the cells should be at 10-30% confluency. Plasmid DNAs should be transfected at 2-5/xg per dish, rather than the 10-1000 ng per dish used for NIH cells. Incubate the precipitated DNA with the recipient cultures for 5-8 hr, and glycerol- shock the cells for only 45 sec. Three days after the transfection, split the entire contents of each transfected 60-mm dish directly into a 100-ram dish to monitor focus formation. For G418 selection, split at 1 : 20 or so, as cells are highly adherent to one another and will peel up in sheets if they become confluent before nonresistant cells are killed off. Cells can be split directly into G418 or changed the day after passage. Feed the cultures twice a week with fresh growth medium until transformed loci have developed. In general, foci appear on Rat-1 cells approximately 1 week after they would appear on NIH 3T3 cells. As Rat-I cells exhibit greater density-dependent growth inhibition than NIH 3T3 ceils, and therefore present a flatter background on which the foci develop, transformed foci can be readily visualized and quantitated by fixing (10 rain in a 10% acetic acid/10% methanol solution) and staining the dishes (10 min) with 0.4% crystal violet in 10% ethanol [prepared from a 1% stock solution and filtered through a Whatman (Clifton, N J) No. 1 filter before use]. Stained dishes should be rinsed in deionized or distilled water gently but thoroughly before air drying in an inverted position. Figure 3 shows an example of Rat-1 cells stained for visualization of foci.

Oncogene Cooperation Assay

Because oncogenesis is a multistep process, it is not surprising that some genes with transforming potential require coexpression with another oncogene in order to transform cells in vitro. 5'23'24 Similarly, some onco-

21 D. J. Capon, E. Y. Chen, A. D. Levinson, P. H. Seeburg, and D. V. Goeddel, Nature (London) 3@2, 33 (1983).

22 G. Patel, M. J. MacDonald, R. Khosravi-Far, M. M. Hisaka, and C. J. Der, Oncogene 7, 283 (1992).

23 T. Hunter, Cell (Cambridge, Mass.) 64, 249 (1991). 24 H. E. Ruley, Cancer Cells 2, 258 (1990).


FZG. 3. Focus formation on Rat-I cells. Rat-1 cells were transfected with either normal or oncogenic forms of ras, then maintained in growth medium, and the entire dishes were stained with crystal violet for visualization of foci at 18 days after transfection (see Transfec- tion of Rat-1 Cells). In Rat-1 cells, only the oncogenic form of H-ras (b) H-ras(61L)] produces foci; these appear as dense clusters of morphologically transformed, proliferating cells that grow over the very flat background of normal, untransformed cells [compare to (a) H-ras(wt)]. Because Rat-1 cells exhibit such a flat morphology, staining followed by visual examination is sufficient for accurate quantitation of foci.

genes can transform "preneoplastic" NIH 3T3 cells, but not primary cells, by themselves. For example, oncogenic ras alone is sufficient to transform NIH 3T3 cells, but in primary rat embryo fibroblasts morphological transformation and focus formation occur only when ras is cotransfected with a variety of complementing oncogenes such as adenovirus Ela, myc, and simian virus 40 (SV40) large T antigen. To test whether a given gene can cooperate with another oncogene to produce cellular transformation, transfection analyses are done with recipient cell lines that require coexpression of at least two oncogenes for transformation. Primary rat embryo fibroblasts or the REF52 rat embryo fibroblast cell line are most commonly used for these assays.

Maintenance o f S tock REF52 Cells

The REF52 cells are maintained in DMEM supplemented with 10% fetal calf serum and antibiotics, z5 The cells are very flat and adherent but, unlike Rat- 1 cells, are not very tolerant to overtrypsinization. They should

25 B. R. Franza, K. Maruyama, J. I. Garrels, and H. E. Ruley, Cell (Cambridge, Mass.) 44, 409 (1986).

[ 2 4 ] BIOLOGICAL ASSAYS FOR C E L L U L A R T R A N S F O R M A T I O N 287

therefore be passaged by quick and careful rinsing in 0.5% (10 x ) trypsin/ EDTA, trypsinization in 0.25% (5 x) trypsin/EDTA, and immediately resuspended in at least 3 times the volume of growth medium (to dilute the trypsin) before splitting at 1 : 10. To maintain flat morphology, passage at no more than 1 : 10, and do not allow the cells to become overgrown. As for NIH 3T3 cells, REF52 cell stocks should be frozen in large quantities, subcultured just prior to confluence, and each aliquot discarded after several passages.

Transfection of REF52 Cells

The transfection procedure is very similar to the one described above for NIH 3T3 cells, except that the quantity of plasmid DNA to be transfected is much higher, and all cells must be drug-selected. Therefore, it is generally desirable to have the two oncogenes to be tested be in expression vectors that contain the same selectable marker. Plate cells in 100-mm dishes such that they will be 70-80% confluent on the day of the transfection. Mix the cooperating plasmid DNAs (2-10 /xg each) with carrier DNA, if necessary, to a total of 14-20/xg, in a volume of 500/zl HBS. As an example, oncogenic ras and SV40 large T antigen can be used at l0 /zg each, obviating the need for carrier DNA. Add 50 tzl of 1.25 M CaCI 2 and vortex immediately. After 40 min at room temperature, add the precipitated DNA to the recipient cultures and incubate for approximately 6 hr at 37 °. Following a 2-min glycerol shock, feed the cells with growth medium and incubate at 37 °. On the day after the transfection, select drug-resistant cells by splitting each dish 1 : 3 into growth medium supplemented with the appropriate drug, for example, G418 (200-400/~g/ ml; see below) or hygromycin B (100-400/~g/ml), making replicate plates if desired. Feed cells twice a week. REF52 cells grow much more slowly than do NIH 3T3 cells, and drug selection will also be much slower. It may therefore take 3-4 weeks before colonies begin to appear. If both plasmid DNAs contain drug resistance genes, there may be more than one type of morphological change, reflecting a mixed cell population in which cells express only one type, or both types, of cooperating oncogene.

In Vitro Parameters of Cellular Transformation

Altered in Vitro Growth Properties

Although a tumor cell can be distinguished from its normal counterpart by a wide range of phenotypic alterations, only a few of these properties provide reliable indicators of malignant transformation.~'2 Among these,


loss of density-dependent growth, enhanced growth rates, a reduction in the requirement for serum growth factors, and the ability to proliferate in suspension are properties commonly associated with the transformed phenotype induced by most oncogenes.

The assays are performed after first establishing cells stably expressing the gene of interest. Because the frequency of transfected cells that have stably integrated the introduced DNA is less than 100%, the use of dominant selectable markers is required to isolate only cells harboring the desired gene. This is best accomplished by using expression vectors that also contain drug-selectable markers, for example, aminoglycoside phosphotransferase (neo r) or hygromycin B phosphotransferase (hygr). 26,27 Vec- tors driven by promoters such as murine retroviral long terminal repeats (LTRs), the cytomegalovirus (CMV) promoter, or the/3-actin promoter work well in NIH 3T3 cells. 28 As controls for these assays, parallel cell lines should be established by transfection with the expression vector alone, to produce an untransformed cell population, and by transfection with a well-characterized oncogene such as mutated ras or viral src, to produce a transformed cell population. For NIH 3T3 cells, transfection by a calcium phosphate precipitation technique is the most common method for introducing the recombinant plasmid. For certain genes of interest or for other cell lines, alternative techniques such as retrovirus infection, lipofection, or electroporation may be preferable. 12 Once stable populations have been established, either pools of drug-selected cells or individual clonal populations, if characterized for expression of the transfected genes, can be used for the biological assays described below.

Accelerated Growth Rates. Many cellular oncogenes promote an enhanced rate of cell division of NIH 3T3 cells, which can be measured by assaying the increase in cell numbers during the exponential growth of subconfluent cultures. This assay can be combined with the saturation density assay for evaluating the loss of density-dependent growth inhibition and/or with the serum growth factor dependence assay described below.

Elevated Saturation Densities. The NIH 3T3 line and other established rodent cell lines used for transformation assays are subject to density- dependent growth inhibition and therefore cease to proliferate when confluent. Most oncogenes trigger a loss of this property, so that cells expressing the products of these genes continue to proliferate even after reaching

56 p. j. Southern and P. Berg, J. Mol. Appl. Genet. 1, 327 (1982). 27 B. Sugden, K. Marsh, and J. Yates, Mol. Cell. Biol. 5, 410 (1985). 28 M. Kriegler, "Gene Transfer and Expression: A Laboratory Manual." Stockton, New

York, 1990.

[ 2 4 ] B I O L O G I C A L ASSAYS F O R C E L L U L A R T R A N S F O R M A T I O N 289

confluence, thereby achieving significantly higher cell densities at saturation.

Reduced Serum Growth Factor Requirements. Compared to untransformed cells, the proliferation of transformed cells is generally less dependent on serum growth factors. The NIH 3T3 cells are usually maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum. For untransformed NIH 3T3 cells, the use of growth medium supplemented with low levels (0.5-2%) of calf serum results in a significantly reduced, or even zero, rate of cell proliferation, whereas oncogene- transformed NIH 3T3 cells can proliferate quite well at these low serum levels.

Establishment of Stable Cell Lines. For each plasmid DNA to be expressed, transfect a single 60-mm dish of NIH 3T3 cells or Rat-1 cells as described above under Focus-Formation Assays, using an expression construct with a drug-selectable resistance marker such as neomycin or hygromycin. Alternatively, if the expression vector lacks a selectable marker, it can be cotransfected with another plasmid that contains the drug resistance gene. This can be done by cotransfecting 2 txg of the oncogene-containing plasmid with 20 ng of the marker plasmid (100 : ! ratio), which usually results in a mixed population in which approximately 50% of the drug-resistant cells have also acquired the nonselected oncogene-containing plasmid.

At day 3, split the transfected cells at 1 : 10 into 100-mm dishes. At day 4, change the medium to growth medium supplemented with the selected drug. For neomycin selection of NIH 3T3 cells, G418 (e.g., Genet- icin, GIBCO-BRL, Gaithersburg, MD) is commonly used at a concentration of 400 txg of active drug per milliliter of growth medium. The cultures should be fed approximately twice a week, or when there are large numbers of sloughed cells. Non-drug-resistant cells begin to die off by day 4 or 5 and should be completely gone by day 9 or so, at which time the appearance of drug-resistant colonies should be quite obvious. When all the drug- sensitive cells have sloughed off, cultures can be returned to regular growth medium if desired. When colonies are fairly dense but not confluent, they can be picked individually or trypsinized off the dish en masse and passaged as pools of drug-resistant cells. The same procedure can be used to select drug-resistant cells that are transfected with hygromycin- resistant vectors by selecting the cells in growth medium supplemented with 400 txg/ml hygromycin B. Both G418 and hygromycin are prepared as I0 x stocks in either 100 mM HEPES (pH 7.2) or serum-free DMEM, respectively, and stored at - 20°. ~5'16

The expression of some gene products is not well tolerated, such that cells which have either low or no protein expression may be preferentially


selected. Therefore, it is desirable (and, in the case of cells which are not morphologically transformed, critical) to test the selected populations for expression of the gene of interest. Ideally, such tests should be performed by immunoblotting or immunoprecipitation at the protein level; if this is not possible, however, mRNA blotting should be performed. In some cases, it is possible to make cells tolerant for the expression of certain proteins by cotransfection with small amounts of an oncogene, such as 10 ng of oncogenic r a s . 29

Determination of Growth Rate, Saturation Density, and Serum Dependence

Trypsinize cells from confluent cultures, seed at 1 × 105 cells per 60- mm dish in growth medium supplemented with either 10 or 1% calf serum, and incubate at 37 ° (day 0). At 2-day intervals for 14 days, determine the number of cells per triplicate dish. Feed cells with fresh growth medium every 2 or 3 days during the assay period. At the appropriate day, harvest the cells by trypsinization and resuspend in phosphate-buffered saline for determination of cell number by Coulter counting or with a hemacytome- ter. Growth rates are determined by calculating the doubling time from the initial slope of the logarithmic growth curve before saturation occurs. The saturation densities are the cell densities achieved during the final, plateau phase of growth. Serum dependence is determined by comparing the growth rate of the untransformed versus transformed cells using growth medium supplemented with different serum levels.

Anchorage-Independent Growth

One of the best in vitro indicators of malignant growth potential is the ability of cells to grow in an anchorage-independent environment.~,2 Whereas most normal cells require adherence to a solid substratum for growth and proliferation, many tumor cells have lost this property and can grow either in stirred suspension cultures or suspended in semi-solid media such as agar or methylcellulose. 3°'31 Growth in agar suspension is the most common assay, although the presence of sulfated polysaccharides in some agar preparations may result in a more stringent assay than that with methylcellulose. The limitations of the assay are reflected in the observations that some normal cells do grow in suspension, and that many human tumor cells fail to grow in suspension. However, at least in NIH 3T3 cells, this assay is very useful for the analysis of transforming potential. 17

29 A. D. Cox, M. M. Hisaka, J. E. Buss, and C. J. Der, Mol. Cell. Biol. 12, 2606 (1992). 30 I. MacPherson and L. Montagnier, Virology 23, 291 (1964). 31 R. Risser and R. Pollack, Virology 59, 477 (1974).

[ 2 4 ] B I O L O G I C A L ASSAYS F O R C E L L U L A R T R A N S F O R M A T I O N 291

Procedure. For each cell line, single cell suspensions are prepared in duplicate, ranging in 10-fold increments from 102 to 10 4 cells per 60-ram dish, in a 0.33% top agar suspension, overlaid onto a 0.5% agar bottom layer, and grown at 37 ° for colony formation.

Use bacto-agar (DIFCO, Detroit, MI) to prepare a 5% (w/v) (I0 × ) agar stock in distilled water. Boil to dissolve the agar, autoclave to sterilize, and store in 50-ml aliquots at room temperature. At the time of the assay, melt the agar stock and allow to cool in a 45 ° water bath. For each dish, 1.1 ml of 10 × stock will be needed, but always start with extra to account for loss in pipetting and in case of accidents. Warm the necessary amount of NIH 3T3 growth medium (i.e., DMEM + 10% calf serum + antibiotics) ( - 7 ml for each bottom layer and 4 ml for each top layer) to 45 °, then add to the melted agar stock to a final concentration of 0.5% agar. Incubate at 45 ° to prevent the agar from solidifying. Because serum growth factors and antibiotics added to the growth medium are sensitive to heat, the following steps should be done promptly.

To prepare the bottom agar layers, pipette 7 ml of the 0.5% agar/ growth medium into each 60-mm dish and allow to solidify at room temperature. To generate a single cell suspension of each cell line to be tested, trypsinize the cells carefully and thoroughly, determine the cell number, then pellet by centrifugation and resuspend in growth medium at 2 × 10 ~ cells/ml. Prepare sequential 10-fold dilutions of the cell suspension to 2 x 10 4, 2 x 103, and 2 × 102 cells/ml. Place a 2-ml aliquot of each dilution of cells into a 15-ml centrifuge tube. To seed the single cell suspensions into the agar, briefly warm the cell suspensions to 45 ° just before adding the agar solution, then add 4 ml of the warm 0.5% agar/growth medium (final concentration, 0.33% agar), mix gently by pipetting to suspend the cells uniformly, then transfer 1.5 ml per dish to the hardened 0.5% agar base layer. Incubate at 37 ° in a humidified 10% CO2 environment. Feed cells twice a week by dropwise addition of growth medium (3 ml per 60-mm dish).

Untransformed NIH 3T3 cells fail to form any colonies under these culture conditions, whereas ras-transformed cells form progressively growing colonies within 1 week (Fig. 4). Score for the presence and frequency of colonies after 2 weeks.

Tumorigenic Growth Potential

Although the assays described above provide the some of the best in vitro approaches to identify growth properties deregulated as a consequence of expression of a particular oncogene, no one parameter, or any combination of these parameters, is an infallible predictor of malignant


FI~. 4. Growth of colonies in soft agar. NIH 3T3 cells were transfected with normal or oncogenic forms of ras transfectants selected in G418, and G418-resistant cells analyzed for the ability to grow in semisolid medium (see Anchorage-Independent Growth). Cells transfected with normal ras do not proliferate in 0.33% agar (a), whereas cells transfected with oncogenic ras [H-ras(61L), (b)] proliferate to form colonies that are visualized under the light microscope (4 × ) as spheres of proliferating cells, one of which is shown here.

growth potential. Currently, the best assessment of the malignant nature of a cell is provided by the use of immune-suppressed or immune-deficient experimental animal models for tumor formation. Most commonly, cells to be tested are injected subcutaneously into immunocompromised, geneti- cally athymic nude mice, followed by observation of the animals for tumor formation. Parameters informative for relative transforming potential include not only the frequency of tumor generation (number positive/number injected), but also the size of tumors induced and the latency periods of tumor induction.~5,~6

The major limitations of the assay system are the facts that many human tumor cells do not form tumors when injected subcutaneously into nude mice and that this procedure often provides an unreliable indicator of metastatic potential. Nevertheless, this assay constitutes the most versatile and informative in vivo system currently available, and it has been widely applied to oncogene-transformed NIH 3T3 cells as well as to tumor- derived rodent and human cell lines. 1~,16

Procedure . At 6-8 weeks of age, athymic nude (nu/nu) mice are inoculated subcutaneously with the cells to be tested for transforming potential. As mycoplasma contamination can drastically alter the tumorigenic potential of cells injected into nude mice, all cell lines to be tested should first be monitored for mycoplasma contamination. Using sterile technique, harvest mycoplasma-free cells by trypsinization or EDTA release from log-phase cultures, wash once, and suspend in serum-free growth medium or PBS (phosphate-buffered saline, pH 7.4). Inoculate 0.2-ml suspensions of cells containing 1 x 10 ~ to 1 x 106 cells subcutaneously into the dorsal

[ 2 4 ] BIOLOGICAL ASSAYS FOR CELLULAR TRANSFORMATION 293

flanks left and right of the midline. The needle should be at least a 21- gauge needle to avoid damage to the inoculated cells, but preferably not more than 18-gauge to avoid trauma to the mice. At least three, but preferably four or more, mice should be inoculated with each cell line, and both positive and negative control cell lines should be included in each experiment. One mouse can serve as the host for two different cell lines if care is taken to keep the injections physically separate. The mice should be monitored for tumor formation twice a week.

Tumor sizes are commonly reported T M as tumor diameter, measured with calipers through the skin of the intact animal; tumor volume, usually calculated by multiplying tumor diameters measured in different dimensions; or tumor weight, measured by weighing the excised tumor after sacrifice of the tumor-bearing host. To avoid excessive discomfort to the animals, all tumor-bearing hosts should be sacrificed by the time the tumor reaches a maximum diameter of 1 cm, and preferably well before that time. Although some transformed cells show rapidly progressive tumor growth as soon as 1 week after injection, depending on the size of the inoculum, tumor-negative animals should be maintained for up to 3 months, as the latency of tumor induction can vary significantly with different oncogenes and different expression systems. However, many lines of untransformed NIH 3T3 cells will form slow growing nodules after as little as 6-8 weeks, necessitating the inclusion of these cells as a negative control end point. It is always desirable to use as few cells as possible that will still give a positive result. In the case of transformed NIH 3T3 cells, this can be as few as 5 x 104 cells. For some human tumor cell lines, more than 10 7 cells and several months may be required for tumor growth.

If desired, following sacrifice of the tumor-bearing animal, the tumors may be excised using sterile technique, minced finely to form explants, resuspended in growth medium, and plated for in vitro passage and characterization. The medium should be changed as soon as the explants (or single cells) are attached. Cells will begin to migrate from the explants within a couple of days, at which time the explanted tissue should be aspirated from the dish or flask, and the migrated cells henceforth treated as usual for the parent cell line.

Summary

A number of standard and widely applied procedures have been used to determine whether expression of a particular gene triggers the growth alterations that are characteristic of most oncogenes. The assays have been used extensively to evaluate the transforming potential of a wide

294 CROWTn AND TRANSFORMATION [24]

variety of genes that encode tyrosine or serine/threonine k i n a s e s , 32-34

small and heterotrimeric GTP-binding signal transduction regulators, 35,36 and nuclear transcription f a c t o r s , 37 among others. Therefore, the growth- promoting characteristics of a particular gene can be compared with the properties of other genes that have been characterized by the same assays.

The assays described do not represent a complete evaluation of the transforming activity ofa gene. Failure to detect growth-promoting activity in any of the assays does not definitively eliminate the possibility that a particular gene is an oncogene. Specialized assays that use (nonfibroblast) recipient cells more closely approximating the likely environment of the gene of interest may provide better approaches for subsequent studies. Other biological assays for transforming potential 1,2 include measurements of the adhesion properties of cells on different substrata, the ability to grow on confluent monolayers of normal cells, the ability to invade into various artificial tissue matrices, and transgenic animal models. 38 Finally, more specific assays for biochemical alterations that reflect the transformed state can also be employed. For example, as discussed in [23] in this volume, one widely used biochemical measure of transforming potential employs transcriptional activation of genes whose promoters contain so- called oncogene-responsive elements. This, as well as other biochemical assays, can be applied to complement the biological studies described in this chapter.

Acknowledgments

We thank Buddy Weissman for helpful comments on the manuscript and Steve Frisch for advice on REF52 transfection assays. Our research is supported by grants from the National Institutes of Health to A.D.C. (CA61951) and to C.J.D. (CA42978, CA52072, and CA55008). C.J.D. is the recipient of an American Cancer Society Faculty Research Award.

32 M. Cross and T. M. Dexter, Cell (Cambridge, Mass.) 64, 271 (1991). 33 L. C. Cantley, K. R. Auger, C. Carpenter, B. Duckworth, A. Graziani, R. Kapeller, and

S. Soltoff, Cell (Cambridge, Mass.) 64, 281 (1991). 34 S. A. Aaronson, Science 254, 1146 (1991). 35 M. Barbacid, Annu. Rev. Biochem. 56, 779 (1987). 36 j . Lyons, C. A. Landis, G. Harsh, L. Vallar, If. Grunewarld, H. Feichtinger, Q.-Y. Duh,

O. H. Clark, E. Kawasaki, H. R. Bourne, and F. McCormick, Science 249, 655 (1990). 37 B. Lewin, Cell (Cambridge, Mass.) 64, 303 (1991). 38 j. M. Adams and S. Cory, Science 254, 1161 (1991).

[ 25 ] MICROFLUORIMETRY AND CALCIUM IMAGING 297

[25] M o n i t o r i n g of R e c e p t o r - M e d i a t e d C h a n g e s in I n t r ace l l u l a r C a l c i u m at t h e Ce l lu l a r a n d Subce l lu l a r L e v e l

b y M i c r o f l u o r i m e t r y a n d I m a g i n g

By S T E P H E N R. R A W L I N G S , . l E A N - M A R C T H E L E R ,

a n d W E R N E R S C H E E G E L

Introduction

Changes in levels of cytosolic ions following G-protein-linked receptor activation can be measured at the single-cell level with fluorescent probes. The most widely used are Ca 2+ probes that are derived from known specific Ca 2+ chelators such as EGTA. These have a high selectivity for Ca 2+ over other divalent cations, and the binding of Ca 2+ to the probe leads to a conformational change resulting in altered fluorescence. For the first Ca 2+ probe of this type available, quin-2, I the Ca 2+ complexed form had simply a higher quantum yield, and a rise in the cytosolic free Ca 2+ concentration, [Ca 2 +]i, was manifest as an increase in fluorescence. Quin-2 thus made it possible to monitor [Ca2+]i in cell populations. Calibration procedures allowed a semiquantitative assessment of [Ca2+]i. The values obtained reflected the average change in the cell population.

Quin-2 could not be applied to the measurement of [Ca2+] i in single cells, since it has essentially a single excitation and emission wavelength optimum. Thus it was impossible to make a reference measurement at another wavelength with which one could correct for nonspecific changes unrelated to [Ca2+]i. In microfluorimetry such changes are frequent, and they can be due to the loss of the probe by leakage out of the cells or by photodestruction (bleaching) or due to cell movements and shape changes. Therefore, monitoring of [Ca2+] i at the single-cell level became possible only when probes were derived for which Ca 2+ binding led to a spectral change, z As is explained below and in Fig. 1, by calculating the ratio between fluorescence intensities at two selected wavelengths one may eliminate nonspecific changes. The dyes for which this is possible are referred to here as dual-wavelength dyes.

This chapter briefly reiterates important aspects of the use of dual- wavelength dyes. In particular, we address the implementation of image

t R. Y. Tsien, T. Pozzan, and T. J. Rink, J. Cell Biol. 94, 325 (1982). 2 G. Grynkievicz , M. Poenie, and R. Y. Tsien, J. Biol. Chem. 260, 3440 (1985).


298 IoNs AND CHANNELS [25]

o

o

\ - . low Eca2 ,i

J ~ ~ high [Ca2+]i J

)H ~i )~2

excitation wavelength, )~

FIG. 1. Scheme showing the use of dual-wavelength dyes. Shown are the excitation spectra of Fura-2 fluorescence at low Ca z + and high Ca 2 +. The manner in which the spectral shift on Ca 2÷ binding of Fura-2 is exploited is explained in the text.

processing procedures for the assessment of cytosolic free [Ca2+], that is, [Ca2+] i imaging. 3

Dual-Wavelength Dyes for Cytosolic Free Calcium Ion

The principle of the application of dual-wavelength dyes is illustrated in Fig. 1. The example chosen is the most widely used Ca 2÷ probe Fura-2. z The fluorescence of this probe reflects Ca 2+ binding by a shift in the excitation spectrum. The optimal excitation wavelength is shifted from h z to the shorter wavelength ht as the probe chelates Ca z+. A change from low to high [Ca 2+] increases fluorescence at h~, whereas it decreases at hz. In between h 1 and hz there is a Ca2+-independent wavelength h i (the isosbestic or isostilbic h). Changes unrelated to Ca z+ chelation cause a parallel change in the fluorescence excited at any wavelength; the ratio between the fluorescence recorded at wavelengths h~ and h z ( F t / F z) remains constant; Ca z+ chelation changes the ratio F~/F 2 . The latter, rather than an absolute value of fluorescence, is then chosen to represent correctly the changes in [Ca 2+] monitored by the probe.

The same principle applies to dual-emission wavelength dyes such as the Ca z+ indicator indo-l,Z and the pH indicator carboxy SNARF-1.4 The spectral properties of the fluorescence of the various dyes is crucial when

3 R. Y. Tsien and M. Poenie, Trends Biochem. Sci. U, 450 (1986). 4 S. Bassnett, L. Reinisch, and D. C. Beebe, Am. J. Physiol. 258, C243 (1990).

[25] MICROFLUOR1METRY AND CALCIUM IMAGING 299

setting up the monitoring. For further details of the range of fluorescent probes and their applications, see the handbook of fluorescent probes. 5

Cell Preparation and Dye Loading

Dye Loading of Intact Cells with Cell-Permeant Ester. Exposure of intact cells to Ca 2÷ probes in the esterified form (e.g., Fura-2/AM) results in accumulation of the dye in the cytosol. The lipophilic esters penetrate cells rapidly and are cleaved by cellular esterases, with the resulting hydrophilic free form of the dye being trapped within cells.

Dye loading with esters requires careful elaboration of experimental conditions, to make sure that the dyes are completely deesterified and do not accumulate in internal organelles (endoplasmic reticulum, mitochondria) rather than the cytosol. Among the pitfalls of ester loading is the possibility of cell entry by endocytosis (rather than diffusion). This can be eliminated by careful solubilization of the dye esters using, for example, the detergent Pluronic F-1275 (Molecular Probes, Eugene, OR 97402), as well as by lowering the temperature.

Dye Loading with Free Form. To include the free forms, cells have to be permeabilized, microinjected, or subjected to patch clamp techniques. Loading in this manner, one is certain that the proper probe is in the cytosol. However, all these procedures may interfere with cell homeostasis.

Microscope

Monitoring of [Ca 2 + ]i with fluorescent probes on inverted microscopes requires a preparation of cells or tissue on glass coverslips in order to use high numerical aperture immersion objectives. A high aperture is crucial for good excitation and efficient light gathering. Note that, for optimal results, the immersion oil has to be pure and devoid of fluorescence, and glycerol has to be kept dry. When working with live tissue sections (slices), upright microscopes and water immersion objectives are used.

Fluorimetry is performed with the epifluorescence mode. As is shown in Fig. 2, a dichroic mirror deviates the excitation light beam into the objective, which serves as a collimator. Fluorescence light is monitored after passage of the same dichroic mirror. Dichroic mirrors are characterized by their critical wavelength hcrit. Light with wavelengths shorter than h~r~t is reflected, wavelengths longer than k~r~t pass. The hcrit values of

5 R. P. Haugland, "Molecular Probes Handbook of Fluorescent Probes and Research Chemi- cals." Molecular Probes Inc., Eugene, Oregon 97402.

300 IONS AND CHANNELS [25]

dichroic mirrors are chosen between the excitation and the emission max- ima of the respective dye, to permit efficient excitation as well as recovery of the maximum of the fluorescence light.

Dual-Excitation Microfluorimetry and Imaging

At the single cell and subcellular level, [Ca2+] i can be monitored either by alternating between two wavelengths for excitation (dual-excitation microfluorimetry) or by discriminating emitted fluorescence into two wavelength sectors measured separately (dual-emission microfluorimetry). In either instance, fluorescence light can be detected with a simple detector or with a camera. In the latter case, digital image processing (imaging) is involved.

The scheme in Fig. 2 presents the essential elements required for dual- excitation microfluorimetry. These are a light source, monochromators or filters, a wavelength alternator, a microscope with epifluorescence configuration, detectors, and data acquisition devices.

Light Sources

A stable light source with a wide spectrum, in particular providing good illumination in the UV range, is essential. The most versatile are xenon arc lamps since they have a broad spectrum covering well the excitation wavelengths for Fura-2 and other widely used probes. Mercury lamps have a less uniform spectrum, which makes it more difficult to obtain optimal illumination.

Excitation Wavelength Selection and Alternation

Using Monochromators. Selective excitation of fluorescence requires a monochromatic light source. The most versatile equipment for fluorimetry includes monochromators for excitation light. Within these, the white lamp light is dispersed by a grating or a prism into monochromatic components. A fraction of this "rainbow" is then directed toward the specimen. Monochromators allow the use of the optimal excitation wavelength for any application by recording excitation spectra and choosing the best wavelength.

Dual-excitation work requires two monochromators. Excitation is al- ternated with a chopping mirror, with choppers or with shutters. Any arrangement has particular faults and merits. In choosing a particular option (i.e., a given instrument), one should consider the limitations in speed, the ease of optical alignment (difficult with chopping mirrors),

[25 ] MICROFLUORIMETRY AND CALCIUM IMAGING 301

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302 IONS AND CHANNELS [25l

potential vibrations and noise (high with shutters), and the precision and ease of synchronizing the output (see below).

Using Filters. For well-known applications such as [Ca 2 ÷ ]i monitoring with Fura-2, a set of filters can fully substitute for the monochromators. The filter set usually consists of an interference filter selecting a band of wavelengths 5 to 10 nm in width and a barrier filter eliminating wavelengths longer than a critical value to correct for the imperfection of the interference filters. Dual excitation is obtained by mounting filters on a wheel or again by the use of a dual light pass with shutters.

In summary, dual-excitation fluorimetry with defined dyes can be optimally performed with filters, which are less expensive and easier to handle. However, monochromators provide the advantage that new dyes can be immediately applied, that is, before the optimal filter sets have been devised and become commercially available.

Fluorescence Light Detection

Photomultiplier Tubes versus Cameras. The light levels of fluorescence produced in [Ca2+] i monitoring are low. Detection requires sensitive photomultiplier tubes (PMTs) or high-sensitivity video cameras. For PMTs, the detection limits are not critical. In general, a fairly standard PMT will suffice. For cameras, the problem is severe, since the light from a single cell which contains all the relevant information is now spread over hundreds of single picture elements (pixels), and in order to generate a reasonable image signal the number of fluorescence photons per pixel has to be clearly larger than the background noise of the camera.

Intensified cameras come with various technologies. Fundamentally two strategies are engaged. (1) A camera may be built which accumulates photon data on a cooled charge-coupled device (CCD) chip, and readout is varied according to light levels. For high levels one reads out in short intervals; for low levels one reads in long intervals (e.g., Coolview from Photonic Science, Robertsbridge, East Sussex, UK). (2) An image intensifier converts the incoming photons to higher energy photons prior to reaching the detector, increasing their chances of detection. Intensifier technology is based either on silicon targets or on microchannel plates. Silicon targets increase the "lag," that is, the time during which the detector will carry over remanent pictures to further frames; microchannel plates generate a characteristic noise and require efficient on-line filtering. The optical coupling between intensifier and detector is crucial, and fiber optics appears as the best solution at present (e.g., Darkstar series, Pho- tonic Science, Robertsbridge East Sussex, UK). A whole range of intensified cameras can be obtained (e.g., from Hamamatsu Ltd., 91781 Wissoux, France, or Photonic Science), but the detection wavelength optima are

[25] MICROFLUORIMETRY AND CALCIUM IMAGING 303

often in the red and IR, since many products are geared to night vision. As the arguments in favor of any type of camera are complex, the reader is referred to a previous comprehensive treatment of the problem. 6 In general, the type of intensified camera should match the priorities of the user (speed versus spatial resolution) as well as the concept of image processing engaged for calculating ratios. Therefore, camera and image processing device have to be chosen together.

Synchronization

Whatever the detector, in order to perform the ratio calculations, the data acquisition system has to be connected to the wavelength alternator for synchronization (or coding). Synchronization is the crux of the problem of dual-excitation imaging. Three solutions have been found to the problem: (1) coding the images, storing them separately, and calculating ratio images off-line; (ii) taking only a single reference image at wavelength hi, recording at h2, and producing ratio images always using the same reference (pseudoratioing); (iii) interconnecting camera synchronization and the wavelength alternator, such that wavelength alternation occurs in synchrony with the TV signal, permitting rapid on-line ratio images. Even for the simple data generated by a PMT, this is not a trivial problem and requires a special interface which generates two virtual channels for the fluorescence excited at either wavelength.

Image Processing

Calculating [Ca2+]i maps for a standard size image (i.e., 528 × 792) requires that a ratio is calculated for 40,000 paired values. Since the structure of the data is constant, fixed array (parallel) processors are commonly used. With these, on-line ratio calculation becomes possible at standard video rates. Coupled with efficient synchronization and a microchannel plate intensified CCD camera (Photonic Science), on-line ratioing produces a practical time resolution of about 10 sec-i; in other words, 10 ratio images with reproducible kinetic development can be obtained from data previously stored in the analog form with standard TV technology. 7,8

Video Mixing Techniques. A remarkable solution has been proposed for excitation ratio imaging. 9 Gustafson and Magnuson devised a proce-

6 R. Y. Tsien and A. T. Harootunian, Cell Calcium 11, 93 (1990). 7 J.-M. Theler, C. B. Wollheim, and W. Schlegel, J. Receptor Res. 11, 627 (1991). 8 A. J. O'Sullivan, T. R. Cheek, R. B. Moreton, M. J. Berridge, and R. D. Burgoyne,

EMBO J. 8, 401 (1989). 9 M. Gustafson and K.-E. Magnusson, Cell Calcium 13, 473 (1992).


dure which makes digital ratio calculations obsolete by recording and video mixing. In brief, images collected and hi and h2 are directed toward frame buffers which are read out continuously, one channel in red, the other in green. The two analog video signals are mixed such that [Ca2+]i changes affect the hue, whereas the total fluorescence intensity determines the brightness. With this comparatively simple procedure valuable [Ca 2÷ ]i image information can be generated with high time resolution at moderate cost.

Dual-Emission Microfluorimetry and Imaging

Wavelength-Selective Light Gathering

A general scheme for the monitoring of dual-emission dyes is presented in Fig. 3. A single excitation wavelength has to be produced with either an interference filter or a monochromator. Again, as with dual excitation, the microfluorimetry is performed in the epifluorescence mode, using a dichroic mirror to deviate the excitation light beam, collecting the fluorescence light after passage of the same dichroic mirror. To exploit the wavelength shift, dual-emission systems need a wavelength discriminator. This is essentially a second dichroic mirror. The latter splits the fluorescence light into two spectral sectors, light with the shorter wavelengths (h < hcrit ) being reflected off, light with longer wavelengths (h > hcrit ) passing the mirror. Barrier or interference filters placed in the two light paths improve the selectivity of the wavelength discrimination. The intensity of each sector is then monitored with a separate PMT (PMT1 and PMT2) or directed toward a part of the TV image.

Dual-Emission Imaging

A simple system for dual-emission ratio imaging for the Ca 2+ probe indo-1 has been developed (patented by Hamamatsu). This contains a wavelength discriminator which puts the two images collected in the two wavelength sectors side by side. Given the selection of wavelengths, the geometrical distortions produced by camera and wavelength discriminator optics are not the same for the two sectors of fluorescent light. A device that corrects, in real time, the geometrical distortions allows ratio images to be subsequently produced by the image processor.

Imaging versus Single-Cell Monitoring with Photomultiplier Tubes

Monitoring receptor-induced changes in [Ca2+]i has advanced in two major steps: from measurements on cell populations to single-cell record-

[25] MICROFLUORIMETRY AND CALCIUM IMAGING 305

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ings and from single-cell recordings to imaging. For most questions in signaling biochemistry, the first step has been a decisive one. Single-cell recordings have taught us essential new elements in [Ca2+] i signaling, most notably the fact that [Ca 2÷ ]i changes occur frequently as oscillations. The [Ca2+] i imaging technique has shown that [Ca2+] i changes are widely variable within a single cell, in particular in those cells which have a compartmentalized morphology. Thus, in neurons, changes in dendrites and cell bodies may occur independently of one another, and [Ca2+]i gradients can be maintained over long periods. Imaging has shown [Ca2+]i waves. For receptor-coupling studies, [Ca2+] i imaging is less essential. Often, results produced at the single-cell level with the simpler PMT technology will suffice to answer the questions addressed.

In choosing which option to pursue, one should consider the context in which the data on [Ca2+]i will be treated. If, for example, [Ca2+] i is monitored in combination with electrophysiological recordings obtained in a whole-cell mode, it is appropriate to use PMT technology and to collect data relevant for the same single cell. In contrast, if the [Ca2+] i signal is being considered in a context of further morphological elements such as the cellular redistribution of proteins or electrical recordings on selected structured parts of large neurons, imaging appears better suited.

It should be noted that single-cell [Ca2+]i imaging data can be produced very easily from stored data. Simultaneous recordings from a group of cells can be exploited to resolve the pattern of each individual cell and to provide very strong correlative data.

Dual Excitation versus Dual Emission

Dual-excitation systems are in general more complex. Wavelength alternators inherently require moving parts and the discontinuous signal special treatment. Furthermore, the discontinuity of the signal implies that ratios are formed for consecutive periods. If the wavelength alternation is slow relative to the kinetics of the [Ca2+] i signal, this may induce systematic errors.

In contrast, dual-emission systems are easier to set up. Wavelength discriminators are now commercially available as sets which include well- matched filters and dichroic mirrors. Mounted on standard cubes, these can be easily exchanged when changing applications (e.g., from indo-1 to carboxy SNARF-1). Dual-emission systems produce two continuous signals which can be acquired with simple means (chart recorders, computer boards, etc.) and ratios are real, that is, formed from data acquired simultaneously. With a simple electronic device, it is possible to treat the

[ 2 5 ] MICROFLUORIMETRY AND CALCIUM IMAGING 307

signals in analog form and produce an analog ratio signal output, which reflects directly the changes in [Ca2+]i.

In favor of dual excitation, however, is the fact that the light levels at the two wavelengths are comparable; in contrast, there is often a marked discrepancy between the light levels exploited in the two sectors for dual- emission recordings. For PMT monitoring, this leads to a large discrepancy in the noise between the two channels; for imaging, this requires that the intensity of one of the sectors has to be reduced with neutral density filters, sacrificing valuable fluorescent light.

Confocal Cytosolic Free Calcium Ion Imaging

A limited number of groups have begun to use confocal fluorescent microscopy for imaging of changes in [Ca:+]i in single cells (e.g., Hernan- dez-Cruz et al. , 1° Lechleiter et al.l l) . This technique has a number of advantages over standard microscopy for such experiments. First, the spatial resolution is improved because of the narrower depth of field and the elimination of out-of-focus elements in the image. This generally leads to an improved axial as well as lateral resolution. Second, the temporal resolution of the image collection can be enhanced by the use of the line- scanning mode (see below). Third, errors in measurement due to variations in path length are minimized since a relatively constant volume is sampled.

Confocal microscopy demands a point source of excitation light usually provided by a laser. Because of the inherent nature of the technique, each image point is very small and is defined as a voxel [a pixel in three dimensions (3D)]. The sample is scanned in both the x and y dimensions to produce an array of voxels, which are then reconstructed as an image using computer software. The temporal resolution of an image is thus generally defined by the scanning speed, which for many confocal microscopes is relatively slow (taking many seconds to produce an image). Scanning in the x dimension is much faster than that in the y dimension, and so limiting the scanning to a single line can produce very rapid images, admittedly limited in the y direction to the size of a single voxel. For a good example of this, see Hernandez-Cruz et al., 1° who used a line- scanning mode to follow the influx of Ca 2 ÷ into an isolated neuron at a time resolution of less than 5 msec.

The usual light source for confocal microscopy is an argon laser producing excitation wavelengths in the visual spectrum (UV lasers exist, but they are expensive and inconvenient to use). This means that fluorescent

to A. Hernandez-Cruz, F. Sala, and P. R. Adams, Science 247, 858 (1990). i1 j. Lechleiter, S. Girard, E. Peralta, and D. Clapham, Science 252, 123 (1991).

3 0 8 IONS A N D C H A N N E L S [ 2 6 ]

indicators with relatively long excitation wavelengths need to be used. Examples of such dyes for Ca 2÷ include fluo-3, Calcium Green, and Fura Red. Unfortunately, these dyes generally have a single excitation and a single emission wavelength, making them unsuitable for the standard ratiometric measurements discussed earlier. However, the major disadvantage of using confocal microscopy for imaging of changes in intracellular ion concentrations remains the high cost of setting up such a system.

[26] Combina t ion of Microf luor imetr ic Moni tor ing of Cytosolic Calc ium and p H with Pa tch C lamp

Electrophysiological Recordings in Neut rophi l Granulocytes

By NICOLAS DEMAUREX, STEPHEN R. RAWLINGS, KARL-HEINZ KRAUSE, MARISA E. E. JACONI,

P. DANIEL LEW, and WERNER SCHLEGEL

Introduction

Receptors linked to G proteins trigger multiple signaling pathways, many of which change ion fluxes. The G proteins can also have direct actions on ion channel activity. To distinguish between direct and indirect channel regulation, it is necessary to measure ion channel activity and ion fluxes of cells in the context of second messenger changes. For intracellular messengers that can be monitored efficiently with fluorescent probes, it is appropriate to combine the electrophysiological recordings with microfluorimetry. Electrical recordings are most often obtained with the "tight seal" or patch clamp technique.~ In the whole-cell configuration, the patch clamp technique also allows the application of second messengers to the cytosol of the cell. This chapter describes how cytosolic Ca 2÷ ([Ca2+]i) and pH can be monitored with the fluorescent probes indo-12 and carboxy SNARF-1, 3 respectively, in human neutrophils and HL-60 cells during electrophysiological recordings.

10. P. Hamill, A. Marty, E. Neher, B. Sakman, and F. Sigworth, Pfluegers Arch. Eur. J. Physiol. 391, 85 (1981).

2 G. Grynkiewicz, M. Poenie, and R. Y. Tsien, J. Biol. Chem. 260, 3440 (1985). 3 S. Bassnett, L. Reinisch, and D. C. Beebe, Am. J. Physiol. 258, C243 (1990).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 238 All fights of reproduction in any form reserved.

[26] COMBINED PATCH CLAMP/MICROFLUORIMETRY TECHNIQUE 309

Apparatus

Microfluorimetry

Microscope. We use a Nikon Diaphot inverted microscope (NIKON AG, 8700 KOssnacht, Switzerland) with epifluorescence optics. The inverted microscope allows the use of high numerical aperture (>1.2) oil or glycerol immersion objectives, thus increasing the efficiency of fluorescence light capture. The top of the recording chamber remains readily accessible for bath solution changes and the approach of micropipettes. Our preferred objective for microfluorimetric measurements is the Nikon UV/CF 40x, with a numerical aperture of 1.3. Although nonquartz optics attenuate the excitation light intensity, especially for wavelengths less than 340 nm, this is not a severe problem since we have to reduce the light intensity still further with neutral density filters (see below). Thus, quartz objectives, which are expensive and generally do not have the equivalent optical quality of standard fluorescence objectives, are not necessarily a prerequisite for such a system. We did, however, purchase the microscope with a quartz collector lens in the objective turret, giving us flexibility if, in the future, we need to use dyes, like Fura-2, that require shorter excitation wavelengths.

Cells may be viewed in transmitted light, although we found that Hoff- mann interference modulation contrast optics 4 significantly improve the visualization of the cell for making the patch clamp recordings, while only slightly reducing the efficiency of the microfluorescence measurements. A 400x magnification (oculars are 10×) is sufficient to perform patch clamping under good visual control and offers a field of view large enough to select the appropriate cell among a population of approximately 30 cells. Using higher magnification provides more visual comfort, but more time is spent in the search of the appropriate cell. There are also other problems associated with using higher power objectives, including significant photobleaching of the fluorescent probes.

Dual-Emission Florimetry. The principle of dual-emission microfluorimetry and its implications have been described in detail elsewhere in this volume) The light source for epifluorescence is a 100-W mercury arc lamp, which has a strong emission line at 365 nm. This is appropriate for measurements with the calcium-sensitive dye indo-1, whose excitation maximum is at 360 nm. Mercury lamps have a highly nonuniform spectral output which restricts their use to dyes whose excitation spectra

4 R. Hoffman, J. Microsc. 110, 205 (1977). 5 S. R. Rawlings, J.-M. Theler, and W. Schlegel, this volume [25].


fits with one of the emission lines. A xenon lamp would be more versatile.

The intensity of excitation light must be optimized to produce a good signal without excessive photobleaching of the dye. For indo-1 recordings the excitation light is attenuated 256 times using two neutral density filters (16x16); for the pFI dye carboxy SNARF-1, attenuation is 32-fold (16x2). The significant attenuation required for indo-1 fluorescence is due to its excitation maximum lying on one of the strongest emission lines of the mercury lamp. The appropriate excitation wavelength is selected by an interference filter (~ = 355 --- 5 nm for indo-1,515 -+ 10 nm for carboxy SNARF-1), and the beam is reflected to the stage by a dichroic mirror (hcrit of 380 and 540 nm for indo-1 and carboxy SNARF-1, respectively). The emitted light collected by the objective is directed to the same dichroic mirror, through which the light with wavelengths longer than hcrit passes. Fluorescence may be viewed through the eyepiece or diverted toward the photometers. A pinhole diaphragm positioned further along the optical path allows the selection of a field area slightly larger than the cell studied.

The fluorescence light to be monitored passes a wavelength discriminator consisting of a second dichroic mirror (hcrit = 455 or 610 nm for indo- 1 and carboxy SNARF-1, respectively). For indo-l, the reflected and transmitted light passes barrier filters (h > 405 nm, h > 480 nm, respectively). For carboxy SNARF-1, the reflected and transmitted light passes interference filters (~, = 580 -+ 20 and 610 --+ 20 nm, respectively). The light at the two wavelengths is directed to two photometers (Nikon P1) (see Fig. I), and the resulting analog signals are sent to the data acquisition system.

Electrophysiology

For patch clamp recordings a LIST EPC-7 amplifier (List Medical, Darmstadt, Germany) is used. The headstage is mounted on a Zeiss micromanipulator (Carl Zeiss AG, Ziirich, Switzerland), which offers motor- controlled fine movements. We found this electronic micromanipulator to be more precise and easier to use than hydraulic micromanipulators, which tend to drift slightly, often resulting in the premature termination of a recording. The Zeiss micromanipulator is fixed to the Nikon microscope by adapting the fixation bar normally used to attach hydraulic micromanipulators. To reduce vibrations, the microscope, the optical elements for microfluorimetry, and the photometers are mounted on a large (120 x 80 cm) floating table (Vibraplane, Kinetic System, Roslindale, MA 02131). A Faraday cage, produced locally and covered with black material, protects the setup from electrical noise and ambient light. Any equipment


| ,'~,'~ ~indo.,~ ''~ ] IPATCH-CLAMP[ ,

I I COMPUTER RECORDER

FIG. 1. Diagram of combined patch-clamp/microfluorimetry setup. The purpose of the apparatus is to manipulate cell membrane potential, as well as the intracellular and extracellular environment of a single neutrophil, and to monitor cytosolic ions (Ca ?+, pH) with fluorescent (dual emission) probes and ion fluxes with electrical measurements. The cell membrane potential is imposed by a pulse generator (stimulator). The extracellular environment is manipulated via a superfusion pipette or a bath exchange system. The intracellular environment is controlled via the patch pipette (i.e., introduction of membrane-impermeant second messengers). Fluorescence discriminated into two wavelength sectors (F~5 and F4s0 for indo-1) by a dichroic mirror (hatched box) is detected by two photomultipliers (PMT1, PMT2), and the electrical signal by a patch clamp amplifier. The analog PMT and amplifier output is converted to a digital form (A/D converter) and handled by a computer. The electrical signal from the patch clamp is visualized on-line using an oscilloscope. For long time course recordings requiring high resolution, the electrical signals can be recorded by a tape recorder.

OSCILLOSCOPE

producing electrical noise is kept outside of the Faraday cage, including the power supplies for the microscope lamps, the photometer controllers, the thermostat controller, and the joystick drive for the micromanipulator.

The voltage pulses that are applied for the study of ionic currents are provided by a programmable, eight-channel stimulator (Master-8, AMPI, Jerusalem, Israel). There are now a number of computer software pack- ages on the market that can deliver a broader variety of pulses and ramps. However, for certain purposes, including the establishment of the patch clamp recording, we have found it useful to have a stimulator separate from the computer system.


A standard oscilloscope provides an excellent means of monitoring the experiment on-line. The stimulation protocols can be monitored after modification by the patch clamp amplifier, and any changes in the recording conditions can be quickly detected, allowing the appropriate measures to be enacted. Certain analog and digital storage oscilloscopes have the facility to store multiple sweeps on the screen, which is useful for monitoring drug effects on ionic currents or for single-ion channel recordings. Further elements necessary for the experiments, but not described in further detail here, include a pipette puller and a microforge for fire polish- ing of pipettes. 6

Data Acquisition

The simultaneous acquisition of ionic currents and single cell fluorescence is a challenging task. Using a computer system requires real-time interface hardware with at least four input channels, since one wants to record simultaneously the outputs of the two photometers, the applied voltage protocol, and the whole-cell or single-ion channel currents. An additional input channel connected to the bath-exchange system allows the different changes of solutions to be recorded.

One problem with data acquisition in such a system is that the temporal nature of ionic currents and of calcium- or pH-dependent fluorescence changes are different. Most ionic channels have rapid kinetics of activation and deactivation, thus requiring acquisition at high time resolution. In contrast, most events monitored with the fluorescent dyes have a relatively slow and long-lasting time course. Recording the whole of the experiment at a collection frequency that can faithfully capture fast ionic channel kinetics would produce very large data files (potentially causing problems for data storage), and a significant part of the data may be superfluous. Thus, when making combined measurements one wants long recordings (over many minutes), at a relatively slow collection frequency, while being able to make short, high-frequency acquisitions during selected periods. For this purpose we use two independent acquisiton systems running in parallel on a PC-compatible microcomputer (IQUE, 486SX/33 MHz). One is dedicated to a low time resolution acquisition, the other to a stimulus- triggered fast acquisition. Both systems function in the Microsoft Windows environment which allows "multitasking," that is, the simultaneous control of both acquisitions. The low time resolution system (Acqui, SICMU, University of Geneva) records, at a capture rate in the millisecond range, up to 16 channels with 12-bit resolution. It is used to monitor changes in

6 j. L. Rae and R. A. Levis, this series, Vol. 207, p. 66.


[Ca2+]i or pH on-line in combination with the changes in transmembrane currents and membrane voltage. Storing the [Ca2+]i/pH and the current signals together in the same data files allows an easy assessment of correla- tions between the variables simply by plotting the traces against one another. The examples given below illustrate the importance of this feature. The fast acquisition system (Daqsys, SICMU, University of Geneva) can record 4 channels at frequencies up to 100 kHz. It is used as a classic electrophysiology acquisition system, coupled to the stimulator.

An alternative solution to the data acquisition problem is to use a tape recorder (e.g., DTR-1201 Biologic, 38640 Claix, France). This provides a convenient backup, as the entire experiment may be stored at high time resolution (up to 50 kHz) on a single tape. Thus, the events that the computer-based system has failed to record or has recorded at too low a time resolution can afterward be played back through one of the acquisition systems described above. The tape recorder is also very convenient for measurements of single-ion channel currents since long recordings at high time resolution are often required for statistical analysis. A single tape stores the equivalent of 1.4 GBytes of data, a mass of information difficult to store on the hard disk of a microcomputer.

Control o f Extracellular Environment

The experimental chamber has been described previously. 7 It consists of a petri dish with a circular hole onto which a coverslip is fixed either with silicone grease or with a nontoxic bicomponent silicon elastomer (Sylgard 184, Dow Corning, 6198 Seneffe, Belgium). To alter the extracellular ionic conditions, we use a bath exchange system that can rapidly and completely exchange the chamber contents during a recording. When connected to a patch clamp setup, such an exchange system should avoid turbulence to preserve the stability of the recording. It should also not pick up electrical noise, which can be solved by placing the whole installation inside of the Faraday cage. Our system, produced by a local work- shop, consists of six pieces of silicon tubing connected to a Plexiglas cube which functions as a "passive" mixing chamber. Flux through the input tubing is driven by gravity and controlled by individual electrically controlled micropinches. The mixing chamber is put close to the recording chamber to reduce the dead space, and solutions are exchanged in less than 5 sec. The electrical switches of the micropinches can be connected to the acquisition system for precise timing. In addition to this bath- exchange system we found it convenient to apply agonists through a

7 W. Schlegel, J. Recept. Res. 8, 493 (1988).


superfusion pipette located close to the cell (Fig. 1). This allows repetitive application of hydrophobic agonists or toxins that are otherwise poorly washed out of the plastic tubing of the bath-exchange system.

Control of Temperature

The temperature of the experiment is controlled by heating or cooling at 15-cm-diameter, 1-cm-thick aluminum plate enclosing the experimental chamber. An electric sensor dipped into the chamber monitors the temperature, which is controlled by two Pelletier elements fixed to the metallic plate. The advantage of the Pelletier elements is that they allow cooling as well as heating of the plate, allowing experiments in a temperature range of 10 ° to 42 °. Good temperature control requires that the solutions be placed in a water bath and brought to the appropriate temperature before being perfused. The major drawback of this system is that it adds some electrical noise to the recordings. Thus, the temperature control has to be turned off when doing high-resolution recordings.

Overview

The scheme in Fig. 1 illustrates all of the essential elements for a combined dual-emission microfluorimetry/electrophysiology setup as described above. Integrated systems marketed by a single company are now available. Purchasing such a system simplifies installation and often provides for technical backup, but it may not always provide the optimal choice of all the elements. It should be kept in mind that the quality of the "primary signal producers" (optical elements for fluorimetry, patch clamp amplifier) determines the quality of the data. However, convenient and efficient data handling and equipment control are also important assets of a good system.

Combined Recordings

Solutions

In the whole-cell configuration of the patch clamp technique the contents of the patch pipette dialyze the cell interior, allowing definition of the ionic composition of the cytosol and introduction of various agents into the cell. Unfortunately, this also can result in the loss of soluble cytosolic factors, a process that may cause a progressive loss ("rundown") of ionic currents and [Ca2+]i responses to agonists. For instance, in HL-60 cells the rise in [Ca2+]i in response to formylmethionylleucylphe- nylalanine (f-MLP) is lost within 60 sec of achieving the whole-cell config-

[ 2 6 ] COMBINED PATCH CLAMP/MICROFLUORIMETRY TECHNIQUE 315

uration. Adding ATP (1 mM) and GTP (10/zM) to the pipette solution slows, but does not entirely prevent, this loss.

In electrophysiological experiments, it is often necessary to use nonphysiological intra- and extracellular solutions to isolate specific ionic currents. To maximize a specific current, other ion channels are blocked and chemical gradients are optimized. In a typical experiment designed to measure currents through Ca 2+ channels both intra- and extracellular solutions contain nonpermeating ions to replace Na + and K +, the extracellular solution contains a high concentration (10 mM) of C a 2+ o r Ba > (a better charge carrier than Ca2+), and the intraceUular solution has a high buffering power for calcium (10 mM EGTA or BAPTA). Obviously with 10 mM EGTA in the cytosol and 10 mM Ba 2+ in the extracellular medium, the changes in [Ca2+]i c a n n o t be measured with fluorescent dyes. Thus, when performing combined fluorimetric/patch clamp recordings, ionic conditions are chosen that are closer to the physiological situation. An example of the internal and external solutions for combined [CaZ+]i/elec - trophysiology experiments is shown in Table I.

Cell Preparation and Dye Loading

Two procedures can be used to introduce the fluorescent dye into the cytosol of cells: (1) incubation with the acetoxymethyl ester (AM) form of the dye, which permeates through the plasma membrane and is subsequently hydrolyzed by cytosolic esterases into the free acid form, and (2) direct loading of the free dye through the patch pipette. The main

TABLE I PIPETTE AND BATH SOLUTIONS FOR

COMBINED CYTOSOL1C CALCIUM/ ELECTROPHYSIOLOGICAL RECORDINGS

IN HL-60 CELLS

Pipette solution Bath solution (raM) (raM)

140 KC1 140 NaCI 5 NaCI 5 KCI 1 MgCI2 1 MgCI2 1 MgATP 1.1 CaCI2 0.2 EGTA 0.1 EGTA 20 HEPES 20 HEPES 0.05 Indo-1 free acid pH 7.2 pH 7.2 290 mOsm 295 mOsm


disadvantage of the second procedure is that the diffusion of dyes through the patch pipette is relatively slow. In human neutrophils and HL-60 cells under most conditions the time required to achieve a sufficient dye loading exceeded 2 min. Thus, when relying solely on the dye loading from the patch pipette many cellular responses that are subject to run-down may be lost before the [Ca2+]i, or pH changes can be measured. For this reason, we prefer to load cells by incubating them with the AM form of the dye. The pipette solution in general also contains the free form of the dye (see Table I) to prevent a significant loss of the indicator, through dialysis, during the course of an experiment. For [Ca2+]i measurements, we incubate the cells (2 x 107/ml) for 30 min at room temperature with 5 tzM indo-1/AM in a HEPES-buffered (20 mM) Krebs-Ringer solution (pH 7.2). The cells are then washed twice, and kept at room temperature until use.

Dye loading should be verified with a good fluorescence microscope. Although the degree of loading may vary from cell to cell, in a given cell the loading should be homogeneous, indicating that no significant compartmentalization into intracellular organelles has occurred. Compari- son of the fluorescence intensity of cells loaded with the AM form of the dye to the fluorescence measured in cells loaded with the free form of the indicator using the patch pipette gives an estimate of the concentration of the dye in AM-loaded neutrophils and HL-60 cells of between 30 and 50/~M.

Calibration of lndo-I Fluorescence Signal for Cytosolic Calcium Ion

In principle, two methods of calibration can be used for any of the ionic probes. The first is to equilibrate the cytosol with a known extracellular ion concentration using ionophores. The second is to equilibrate the cytosol with the solution in the patch pipette in the whole-cell configuration. Both methods should yield comparable results. Here we describe the calibration recordings made with indo-l. The ratio of emitted indo-1 fluorescence, F4os/F48o, allows the determination of [Ca2+]i according to

[Ca2+]i = K d / 3 ( R - R m i n ) / ( R m a x - R ) (1)

where K d is the dissociation constant of indo-1 (250 nM, determined in vitro2), R the measured F4os/F4so ratio, Rmin the minimum F4os/F48o ratio when the dye is free of calcium ([Ca 2+] < |0 nM), Rma x the maximum F4oJF48o ratio when the dye is saturated with calcium ([Ca z+] > 2 mM), and/3, the ratio of the fluorescence signals measured at horn of 480 nm in the absence of Ca z+ and at Ca 2+ saturation (F480 free/F480 saturated). Note that no dye should be lost, or photobleached, when determining/3.


When calibrating with an ionophore, the calibration constants are determined by exposing the cells to 2 /zM ionomycin in the presence of either 10 mM Ca 2+ (Rma x) o r 10 nM Ca 2+ (Rrnin). Then/3 is determined by measuring the fluorescence at 480 nm after switching from one solution to the other.

When calibrating with the patch pipette, the constants are determined by dialyzing the cell with a solution containing either 10 mM Ca 2+ (Rmax) or 10 mM EGTA (Rmin). Thus/3 is not determined directly, since it is not possible to exchange the pipette solution with our system. However, by equilibrium with an intermediate [Ca 2+] in the pipette, a value for/3 can be obtained indirectly by solving Eq. (1):

gd/3 = [Ca2+](Rmax - R ) / ( R m a x - Rmin) (2)

The Kd/3 value is determined by dialyzing the cell with a [Ca 2+] of 300 nM (9.2 mM EGTA and 5.4 mM CaZ+). This [Ca 2+] is verified using a Ca 2- electrode.

The calibration constants are determined as averages from more than 20 cells sampled. Such constants are determined for each cell type, and they are stored on the computer for off-line calibration of the fluorescence signals.

An alternative to calibrating with constants (/3, Rma x , Rmin) is to establish calibration curves by equilibrating cells at various levels of known [Ca 2+] or pH. This avoids the extreme conditions under which R~a x and Rmi n have to be established.

Autofluorescence, that is, cellular fluorescence observed without any loaded dye, is not normally considered for the calibration. However, it should be ensured that autofluorescence represents a negligible contribution (< 10%) to the fluorescence signals.

Use of Combined Pa tch Clamp/Microf luor imet ry Techn ique in H u m a n Neutrophi ls and H L - 6 0 Cells

Example 1: Translocation of Small Numbers of Ions in Patch-Clamped Cells Detected by Fluorescent Probes

The combination of patch clamp and microfluorimetry was particularly important for the study of Ca 2+ influx in human neutrophils and HL-60 cells. 8 We have used the patch clamp technique to control the cell membrane potential and to microperfuse second messengers of interest. As

8 N. Demaurex, W. Schlegel, P. Varnai, G. Mayr, D. P. Lew, and K.-H. Krause, J. Clin.

I nve s t . 90, 830 (1992).


nM 900

7 0 0 - -

SO0 - -

300 - -

100 - -

mV

+60 E | n s ( l ' 4 ' 5 ) P 3

-60

+ Ca 2+

b nM

700

1 rain

500 - -

300 --

100 --

mV

+60 E

-60

~ 2+ . 2+ + ~ a + INI

Ins (1 ,4 ,5)P 3

C nM

700 --

500 - -

300 - -

1 0 0

mV

+60 E

-60

1 m i n

+ Ca 2+

I n s ( 1 , 4 , 5 ) P

1 rain


the currents associated with Ca 2+ influx in HL-60 neutrophils are very small, the influx is monitored in these studies by microfluorimetry, con- trasting [CaZ+]i changes in the presence versus the absence of extraceUular Ca z+ (Fig. 2a). The Ca 2+ influx is activated by microperfusion of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] (Fig. 2) and is blocked by Ni 2+ (Fig. 2b). The influx is probably stimulated as a result of the depletion of intracellular Ca 2+ stores. 9 The HL-60 cells do not contain voltage-activated Ca 2+ channels, and thus depolarization of the membrane decreased the Ca 2+ influx (Fig. 2c) by reducing the electrical driving force for Ca 2+ translocation.

Example 2: Combined Measurements Allowing Calculation of Whether a Given Current May Account for Observed Change in lntracellular Ion Concentration

Studies from our laboratory have shown that HL-60 neutrophils possess a voltage-activated H + conductance. ~° The parallel monitoring of intracellular pH strongly supported the notion that the observed currents were indeed carried by protons. In voltage-clamped HL-60 neutrophils, depolarization of the cell from the resting potential to +60 mV stimulated an outward current and a concomitant increase in pH i monitored by carboxy SNARF-1 fluorescence. The initial rate of the intracellular alkaliniza- tion was 1 pH unit/30 sec, and the accompanying outward current was approximately 50 pA. Based on cell volume and cytosolic buffering power, the H ÷ currents necessary to support the observed p H i change were calculated and found to be in good agreement with the experimental data. ~° Thus, the magnitude of the above-described outward current corresponds

9 j. W. Putney, Jr., Cell Calcium 11, 611 (1990). to N. Demaurex, S. Grinstein, M. Jaconi, W. Schlegel, D. P. Lew, and K.-H. Krause, J.

Physiol. (London) 466, 329 (1993).

FIG. 2. Monitoring of changes in [Ca2+]i induced by Ins(1,4,5)P 3 . Indo-l-loaded HL-60 cells were voltage-clamped in the whole-cell configuration of the patch clamp technique. The patch pipette contained the solution shown in Table I plus 10/zM Ins(1,4,5)P 3 . Arrows indicate when the whole-cell configuration was achieved, and thus when microperfusion of the cell interior with the pipette contents was begun. The voltage protocols are shown under the [Ca2+]i traces for each experiment. (a) Ins(1,4,5)P3-induced [Ca2÷]i changes in the presence or absence of extracellular Ca 2+ at a constant holding potential of -60 mV. (b) Same experiment as in (a) but with extracellular Ca 2+ and Ni 2+ (5 mM). (c) Effect on [Ca2÷]i of a depolarization to +60 mV during the sustained phase of the Ins(l,4,5)P3-induced [Ca2+]i increase in the presence of extracellular Ca 2+. (Reproduced from The Journal of Clinical Investigation, 90, 830-839, 1992, by copyright permission of the American Society for Clinical Investigation.)


well to the expected H ÷ ion fluxes, suggesting that H ÷ efflux through a voltage-activated membrane conductance is responsible for both the measured changes in pHi and the concomitant outward current.

Example 3: Precise Parameters of Calcium Activation of Ion Channels Studied by Parallel Monitoring of Cytosolic Calcium Ion Concentration and Currents

Studies in human leukemic T cells have used the combined patch clamp/microfluorimetry technique to study the activation of apamin-sensitive K ÷ channels by Ca2+. H Recordings obtained with the combined technique are generally represented by plotting fluorescence and current against time. However, in these studies the authors plotted fluorescence ([Ca2+]i) against current, which allowed them to determine precisely the threshold of activation, the half-maximal activation, and the cooperativity of the channel activation by Ca 2+. Interestingly this channel is activated by Ca 2+ with a very low threshold (-200 nM) and a high cooperativity (Hill coefficient of 5). These properties render the channel exquisitely sensitive to the [Ca2+]i signal of the lymphocytes.

Example 4: Relationship between Changes in Cytosolic Ion, Calcium, and Cellular Function, Exocytosis, Studied Simultaneously in Same Cell

A modification of the patch clamp technique allows the recording of cell membrane capacitance, and thus the monitoring of exocytosis. During exocytosis, secretory granules fuse with the plasma membrane increasing the surface area and capacitance of the cell (for a detailed review, see Lindau and Gomperts12). Using combined recordings of cell membrane capacitance and [Ca2+]i (with the indicator Fura-2), Ni~sse and Lindau 13 demonstrated that GTPyS-stimulated [Ca2+]i transients and the rate of exocytosis had a comparable time course. Suppression of Ca 2÷ transients by inclusion of Ca 2÷ buffers in the patch pipette led to delayed kinetics of exocytosis. These results, and others, demonstrated (1) the role of [Ca2÷]i in GTPyS-mediated exocytosis and (2) the existence of mechanisms that are able to activate exocytosis in the absence of a change in [Ca2÷]i .

11 S. Grissmer, R. S. Lewis, and M. D. Cahalan, J. Gen. Physiol. 99, 63 (1992). 12 M. Lindau and B. D. Gomperts, Biochim. Biophys. Acta 1071, 429 (1991). 13 O. Niisse and M. Lindau, Biosci. Rep. 10, 93 (1990).

[ 2 7 ] C O N F O C A L MICROSCOPY A N D /Ca,C1 3 2 1

[27] G - P r o t e i n - M e d i a t e d P a t h w a y s Assayed by Elec t rophys io logy and Confocal Microscopy

By LISA STEHNO-BITTEL, JEFF AMUNDSON, and DAVID CLAPHAM

In t roduc t ion

The interaction of G proteins with their receptors and effectors is best studied in a simple system in which protein sequences have been identified, receptor types are limited, and activation of the pathway is easily assayed. For the study of G proteins interacting with muscarinic acetylcholine receptors, Xenopus laevis oocytes are excellent models as they meet the criteria listed above. First, many of the G-protein oz subunits from Xenopus oocytes have been cloned. ~,2 Second, functional endogenous muscarinic receptors are not found on defolliculated mature oocytes. 3 Thus, single subtypes of receptors can be expressed, providing a more clear interpretation of the pathway activated. Finally, activation of the muscarinic pathway can be assayed easily by an increase in intracellular calcium. We use two modalities to measure changes in intracellular Ca 2+ in oocytes following G - protein stimulation: Ca 2 + imaging with confocal microscopy and monitoring the Ca 2 +-activated CI- inward c u r r e n t (1Ea,E l). The purpose of this chapter is to describe techniques and appropriate conditions for monitoring G-protein-mediated pathways using confocal microscopy and electrophysiology.

Typical m2 and m3 Muscarinic Responses

Muscarinic acetylcholine receptors are present in heart, smooth muscle, neurons of the central and peripheral nervous system, and a variety of exocrine glands. The five subtypes of muscarinic receptors (ml-m5) can be grouped into two broad categories. Stimulation of the ml, m3, and m5 receptors initiates phosphoinositide hydrolysis through a G protein which interacts with phospholipase C. In contrast, m2 and m4 subtypes inhibit adeylyl cyclase (adenylate cyclase) activity and only weakly stimu-

J. Olate, H. Jorquera, P. Purcell, J. Codina, L. Birnbaumer, and J. Allende, FEBS Lett. 244, 188 (1989).

2 j. Olate, S. Martinez, P. Purcell, H. Jorquera, J. Codina, L. Birnbaumer, and J. Allende, FEBS Lett. 2,68, 27 (1990).

3 j. Lechleiter, R. Hellmiss, K. Duerson, D. Ennulat, D. Nathanial, D. Clapham, E. Peralta, EMBO J. 9, 4831 (1990).



m2

m3

30s

Fie. 1. Typical ACh-induced (10 ~M) responses from two cells expressing either m2 or m3 muscarinic receptors. The m2 tracing illustrates the oscillations in lca,o following application of ACh (arrows) which persist for several minutes. In contrast, the m3 response reaches a greater peak amplitude in a shorter period of time and rapidly returns to baseline.

late phosphoinositide turnover. Xenopus oocytes expressing m3 (phosphoinositide pathway) or m2 (adenylyl cyclase pathway) muscarinic receptors have different acetylcholine (ACh)-induced Ca 2+ responses. 4'5 We have chosen to study m2 and m3 receptors as representatives of the two subtype groups.

Earlier work from our laboratory provides evidence that the m2 and m3 receptors transduce their signals via two different G-protein pathways in Xenopus laevis oocytes. 3'5'6 The ACh-induced stimulation of m2 results in a slow fluctuating increase in Ica,c~ that begins 10-30 sec following the application of ACh (Fig. 1). 3 The average peak of the Ic~,cL current during m2 activation is less than 1 tzA in amplitude. The m2-elicited response is nearly abolished by pertussis toxin pretreatment. 3'6 In contrast, the ACh application to m3-expressing oocytes results in a rapid, transient increase in Ica,c~ which returns to baseline quickly (Fig. 1). The typical peak current for m3 activation is 3-5/zA and is insensitive to pertussis toxin pretreatment. Subsequent applications of ACh fail to elicit additional responses, presumably because of depletion of Ca 2+ stores and/or receptor desensiti- zation. 3

Digital imaging of free intracellular Ca z+ using confocal microscopy illustrates differences in the Ca 2+ release pattern in m2- and m3-expressing oocytes. Responses mediated by m2 receptors require a higher ACh concentration (50 times more) than m3 responses) When activated, m2 receptors produce multiple foci of calcium increases in the cell which vary

4 j. Lechleiter, S. Girard, E. Peralta, and D. Clapham, Science 252, 123 (1991). 5 j. Lechleiter, S. Girard, D. Clapham, and E. Peralta, Nature (London) 350, 505 (1991). 6 j. Lechleiter and D. Clapham, Cell (Cambridge, Mass.) 69, 1 (1992).

[27] CONFOCAL MICROSCOPY AND ICa,CI 323

widely in number and distribution (Fig. 2). Like the delay between the ACh application and the increase in lCa,Ct in m2-expressing cells, the initiation of Ca 2 + waves is also delayed. In contrast, m3 activation results in a large wave of high intracellular Ca z + which develops immediately after the ACh application (Fig. 2). The wave travels across the cell and may envelope the entire cell. The high Ca 2+ levels return to near-baseline values within minutes. Large, single Ca 2+ waves, typical of the m3 response, are rarely seen in m2-expressing cells. To validate these observed differences in lca,cl and in the visual Ca z+ release patterns between m2- and

FIG. 2. Typical ACh-induced C a z+ release patterns from two cells expressing either m2 or m3 muscarinic receptors. The top tracing illustrates typical release patterns of Ca 2 ÷ during m2 activation as monitored by changes in fluorescence in Calcium Green using confocal microscopy. Several loci of Ca 2÷ release are noted. The lower tracing shows the typical m3 response of a single large wave of Ca 2+ increase that envelopes the entire cell within seconds. Images were formed by stacking 500 (m2) or 100 (m3) sequential optical slices (420 × 420 x 40 txm) which are volume rendered. Background fluorescent emission taken from the average image of 20 individual scans of the oocyte prior to stimulation was subtracted from each image shown. Representative optical slices of individual scans are shown below each volume rendered image.

324 IONS AND CHANNELS [2 7]

m3-expressing oocytes as different G-protein pathways, several control experiments are described below.

Methods

Electrophysiology

Oocytes are removed from animals and defolliculated manually with a pair of fine forceps. We have attempted enzymatic defolliculation with collagenase but find the percentage of viable cells remaining following treatment undesirably low. Cells are stored in L-15 supplemented medium with 5% horse serum and pH of 7.6 (data on effects of horse serum are presented below) at 19 °. Cells are placed in fresh medium once a day. Oocytes are injected with mRNA and antisense DNA via a Drummond (Broomall, PA) Nanoject. Injection pipettes are pulled from Drummond 10-/zl tubes on a vertical puller (David Kopf Instruments, Tujunga, CA). Tips are broken with fine forceps to a diameter of 14-16/~m and baked for 4 h at 300 °. Transcripts (100 ng/oocyte) and antisense oligonucleotides (0.3 /~g//zl) are injected in 50-nl aliquots into the cytoplasm. Receptor transcripts are injected 2 days prior to voltage clamp and antisense oligonucleotides 4 days prior to voltage clamp. Current measurements are amplified by Turbo TEC 01 (npi) using the two-electrode configuration. Currents are filtered at I kHz. Electrodes are pulled using a horizontal puller (Sutter, San Francisco, CA) to resistances of 1 and 6 Mr/for current and potential electrodes, respectively. Electrodes are filled with 2 M KC1. Data are stored and analyzed with Axobasic software (Axon Instruments, Foster City, CA). Current-voltage relationships are measured with a ramp protocol ( - 8 0 to 80 mV in 500 msec). The bath solution for recordings is Barth's medium. Acetylcholine (Sigma, St. Louis, MO) is added directly to the bath, and ACh-induced responses are measured at a constant membrane potential of - 70 mV.

Confocal Microscopy

Methods for preparation of oocytes for confocal microscopy have been presented elsewhere.3-5 For images obtained in Fig. 2, oocytes are injected with 47 nl of 0.25 mM Calcium Green (Molecular Probes, Eugene, OR) in hexapotassium salt form (assuming a 1-ml cell volume, the final concentration of dye is 12.5/zM). The dye is allowed to equilibrate in the cell for 30 min. Calcium Green is more resistant to photobleaching than fluo- 3 when scanning at wavelengths of 480 nm. The potassium salt form of Calcium Green can be injected into cells, providing a more accurate esti-

[27] CONFOCAL MICROSCOPY AND ICa,C1 325

mate of intraceUular dye concentrations compared to the cell-permeant acetoxymethyl ester forms of calcium dyes. 7 Calcium dyes are commercially available in high molecular weight dextran-linked forms (up to 70,000). Dextran-bound dyes have an advantage over lower molecular weight dyes in not rapidly diffusing intracellularly. At high concentrations low molecular weight dyes may themselves be the propagating species creating an artificially measured spatial change in Ca 2 +. At low dye concentrations (10-20 IxM) there is no difference in the development, propaga- tion, regeneration, or other characteristics of the waves when using dextran forms compared to the free salt. 8 The salt form of Calcium Green does however yield a stronger fluorescent signal.

Oocytes are placed in a small chamber (total volume 1/zl) filled with Barth's solution. The cell must be held in place in some manner so that ACh application and bath perfusion do not move the oocyte. Small suction pipettes can be used to hold the cell in place, but we have found it easier to affix a small piece of 760 txm polyethylene mesh (Spectrum) to the coverslip. The mesh gives enough lateral support so that the oocyte will not move when the bath is changed or when agonists and antagonists are added.

Varying concentrations of ACh are added to the bath, and sequential images are taken of a single confocal slice about 40/~m thick (10 x objective) and generally 600 by 400 /xm in the x,y plane. The muscarinic- mediated Ca 2+ responses may be observed only from the peripheral 20 or 30 t~m of the oocyte owing to the number of large yolk platelets. Analysis is performed on a Silicon Graphics workstation using Analyze software (Mayo Foundation, Rochester, MN). Examples of m2 and m3 responses to ACh are shown in Fig. 2.

Calcium-Sensitive Chloride Current

Calcium sensitive CI- channels are selective for CI- and are open more of the time when Ca 2+ binds to the channel. Prior to using Ica,c~ as an assay for activation of G-protein-mediated pathways, we felt several criteria had to be met. First, Ica,c~ should be the predominant current at the membrane potential used in the protocols. Second, muscarinic receptor expression in Xenopus oocytes should be stable for several days. Third, Ica,cl should not be activated directly by pharmacological stimuli independent of increases in internal Ca 2 +. Fourth, block of the G-protein pathway should abolish ACh-induced changes in ICa,CI" These are some of the

7 G. Grynkiewicz, M. Poenie, and R. Tsien, J. Biol. Chem. 260, 3440 (1985). S. Girard, J. Amundson, and D. Clapham, submitted for publication.


O O

o

•90 10 30 50 70 90 _~' / I Vm (mY)

-1

-2

FZG. 3. Whole-cell current-voltage relationships for m3-expressing oocytes. Oocytes were bathed in Barth's solution (control), and the whole-cell current reversed at -33 mV ((3). When I0 mM EGTA was injected into oocytes prior to voltage clamp the reversal potential shifted to - 6 0 mV (rq). Activation of m3 receptors by ACh (5 /zM) increased intracellular Ca 2+ and the whole-cell current amplitude (~7). The m3-activated current reversed at -23 mV.

criteria previously used to verify the whole-cell Ca 2 +-activated K + current as an assay for intracellular Ca 2+ concentrations in smooth muscle cells. 9

Dominant Whole-Cell Current as Calcium-Dependent Chloride Current

Defolliculated Xenopus oocytes display a native whole-cell current reversing at - 3 3 mV (Fig. 3, circle) which is slightly more negative than the calculated equilibrium potential for CI- of - 2 0 mV. 10 It is likely that the native whole-cell current is influenced by both Ica,C~ and potassium

9 L. Stehno-Bittel and M. Sturek, J. Physiol. (London) 451, 49 (1992). l0 K. Kusano, R. Miledi, and J. Stinnakre, J. Physiol. (London) 328, 143 (1982).

[27] CONFOCAL MICROSCOPY AND Ica,cl 327

current (IK) since preventing/Ca,CI activation shifts the reversal potential of the whole-cell current ( - 6 5 mV) in the direction of the equilibrium potential for K ( - 100 mV) l° (Fig. 3, square). The Ica,cl is blocked when intracellular Ca 2÷ is lowered by injecting oocytes with EGTA (10 mM intracellular concentration) approximately 1 hr prior to voltage clamp. In contrast, activation of the muscarinic receptor (ACh, 5 ~M) results in an increase in the amplitude of the whole-cell current (from 0.5 to 3.7/xA at 80 mV, Fig. 3, triangle), and the reversal potential shifts to - 23 mV which is near the equilibrium potential for CI- ( - 20 mV). ~° As it appears that the native current is partially influenced by K ÷ channels we chose a holding potential near the calculated K ÷ equilibrium potential with which to assay the muscarinic response ( - 7 0 mV) since the K ÷ current should be very small and the predominant current will be Ica.CV Thus, the first criterion for use of Ica,c~ as an indicator of the muscarinic receptor-activated G-protein pathway has been met.

Receptor Expression Stability

The second criterion for the use of Ica,cl as an assay for the G-protein pathway is that the muscarinic receptor expression in oocytes is stable for several days. Figure 4 illustrates that the oocyte response to ACh is extremely stable in muscarinic receptor-expressing cells. All cells are removed from the animal and defolliculated manually 2 days prior to any voltage clamp (day 0). Transcripts for m3 receptors are injected into each group of cells 2 days prior to voltage clamp. By using this protocol the number of days between m3 mRNA injection and voltage clamp are held constant. The amplitude of the ACh-induced whole-cell current is shown in Fig. 4 for I week. The mean values range from 4.1 -+ 0.6 to 3.2 -+ 0.5 /~A from a total of 303 cells. Thus, there is no difference statistically between the responses for 1 week.

Viability of the cells for 1 week is completely dependent on the presence of 5% horse serum (GIBCO, Grand Island, NY) in the storage medium. Without horse serum, cells remain healthy for only 3-4 days. We have defined healthy cells as those with clearly pigmented animal poles, intact membranes, and negative resting membrane potentials. One day after defolliculation and injection of mRNA there is no difference in the percentage of viable cells between those stored with or without horse serum (approximately 94% for both groups). By the seventh day 79% of the cells kept in horse serum are healthy (n = 74 cells), whereas less than 5% are viable when stored without horse serum (n = 67). Oocytes must be removed from the horse serum-containing medium several hours prior to voltage clamp. When horse serum is removed from the medium only


4 < :3.

3

• N 2

0 2 4 6 DAYS

FIG. 4. Whole-cell current peak amplitude during ACh activation of m3. Oocytes were injected with transcript for m3 receptors 2 days prior to voltage clamp. Peak ACh-induced current amplitudes are shown. There is no statistical difference between the responses on any of the days (p -< 0.05).

5 hr prior to voltage clamp the mean ACh-induced response is at tenuated to 0.7 - 0.1 ~A (n = 5) versus cells injected at the same time but removed from horse serum 1 day prior to voltage clamp (2.9 - 0.2 tzA, n = 6). It has been suggested previously that horse serum interferes with cell surface responses. N Oocytes stored properly will remain healthy and express muscarinic receptors for 1 week, thus fulfilling the second criterion.

No Direct Effect by Pharmacological Agents

The third criterion for use of Ica,cl in oocytes as an assay for G- protein-mediated pathways is that the current is not affected directly by pharmacological agents applied during the course of the experiments. For all experiments discussed in this chapter ACh is the agent used to activate the muscarinic response; therefore, it is important to rule out direct effects of ACh on Ica,o. Cells without prior injection of mRNA for muscarinic receptors fail to respond to ACh even at concentrat ions of 100/~M. The mean increase during ACh application in cells not injected with muscarinic

li M. Quick, J. Naeve, N. Davidson, and H. Lester, BioTechniques 13, 360 (1992).

[27] COr~FOCAL MICROSCOPY AND Ica,Cl 329

receptors is less than 100 nA (n = 21 cells). Therefore, the native Ica,c~ is not affected directly by ACh. Importantly, these experiments show that defoUiculated Xenopus oocytes do not contain functionally active muscarinic receptors. Activation of the cells expressing muscarinic receptors but with low intracellular free Ca z+ (prior injection of 100 ~M EGTA) also fail to respond to ACh (Fig. 5). Water-injected oocytes expressing m3 show an increase in Ica,ct to 4.6 + 1.0/.tA (n = 23), whereas 10/xM EGTA decreases the peak current to 1.7 +- 0.4/zA (n = 9). Injection of 100 /zM EGTA attenuates the muscarinic response further to 0.1 /zA (n = 9). Therefore ACh-induced activation of Ica,cl is dependent on expression of muscarinic receptors and on intracellular Ca 2+ , whereas ACh does not directly alter the native whole-cell current.

Block of G Proteins to Abolish Ica,c t Response

If lca,c~ is an accurate assay of activation of the G-protein pathway in oocytes, then blocking G-protein synthesis should block the ACh-induced response. Blocking the expression of selected genes can be achieved with antisense deoxyribonucleic acids. Oligonucleotides are typically short DNA sequences that are complementary to target mRNA segments bind-

5

o

~d z

d~

0 H20 10 IIM EGTA 100 laM EGTA

FIG. 5. Effects of low intracellular Ca 2÷ on ACh response. Oocytes were injected with m3 transcript 2 days prior to current measurement. Two hours prior to voltage clamp cells were injected with water (HzO) or EGTA for a final concentration of 10 or 100/zM EGTA. Reducing free intracellular Ca 2+ with EGTA attenuated the m3-mediated response.


~ ntisense

n s e

1 pA I 30 sec

FIG. 6. Injection of antisense to Ga subunits attenuates the m3-mediated response. Antisense or sense deoxyribonucleic acids designed to bind to homologous regions of Gc~ were injected into oocytes 4 days prior to voltage clamp. The antisense oligonucleotide attenuates the typical response dramatically from 4.3 to 0.7/~A in this example.

ing with high affinity and inhibiting further translation of the protein. Antisense oligonucleotides injected into Xenopus oocytes result in specific cleavage and end-point degradation of up to 96% of the target endogenous mRNA. 12

We utilize antisense oligonucleotides to block synthesis of all endogenous Ga subunits in Xenopus oocytes. The oligonucleotide referred to as common2 was designed to bind to homologous regions of mRNA for all G proteins (GAG AGT GGC AAG AGC ACC TTC ATC AAG CAG, corresponding to nucleotides 228-260 of Gas). Oligonucleotides manufac- tured by the Mayo Molecular Biology Core Facility are injected into oocytes (0.3/zg//zl, 50 nl) that are voltage clamped 4 days later. Common2 decreases the Ica,cl response to ACh by 71% compared to water-injected cells (Fig. 6). The results confirm that G proteins are essential for the ACh-induced increase in Ica,cl in oocytes expressing muscarinic (m2 or m3) receptors. Inhibition of the response shown in Fig. 6 suggests that the a subunit of a G protein (probably Gqa) is important in signal transduction for m3-mediated responses; however, this work does not rule out the possibility that G-protein fly subunits are involved in muscarinic receptor pathways. Signal transduction by G-protein/3y subunits has been demonstrated previously T M and is implicated in activation of phospholipase

iz S. Shuttleworth and A. Colman, EMBO J. 7, 427 (1988). 15 D. Logothetis, D. Kim, J. Northup, E. Neer, and D. Clapham, Proc. Natl. Acad. Sci.

U.S.A. 85, 5814 (1988). J4 C. Kluess, H. Scheriib, J. Hescheler, G. Schultz, and B. Wittig, Nature (London) 358,

424 (1992).

[27] CONFOCAL MICROSCOPY AND /Ca,CI 331

C-/32. ~5 It is possible that the different outcomes of m2 and m3 activation are due to signals transduced by a (m3) and/33, (m2) subunits. Currently, we are testing this hypothesis.

All the results above suggest that the lc~,c~ is a good assay of G- protein pathways in our system. First, at - 7 0 mV (membrane potential for measuring the muscarinic response) the dominant current is lca,c ~. Second, viable oocytes can be maintained for at least 1 week, which is important for antisense experiments, and the muscarinic response can be elicited for 2-4 days following m2 or m3 mRNA injection. Third, Ica,cl is not activated directly by ACh. Fourth, blocking of Ga subunit synthesis with antisense oligonucleotides abolishes the m3-mediated response. These results support the use of Ica,cl in monitoring muscarinic responses and provide interesting information when measured simultaneously with intracellular Ca 2+ concentrations using confocal microscopy.

Confocal Microscopy

Confocal microscopy is a useful tool in studying muscarinic receptor- initiated Ca 2÷ responses. The schematic in Fig. 7 illustrates the main sections of the microscope, which have been described previously in more detail. 16,17 Two argon laser sources emit light in visible or UV wavelengths. Dichroic mirrors (DM1, DM2, and DM3) reflect or pass the light (depending on the wavelength) to the X-Y scanning modulator, which can scan at different resolutions and rates. The scanning beam passes through a projection lens (custom), telon adapter lens (Bio-Rad, Richmond, CA), and objective lens (Olympus Plan Apo UV 10 x ) and is focused on a diffraction-limited spot on the specimen. The emission light is passed again through the modulator and dichroic mirrors (DMI and DM2). The mirror splits the light beam according to wavelength, and the two filters allow for the detection of specific bands of the signal by each of two photomultiplier tubes (PMTs). The dichroic mirror and the filters are mounted in easily changeable cases and are chosen depending on the emission spectra of the dyes used, The two channels allow for either of the two different dyes to be used simultaneously, or for a ratioing emission spectrum dye to be used. An analog-to-digital converter allows the information to be analyzed and stored on computer with an additional display screen.

t5 M. Camps, C, Hou, D. Sidiropoulos, J. Stock, K. Jakobs, and P. Gierschik, Eur. J. Biochem. 206, 821 (1992).

16 C. Bliton, J. Lech|eiter, and D. Clapham, J. Micros. 169, 15-26 (1993). ~7 M. Burke and D. Clapham, "Computer-Assisted Multidimensional Microscopies." (1992).


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[27] CONFOCAL MICROSCOPY AND ICa,Cl 333

Advantages

As mentioned previously, intracellular free Ca 2+ concentrations increase during activation of both m2 and m3 muscarinic receptors. Fluores- cent indicators which either emit more light (Calcium Green, fluo-3) or change emission spectrum on binding Ca 2+ (indo-1) are used to monitor intracellular Ca 2+ levels. The confocal microscope provides high-resolution visualization of free intracellular C a 2 + because it excludes all information (in this case, fluorescence signal) from planes which are out of focus, resulting in a clear image of a very thin optical slice. This is accomplished by using a collimated illuminating source (a laser) which focuses on a point of the specimen. The emitted fluorescent signal is passed through a "pinhole" aperture near the detector which excludes all the fluorescent signal from the area outside the plane of focus. In addition, illumination and detection are limited to a small area on the specimen at any given time, allowing for higher X, Y resolution. When the area is so small as to be diffraction-limited, the resolution can be greater than that ofa nonconfo- cal, light microscope.~S Thus, the ability to exclude out-of-focus information from the detector provides the greatest advantage of confocal microscopy and allows visualization of sites of Ca 2+ release in oocytes.

A second advantage of the confocal over the light microscope is the ability to use two detectors to measure different wavelengths of light simultaneously (Fig. 7). Most available confocal microscopes contain optics suitable for visible-wavelength light only. Modifications made in our laboratory allow simultaneous collection of information from excitation by UV and visible light. ~6,~7 The optical design for such measurements is complex since the illumination and imaging optics are identical, and excitation and emission beams are both imaged by the same eyepiece and objective. The lenses must be chromatically corrected for all wavelengths of light used.16 Another advantage of such a configuration, described in detail previously, ~6 lies in the ability to activate caged compounds within the cell. Caged compounds, which are inactive until released by UV light, are injected into oocytes prior to voltage clamp. The laser can be used to activate the caged compound [GTPyS, inositol trisphosphate (InsP3), and InsP~S3) while changes in intracellular Ca 2+ are measured with fluorescent dyes. 6 The confocal microscope provides superior temporal and spatial (x, y) selectivity when activating caged compounds.

Two detectors are also an advantage for simultaneously measuring emission from a UV source with different wavelengths. Different excitation wavelengths are used by fluorescent Ca z+ ratioing dyes which allow

18 j. Pawley, "Handbook of Biological Confocal Microscopy." Plenum, New York, 1990.


for determination of free intracellular Ca 2+ .7 To monitor the muscarinic- mediated response of oocytes, the Ca z + ratioing dye indo-1 has been used successfully (S. Girard and D. Clapham, unpublished). Indo-1 is excited by UV light at 330 and 350 nm wavelengths and emits light in the visible range of wavelengths. The use of ratioing dyes provides more quantitative estimates of changes in the free intracellular Ca 2+ concentration.

The third major advantage of the confocal microscope for measuring muscarinic responses in oocytes is that it provides images in a digital format. The images can be collected over a given time period (Fig. 2) or collected from various focal planes. Thus, changes in free intracellular C a 2+ c a n be visualized in real time at a given location or can be measured in varying planes, resulting in three-dimensional (3D) images. These two modes are achieved by collecting serial images of the same x-y slice over a variable time period, or by imaging serial x-y slices while varying the z-axis plane of focus.

Limitations

There are both mechanical and optical limitations with the common commercially available confocal microscope. Most current confocal microscopes are useful in studying large areas at relatively slow time courses such as the G-protein-linked responses in Xenopus oocytes. There are several factors which preclude its use in studying faster or smaller responses. The microscope scanning speed limits the number of pixels per second that can be scanned. To obtain the resolution shown in Fig. 2 a single 768 x 512 image is collected at a frequency of l Hz. Thus, the most appropriate situations for use of confocal microscopy are for slowly developing (several seconds) Ca :+ changes. Speed can be increased by reducing the number of scan lines. An additional problem with confocal measurements of intracellular Ca 2+ is that emission from many of the Ca2+-sensitive dyes declines (bleaches) during illumination. Decreasing laser power decreases dye photobleaching but leads to lower emission signal strength. Partial correction of the problem can be made by using a slower scanning speed or a larger opening of the detector aperture. How- ever, these corrections lead to losses in either temporal or spatial (z axis) resolution. Scanning speed, resolution, and image size are interdependent, and an increase in one leads to a decrease in the others.

Conclusion

Monitoring the muscarinic-mediated G-protein pathways in Xenopus oocytes has been accomplished with the use oflc~,c t and confocal micros-

[28] N-TYPE CALCIUM CHANNEL COMPLEX 335

copy (Figs. 1 and 2). The greatest strength of these assay systems lies in the ability to monitor two parameters simultaneously. In comparing responses of Ca 2+ release patterns with the lEa,C1 within the same cell, subtle differences between the pathways can be identified and studied. Much information has been published on the muscarinic receptor subtypes and the final Ca 2+ response. 3-6 Currently we are focusing our efforts on the different steps between receptor activation and Ca 2+ increases, including identification of G proteins and their effectors. Molecular biology techniques, such as the use of antisense oligonucleotides, will be useful in identifying individual steps in the pathways. The greatest promise lies in further development of new fluorescent probes sensitive to levels of second messengers. For example, development of a ratioing InsP3-sensitive dye would be most useful in separating the roles of Ca 2+ and InsP3. With improvement in microscope speed or sensitivity, responses could be viewed at a much smaller scale, perhaps allowing better correlation between intracellular structure and pathway responses.

[28] Pur i f i ca t ion a n d R e c o n s t i t u t i o n o f N - T y p e C a l c i u m

C h a n n e l C o m p l e x f r o m R a b b i t B r a i n

By D E R R I C K R. W I T C H E R , M I C H E L DE W A A R D , S T E V E N D. K A H L , and KEVIN P. CAMPBELL

Introduction

Voltage-sensitive Ca 2+ channels play important roles in the regulation of intracellular calcium concentrations in cell types as diverse as muscle cells, neuroendocrine cells, and neurons. Calcium that enters the cell through voltage-sensitive channels acts as a second messenger in cellular processes including the initiation of cardiac and smooth muscle excitation-contraction coupling and synaptic vesicle fusion with the plasma membrane leading to neurotransmitter release in neurons. Calcium channels have been classified into four different types (L, N, T, and P), each of which can be identified by pharmacological and biophysical properties. The best characterized voltage-sensitive Ca 2 + channel is the skeletal muscle dihydropyridine (DHP)-sensitive Ca 2+ channel. This channel is essential in the process of excitation-contraction coupling, and it is thought to function both as a calcium channel and as a voltage sensor in excita-

1 B. P. Bean, Annu. Rev. Physiol. 51, 367 (1989).



tion-contraction coupling. 2 The skeletal muscle DHP receptor has been purified and is composed of four subunits, oq (170K), Ctzb (175K), fl (52K), and y (32K), 3 all of which have been cloned? However, a number of a 1 subunits, the pore-forming component of vo|tage-sensitive C a 2 + channels, have been cloned and shown to be products of five genes (A, B, C, D, and E type). 5-7 These a~ subunits share homology with the skeletal muscle al subunit. Thus, molecular biology has provided another means of classi- fying the voltage-sensitive Ca 2+ channels. The L-type Ca z+ channels (C and D type) are also present in neurons, and their channel activity is modulated by dihydropyridines. The P-type Ca z+ channel (A type), first identified in Purkinje neurons, is blocked by to-Aga-IVA, a funnel web spider toxin. The snail toxin to-conotoxin GVIA binds with high affinity and exerts an inhibitory effect on the N-type Ca 2+ channel (B type). These channels are also neuro-specific.

There is a substantial amount of evidence indicating that N-type C a 2+ channels are responsible for the voltage-activated release of neurotransmitters in a variety of neurons. The specific N-type Ca 2+ channel blocker to-conotoxin GVIA directly inhibits presynaptic Ca 2+ currents. 8 oJ-Conotoxin GVIA binding sites have also been localized precisely at active zones in the presynaptic membrane where neurotransmitter release o c c u r s . 9'1° The N-type Ca 2+ channels have been shown to be inhibited by norepinephrine by acting through az-adrenergic receptors. This modulation of the to-conotoxin-sensitive Ca 2+ current is thought to involve G proteins. H-~3 Because the role G proteins play in the modulation of N-type Ca z+ channels may provide a significant mechanism in regulating neurotransmitter release, this chapter focuses on the purification and reconstitution of the N-type Ca 2 + channel from rabbit brain.

T. Tanabe, K. G. Beam, J. A. Powell, and S. Numa, Nature (London) 336, 134 (1988). 3 K. P. Campbell, A. T. Leung, and A. H. Sharp, Trends Nearosci. 11, 425 (1988). 4 R. J. Miller, J. Biol. Chem. 267, 1403 (1992). 5 T. P. Snutch, J. P. Leonard, M. M. Gilbert, H. A. Lester, and N. Davidson, Proc. Natl.

Acad. Sci. U.S.A. 87, 3391 (1990). 6 T. P. Snutch, J. Tomlinson, J. P. Leonard, and M. Gilbert, Neuron 7, 45 (1991). 7 y . Mori, T. Friedrich, M. Kim, A. Mikami, J. Nakai, P. Ruth, E. Bosse, F. Hofmann,

V. Flockerzi, T. Furuichi, K. Mikoshiba, K. Imoto, T. Tanabe, and S. Numa, Nature (London) 350, 398 (1991).

8 E. F. Stanley and G. Goping, J. Neurosci. 11, 985 (1991). 9 S. J. Smith and G. J. Augustine, Trends Neurosci. 11, 458 (1988). 10 R. Robitaille, E. M. Adler, and M. P. Charlton, Neuron 5, 773 (1990). n B. P. Bean, Nature (London) 3411, 153 (1989). lz M. R. Hummer, A. Rittenhouse, M. Kanevshy, and P. Hess, J. Neurosci. 11, 2339

(1991). 13 A. H. Delcour and R. W. Tsien, Science 259, 980 (1993).


The availability of a high-affinity radiolabeled compound, ~zSI-labeled oJ-conotoxin GVIA, for the receptor site on the N-type Ca z+ channel has made this receptor ideal for investigating the structure and function of neuronal Ca 2+ channel complexes. Also it has been demonstrated that the purified DHP receptor from skeletal muscle, reconstituted in artificial lipid bilayers, is directly stimulated by G~, the G protein of adenylyl cyclase (adenylate cyclase).14 Therefore, the reconstitution of the N-type Ca 2+ channel complex should provide a direct method for testing the regulatory role G proteins exert on neuronal Ca 2+ channel activity.

Procedures

Isolation and Purification of Crude Brain Membranes from Rabbit Brain

Rationale. ~zSI-Labeled to-conotoxin binding experiments with crude brain membranes suggest that the receptor is present at the range of 200 to 300 fmol/mg of protein, a level 1000-fold less than the DHP receptor in skeletal T-tubule preparations, thus making the purification of the N-type Ca z+ channel more difficult. Because the DHP-sensitive Ca 2+ channels from skeletal muscle are localized in the T-tubules, dihydropyridine binding can be enriched from homogenates about 5-fold in KCl-extracted skeletal muscle microsomes. Washing of skeletal muscle microsomes with KCI enriches for the DHP receptor by removing extrinsic proteins. Because the ~0-conotoxin-sensitive Ca 2+ channels are widely distributed throughout the brain, no method is available to enrich substantially these channels in purified membrane preparations without drastically reducing the total yield of the receptor. However, KC1 washing of crude brain membranes yields a membrane preparation with twice the Bma x of ~25I-labeled to-conotoxin compared to crude brain membranes (400-600 fmol/mg protein). Thus, KCl-extracted crude brain membranes are used as the starting material for the purification of the N-type Ca z+ channel.

Buffers

Medium I: 50 mM Tris-maleate, pH 7.4 0.5 mM EDTA Medium II: 20 mM Tris-maleate, pH 7.4, 0.303 M sucrose

All buffers contain a cocktail of protease inhibitors including aprotinin (0.5/.~g/ml), benzamidine (100 p.g/ml), leupeptin (92.5 p.g/ml), pepstatin A (0.5/xg/ml), and phenylmethylsulfonyl fluoride (PMSF, 40/~g/ml).

E4 A. Yatani, Y. Imoto, J. Codina, S. L. Hamilton, A. M. Brown, and L. Birnbaumer, J. Biol. Chem. 263, 9887 (1988).


Procedure. All steps are performed at 4 ° . Four fresh whole brains from New Zealand White rabbits (3-4 kg) without the meninges are immediately placed in 200 ml of medium I to remove excess blood prior to homogenization. Approximately 1600 g of brain tissue is homogenized three times in 400 ml of medium I for 30 sec at speed 5 with a Brinkmann PTA 20S Polytron (Westbury, NY). The homogenate is then centrifuged at 17,300 rpm (35,000 gmax) for 20 rain in a Beckman type 45 Ti rotor (Fullerton, CA). The supernatants are discarded. The pellets are resuspended in 400 ml of medium II with 0.6 M KC1 and gently stirred for 20 min. The sample is then filtered through six layers of cheesecloth and centrifuged at 40,000 rpm (186,000 gmax) for 60 min in a Beckman type 45 Ti rotor. Again, the supernatants are discarded and the pellets extracted by gently stirring for 20 min with medium II containing 0.6 M KCI. The sample is filtered through six layers of cheesecloth and centrifuged at 25,000 rpm (72,660 gmax)" The pellets are resuspended in medium II to a protein concentration of approximately 20-25 mg/ml. Protein concentrations are determined by the method of Lowry et al.~5 as modified by Peterson using bovine serum albumin (BSA) as a standard.16

Solubil&ation and Purification o f N-Type Calcium Channel

Rationale. The procedure for purifying the N-type Ca z+ channel from brain was developed based on our knowledge of the purification and subunit composition of the skeletal muscle DHP receptor. The skeletal muscle DHP receptor was purified with the use of ion-exchange chromatography, wheat germ agglutinin (WGA)-agarose chromatography, and sucrose density gradient centrifugation. Because of the low density of o~-conotoxin receptors in brain, an immunoaffinity purification step is needed to obtain useful amounts of the receptor. A number of monoclonal and polyclonal antibodies to the individual subunits of the DHP receptor were tested for the ability to immunoprecipitate the solubilized o~-conotoxin receptor. Monoclonal antibody VD21 against the/3 subunit of the L-type Ca z+ channel immunoprecipitates greater than 85% of the solubilized o~-conotoxin receptor, demonstrating that the N-type Ca 2+ channel complex contains a/3 subunit with a similar epitope as the DHP receptor. However, additional steps are necessary to provide a homogeneous purification of the N-type Ca 2 + channel. The a z subunit of the DHP receptor is highly glycosylated and binds to immobilized WGA. Therefore,

i50. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

16 G. L. Peterson, Anal. Biochem. 83, 346 (1977).


the use of WGA-agarose provides a powerful method for the purification of the N-type Ca 2+ channel.

The WGA-agarose resin along with others, such as heparin agarose, were tested for the ability to bind solubilized 125I-tagged to-conotoxin- labeled receptor from brain membranes. Although WGA-agarose is able to bind approximately 50% of the 125I-tagged to-conotoxin-labeled receptor, the elution conditions of this procedure have been determined to be ineffi- cient for a potential purification step. However, heparin-agarose binds 85 to 90% of the solubilized to-conotoxin receptor, which could be eluted using increasing ionic strength. For large-scale preparations, heparin agarose column chromatography provides a 4-fold purification of the receptor. Finally, the large size of the N-type Ca 2÷ channel complex (21 S) also makes it possible to use sucrose density gradients to increase the purity of the N-type Ca 2+ channel. Thus, the N-type Ca 2+ channel is purified using ion-exchange chromatography, immunoaffinity chromatography, and sucrose density gradient centrifugation.

Reagents. 125I-Labeled to-conotoxin GVIA (1600 Ci/mmol) is obtained from Amersham (Arlington Heights, IL). Digitonin is obtained from ICN (Costa Mesa, CA) and purified as previously described, 17 heparin-agarose is from Sigma (St. Louis, MO), and hydrazide avidin gel is from Unisyn Technologies Inc. Monoclonal antibody VD21-agarose ~8 and anti-46K- agarose are prepared according to the instructions provided with the hydrazide avidin gel with 3 mg of antibody cross-linked per milliliter of swollen gel.

Buffers Buffer I: 10 mM HEPES, pH 7.4, 100 mM NaCI, 0.2 mg/ml BSA Buffer II: 10 mM HEPES, pH 7.4 Buffer III: 10 mM HEPES, pH 7.4, 1.0 M NaCI, 1.0% digitonin

% w/v Buffer IV: 10 mM HEPES, pH 7.4, 0.3 M NaC1, 0.1% digitonin Buffer V: 10 mM HEPES, pH 7.4, 0.4 M NaC1, 0.1% digitonin Buffer VI: 10 mM HEPES, pH 7.4, 0.7 M NaCI, 0.1% digitonin Buffer VII: 50 mM CAPS, pH 10.0, 0.6 M NaC1, 0.1% digitonin

Protease inhibitors are added from stock solutions to the concentrations employed for the purification of the receptor. Calpain inhibitors I and II (2/~g/ml) are also added to the solubilization and elution buffers. These protease inhibitors are included in the preparation to decrease proteolysis of the am subunit (230K) of the receptor.

i7 A. T. Leung, T. Imagawa, and K. P. Campbell, J. Biol. Chem. 262, 7943 (1987). 18 j . Sakamoto and K. P. Campbell, J. Biol. Chem. 266, 18914 (1991).


Prelabeling with lesI-Labeled co-Conotoxin GVIA. To follow the purification of the N-type calcium channel throughout each experimental step, a small portion of brain membranes is labeled with the specific channel blocker ~25I-labeled co-conotoxin GVIA before the solubilization. Two milligrams of KCl-extracted brain membranes is suspended in 2 ml of buffer I, and ~zSI-labeled co-conotoxin GVIA is added to a final concentration of 0.5 nM as previously described? 8 The sample is incubated at room temperature for 60 rain and vortexed periodically. The membranes are centrifuged for 10 rain at 100,000 rpm in a TLA Beckman 100.3 rotor, and the supernatant is removed and discarded. The resuspended pellet, which contains the labeled N-type Ca 2+ channels, is added to the solubilization mixture as described below. The ~zsI-labeled o~-conotoxin GVIA-N-type Ca 2+ channel complex is very stable at 4 ° (half-life >12 hr). Under these conditions, the amount of radiolabeled Ca 2+ channels in 2 mg of brain membranes is sufficient to trace the receptor throughout the purification.

Solubilization. The 125I-tagged co-conotoxin-labeled membrane pellet is homogenized in 1 ml of buffer II by drawing it up and down in a pipette tip. The sample along with approximately 900 mg of brain membranes is added to buffer III to a final volume of 300 ml. The final protein and digitonin concentrations are 3 mg/ml and 1%, respectively. After gentle stirring for I hr at 4 °, the membrane suspension is centrifuged at 35,000 rpm (142,000 gmax)for 37 min in a Beckman type 45 Ti rotor. The supernatant, which contains the solubilized N-type Ca 2+ channel complex, is slowly diluted with 700 ml of buffer II while gently stirring to reduce ionic strength. The final volume of 1000 ml has a protein concentration of 0.9 mg/ml, a digitonin concentration of 0.3%, and an NaC1 concentration of 0.3M.

Heparin-Agarose Chromatography. The solubilized co-conotoxin receptor is passed through a column containing 50 ml of heparin-agarose preequilibrated in buffer IV at a flow rate of 5 ml/min. The heparin-agarose column is extensively washed with 5 column volumes of buffer IV and then 8 column volumes of buffer V. The bound receptor is eluted from the column with buffer VI at a flow rate of 2 ml/min. Aliquots of the eluted fraction are counted in a y counter to determine t25I-labeled co-conotoxin radioactivity.

VD2rAgarose Affinity Chromatography. The fractions containing the co-conotoxin binding activity are pooled (90-100 ml) and incubated overnight at 4 ° with 8 ml of VD2ragarose preequilibrated with buffer II containing 0.6 M NaCI and 0.1% digitonin. After extensive washing, the co-conotoxin receptor is eluted from the VD21-agarose column with buffer VII and neutralized immediately. This step provides greater than 1000-fold enrichment of the receptor complex. The VD2ragarose-purified receptor


periodically contains a variable amount of a contaminating protein (46K) which is removed by preadsorbing the VD2ragarose-eluted receptor with an immunoaffinity resin prepared using polyclonal antibodies raised against the 46K protein. Anti-46K polyclonal antibodies do not immunoprecipitate 125I-labeled co-conotoxin labeled receptor.

Sucrose Density Gradient Centrifugation. The to-conotoxin receptor is concentrated to 0.6 ml in an Amicon (Danvers, MA) ultrafiltration cell using a YM 100 membrane and layered onto a linear 5-30% sucrose density gradient (12.5 ml). Gradients are centrifuged at 4 ° in a Beckman VTi 65.1 rotor for 100 min at 215,000 g. Fractions (0.6 ml) are collected from the top of the gradients using an ISCO Model 640 density gradient fractionator (Lincoln, NE) and counted in a Beckman y counter (Fullerton, CA) counter. The receptor is present in gradient fractions 8-12.

Subunit Composition and Properties. The purified N-type Ca :+ channel complex from rabbit brain consists of several subunits. Figure 1 shows a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the various steps in the purification of the N-type Ca 2÷ channel. Samples from each purification step are separated on a 3-12% linear SDS-polyacrylamide gel and stained with Coomassie blue. The purified N-type Ca 2÷ channel is composed of a 230K subunit (cqB) tightly associated with a 140K subunit (a2, reduced), a 57K subunit (ill and a novel 95K subunit. The positions of these subunits are indicated by the arrows in Fig. 1 (eq, et 2, 95K, and/3). The subunits of the receptor complex comigrate on the sucrose gradient with the peak of ~25I-labeled to-conotoxin binding activity. For comparison, Fig. 1 also shows a Coo- massie blue-stained 3-12% polyacrylamide gel of 25/zg of purified skeletal muscle DHP receptor. The subunits of the L-type Ca 2+ channel complex are indicated by the arrows (al, OrE, /3, and 7). Analysis of the sucrose density gradient fraction with affinity-purified sheep polyclonal antibodies to each subunit demonstrates that all four subunits of the N-type Ca 2+ channel comigrate on the sucrose density gradient and are immunologi- cally distinct. Furthermore, the subunits of the N-type Ca 2+ channel are coimmunoprecipitated as a receptor complex by affinity-purified antibodies against each of the individual subunits/9,2°

Immunoblot analysis shows that affinity-purified polyclonal antibodies to the oJ-conotoxin receptor ot 2 and/3 subunits identify the a2 and/3 subunits of the skeletal muscle DHP receptor. Also, similar to the a2 subunit of the skeletal muscle DHP receptor, the a2 subunit of the o-conotoxin

~9 D. R. Witcher, M. De Waard, and K. P. Campbell, Neuropharmacology 32, 1127 (1993). 2o D. R. Witcher, M. De Waard, J. Sakamoto, C. Franzini-Armstrong, M. Pragnell, S. D.

Kahl, and K. P. Campbell, Science 261, 486 (1993).


CJ-CgTx Receptor Purification Purified DHPR

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205 " - - ~1

116 " - - 0(2 ¢'?' 98 o -",--- 95K × 66

~ 45 36 29 24

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FIG. 1. Gel electrophoresis analysis of N-type Ca 2+ channel purification and purified skeletal muscle DHP receptor. (Left) Samples from each purification step were analyzed by SDS-PAGE and stained with Coomassie blue. The position of the subunits of the N-type Ca 2+ channel are indicated by the arrows (al, ~2, 95K, and fl). (Right) Purified skeletal muscle DHP receptor was analyzed by SDS-PAGE and stained with CooInassie blue. The position of the subunits of the L-type Ca z+ channel are indicated by the arrows (al, a2,/3, and 3). Molecular weight standards are indicated at left. Samples are as follows: membranes, 100/zg of isolated rabbit brain membranes; solubilized, 100/zg of digitonin- solubilized membranes; hep pool, 50/xg of heparin-agarose pooled fractions; pH 10 pool, 8/xg monoclonal antibody (MAb) VD2~, pH 10 eluted pooled fractions; Ab Col Void, 8/xg void volume from anti-46K column; and Sucrose Grad, 30/zg of sucrose gradient fractions 10 and 11,

r e c e p t o r shif ts m o b i l i t y on S D S - P A G E on r educ t i on , y ie ld ing 8 p e p t i d e s o f 24K and 27K. The W G A , w h i c h b inds to c o m p l e x sugars , l abe ls the a 2 subun i t s o f b o t h r e c e p t o r s . N - G l y c o s i d a s e F t r e a t m e n t o f the to -conotox in r e c e p t o r s h o w s tha t the a 2 subun i t as wel l as the 95K subun i t con t a in N - l i n k e d sugars . Af f in i ty -pur i f i ed a n t i b o d i e s to the a~ and 95K subun i t s o f the N - t y p e Ca 2÷ channe l d e m o n s t r a t e tha t t he se subuni t s a re p r e s e n t in the to -cono tox in r e c e p t o r bu t no t in the ske le ta l m u s c l e D H P r e c e p t o r . 2°


Reconstitution for Single-Channel Analysis

Rationale. The purification of two major Ca 2+ channels, the skeletal L type and the brain N type, have now been reported. 2°'zl Although both channels are involved in dissimilar cellular functions (excitation-contraction coupling for the skeletal DHP receptor and transmitter release for the to-conotoxin receptor) each receptor complex is composed of four subunits. However, because the categorization of Ca 2 + channels into each particular group is based on pharmacological and biophysical properties, reconstitution of the purified receptor provides valuable information on the channel activity of the receptor. It further provides a means to distinguish between biophysical properties specifically due to the purified complex and those that might be due to cellular regulation of the native channel. Specific advantages of reconstitution include easier control over the lipid composition and the solutions on both sides of the membrane. These two factors are either impossible or difficult to control for N-type Ca 2+ channels in native membranes since (1) channel activity cannot be studied in excised patches, (2) solutions on the inside of a cell cannot be readily controlled, and (3) N-type Ca 2+ channels are often located in structures which are inaccessible, such as nerve terminals. Therefore, studies on ion-permeation properties and pharmacological regulation of the Ca 2~ channel are considerably easier for reconstituted channels.

Choice of Reconstitution Technique. Three major forms of ion channel reconstitution systems are available: (a) patch clamp of large liposomes 22 or painted membranes 23 in which channels have been preinserted, (b) planar bilayers formed from bulk solutions across apertures 50-300/xm in diameter in Teflon septi, 24 and (c) lipid monolayers. 25 Bilayers formed from monolayers can be used to separate two compartments either of the classic bilayer chamber 25 or at the tip of patch electrodes. 26 In cases (b) and (c), channels can be formed either by preinsertion into vesicles and subsequent fusion of the vesicles with the bilayer or by direct insertion of channels into the bilayer.

We have limited our choice for the method of reconstitution by the expected properties of the purified receptor. The N-type Ca 2+ channels

21 A. T. Leung, T. Imagawa, B. Block, C. Franzini-Armstrong, and K. P. Campbell, J. Biol. Chem. 263, 994 (1988).

22 D. W. Tank, C. Miller, and W. W. Webb, Proc. Natl. Acad. Sci. U,S.A. 79, 7749 (1982). 23 p. Mueller, Ann. N.Y. Acad. Sci. 264, 247 (1975). 24 p. Mueller, D. Rudin, H. T. Tien, and W, C. Wescott, Circulation 26, 1167 (1962). 25 M. Montal and P. Mueller, Proc. Natl. Acad. Sci. U.S.A. 69, 3561 (1972). 26 R. Coronado and R. Latorre, Biophys. J. 43, 231 (1983).


typically have small current amplitudes in the low picoampere range and have fast open-time durations in the millisecond range. 2°,27 Therefore, reconstitution of the receptor in bilayer chambers is problematic because the capacitance of the large membrane severely limits the recording band- width and creates large, complex capacity transients in response to voltage modifications. Although such reconstitutions have been shown for the DHP receptor or the tetrodotoxin (TTX)-sensitive Na + channel, they were mostly done in the presence of drugs (BAY K 8644 and batrachotoxin, respectively) known to affect the open-time constants of both channels. However, compounds with similar functional effects have not been reported for the N-type C a 2 + channel.

Because recordings at the tip of patch pipettes allow higher time resolution and better signal-to-noise ratios, we have chosen to study the N-type Ca 2+ channel by this technique. The tip-dip method presents further distinct advantages: (1) since bilayers at the tip of electrodes cover a smaller surface area, they support stronger voltage gradients (up to 200 mV); (2) contrary to bilayers formed across Teflon holes, tip-dip bilayers do not contain solvents since they are allowed to evaporate from the monolayer; and (3) channels do not need to be preincorporated into vesicles which themselves would form subsequently the basis of the monolayer, and this in turn minimizes considerably the number of protocol steps, which is essential for reducing the amount of proteolysis.

Description of Tip-Dip Technique. In the tip-dip method, the formation of bilayers requires a two-step procedure subsequent to the formation of a monolayer at the surface of the bath solution (Fig. 2). The first step induces the formation of a monolayer by removing the electrode from the bath solution (Fig. 2B), and the second step results in bilayer formation by reimmersion of the pipette, as assessed by the resulting gigohm seal resistance (Fig. 2C). In some cases, however, these procedures are not needed because of direct formation of the bilayer during the first pipette immersion.

For the formation of monolayers, we use a mixture of bovine brain phosphatidylethanolamine (PE) and phosphatidylserine (PS) in a 1 to 1 weight ratio (PE and PS are from Avanti Polar Lipids, Birmingham, AL). The lipids are stored at - 80 ° and dried under a stream of nitrogen. Dried lipids are then dissolved in n-decane at 30 mg/ml. One to three microliters of lipids are then spread on top of the bath solution. Heat-polished electrodes of 7 MI) resistance are used, which are coated with Sylgard to improve pipette capacitance. Under these conditions, the probability of successful lipid formation exceeds 90%. However, not all the membrane

27 M. De Waard, D. R. Witcher, and K. P. Campbell, J. Biol. Chem. 269, 6716 (1994).


A

13

C

STIMULATION

5 mV

RE=7.1 Mohm

iiiiiiiiii!iiiiiii! JL lkLLkLJt I LILt %,LLkLJ LLLJ 1]1

__J 100 pA

20 ms

/ - V ~Llt l LTLI ~'Lll l ' 'Lsk ~llt,lf¢'~\ l lt t, I )I.I l I l I I I l l l l l I RS=5 Gohm

:::5"// ~ _ L L L L L L L L [ . L L I

....... .............. ............. 100 X

Fro. 2. Tip-dip technique. (A) A patch electrode connected to an amplifier is dipped into the bath covered by a monolayer of lipid mixture. The electrode resistance can be measured in response to a 5-mV pulse. (B) Removal of the electrode results in the formation of a monolayer at the tip of the electrode. (C) Placing the electrode back into the solution results in the formation of a bilayer. This causes an important increase in the electrode resistance. The seal resistance, in response to a 5-mV pulse, is measured after a 100-fold increase in the gain of the amplifier.

formations represent true bilayers, as assessed by the rate of successful recordings. This rate is much lower probably because of the combination of real bilayer formation success and channel presence or incorporation rate. We have frequently performed control experiments in the absence of receptor to test for the stability of the bilayer. Constant voltages applied to the bilayers show that under these conditions no channel-like openings are present, owing to stepwise decrements in seal resistance even in the presence of digitonin at concentrations 105- to 107-fold higher than achieved during the reconstitution of the receptor (Fig. 3A).

Guidelines to Ensure Formation o f Good Bilayers. Bilayers formed within the first 10 min result in seal resistances higher than 100 GO that do not give rise to channel incorporation. This is probably due to an agglomeration of lipid layers favored either by the residual presence of solvent or by an incomplete monolayer formation. We avoid making bilayers 40 min after spreading the lipid because of oxidation of lipids occurring at the air-lipid interface. The presence of PS in combination with

346 IONS AND CHANNELS [ 2 8 ]

A

~ g 60 o . j =

• , ~ o 40 ~.~

20 7

8 o

+ I

4 5 11

o , "E

R I -

# CHANNELS

- CHANNELS

DIGITONIN

H EAT-INACTIVATED

TRYPSINIZED

. .~ 1 pA

50 ms

B CONTROL

J , ~A

1 O0 m s

1 pM ¢ o - C g T x G V I A

FIG. 3. Reconstitution of purified N-type Ca 2+ channel complex. (A) The percentage of patches giving rise to channel activity was measured under a variety of conditions: in the presence of 5-20 pmol of receptor, in the absence of channels, in the presence of 0.1% digitonin, and after heat-inactivation or trypsin-digestion of the receptor. Corresponding recordings at right show that channel activity was obtained only in the presence of the receptor. Ionic conditions were as follows: symmetrical 100 mM BaCI2, 10 mM HEPES, pH 7.4. The holding potential was +40 mV (+ channels) and + 100 mV in other cases. Solid lines represent the closed state of the channel. (B) The N-type Ca 2+ channel activity is blocked with 1/zM ¢o-conotoxin GVIA. Symmetrical ionic conditions were used: 100 mM BaC12, 10 mM HEPES, pH 7.4 (electrode), and 1 mM BaClz, 150 mM HEPES, pH 7.4 (bath). The holding potential was - 1 0 inV. Channel activity is blocked 3 rain after the addition of 1 /zM oJ-conotoxin GVIA to the bath.

divalent cations on both sides of the bilayer will ensure membrane stability. The occurrence of artifacts seemed enhanced by reducing either the proportion of PS or the divalent cation concentration to submillimolar levels. Finally, electrode resistance should remain in the range 2 to 10 MO since low-resistance electrodes have more unstable bilayers and high-resistance electrodes have a tendency to be obstructed.

Channel Insertion, The rate and number of channels inserted into the bilayer is best controlled by adjusting the final receptor concentration, the level of pipette immersion, and the subsequent perfusion of the bath medium. Higher receptor concentrations are required for channel incorporation occurring after bilayer formation. Furthermore, the receptor con-


centration must be higher when the channels are added to the cup rather than in the electrode owing to the larger bath volume. In contrast, when channels are incorporated into monolayers before patch formation, lower concentrations are required. This is probably because of a better incorporation into monolayers.

Other factors may affect the number of channels in bilayers. We found substantial differences with different lipid amounts added (I/~l compared to 2 or 4/~1, for example). The lower the amount of lipids, the higher the channel concentration. This probably arises from (1) a displacement of equilibrium between channels in solution and channels in the lipid phase and (2) an increase in surface areas that really are monolayer in nature, which again suggests a better channel insertion into monolayers. Because the purpose of the experiment is to obtain as consistently as possible a single channel in the bilayer, and because parameters such as the exact concentration of channel in the purified fraction, the amount of lipids added, the surface of the extracellular cup, and the tip surface of the electrode are involved, the experimental conditions need to be defined empirically each time.

It is worth noting that extremely small amounts of purified proteins (5 to 20 pmol) are necessary. Based on the concentrations of N-type Ca 2 channels used, experiments performed in a day would require less than 5/~1 of the purified receptor. Because the dilution factor used for recordings of isolated receptors was extremely high, removal of detergent (by polystyrene resins such as Bio-Beads SM-2, Bio-Rad, Richmond, CA) from the purified preparation was not necessary. Figure 3 shows two single-channel recordings that illustrate the specificity of the channel reconstitution. Only bilayers containing intact purified o~-conotoxin receptor are active (symmetrical 100 mM BaCI2, voltage electrode + 40 mV, Fig. 3A). Like the native N-type C a 2+ channel, the reconstituted channel activity is specifically blocked by a bath application of 1/~M ~o-conotoxin GVIA.

Interpretation. In some cases, the number of channels inserted into the bilayer can increase with time. This is true especially when channels are inserted into the electrode where perfusion is not easily feasible. Obviously, there is not a simple bilayer present over the entire area covered by the tip of the electrode. Instead, a lipid bulk at the rim of the electrode may constitute a reservoir where channels can diffuse laterally to decrease or increase the number of functional channels. It is also possible that further lipid or protein diffusion occurs from the surface monolayer into the bilayer along a lipid film present on the glass surface. Increases in lipid amounts at the tip of the electrode would result in the loss of bilayer formation. We have indeed recorded some irreversible increases in seal resistance during the time course of an experiment.


Because these events were more frequent with higher amounts of lipid spreading at the surface of the solution, it does suggest the likelihood of this process. We minimize the occurrence of these events by engaging the electrode deep enough into the solution and by reducing maximally the amount of lipids used (defined as the volume that gives a success rate of at least 50%).

Acknowledgments

We gratefully acknowledge Mike Mullinnix for excellent technical assistance. Kevin P. Campbell is an Investigator of the Howard Hughes Medical Institute.

[29] In fus ion o f G u a n i n e N u c l e o t i d e s t h r o u g h R e c o r d i n g

E l e c t r o d e s for S t u d i e s on G - P r o t e i n R e g u l a t i o n of Ion C u r r e n t s a n d C h a n n e l s

By R O D R I G O A N D R A D E

Introduction

Intracellular infusion of GTP and guanine nucleotides has been extensively used in electrophysiological experiments to pinpoint the participation of G proteins in the regulation of ion currents and channels. Indeed the use of these compounds has become one of the standard criteria used to implicate heterotrimeric G proteins in membrane phenomena. As a result there is a vast literature regarding the use of GTP and hydrolysis- resistant guanine nucleotides in electrophysiological experiments. Al- though identical conditions are rarely used to test these compounds, most procedures are simple variants of a few common themes as discussed below.

Use of GTP and Hydrolysis-Resistant Guanine Nucleotides in Whole-Cell Recording

G-protein-coupled receptors are one of the main types of cell surface receptors mediating cell-to-cell communication. Because transmembrane signaling by these receptors is dependent on the hydrolysis of GTP, this nucleotide is an essential cofactor for a variety of transmembrane signaling mechanisms. In intact cells intracellular GTP concentration is maintained by endogenous metabolic pathways. During whole-cell recording, how-


[ 2 9 ] INTRACELLULAR INFUSION OF GUANINE NUCLEOTIDES 349

ever, there is a rapid exchange of the cytoplasm of the cell and the electrode recording solution. This exchange disrupts intracellular metabolism and can depress the availability of essential constituents for transmembrane signaling, including GTP. As a result, signaling through G proteins can be severely impaired. Therefore the simplest use of GTP is as a cofactor to be included in the intracellular solution of the electrode to maintain G- protein functioning. This is accomplished simply by adding GTP to the intracellular solution. The concentrations used for this purpose typically range from 100 to 500 IxM and therefore are 2 to 10 times higher than estimated normal intracellular GTP concentrations.

The ability of whole-cell recording to inhibit G-protein signaling by depleting intracellular GTP can be used to demonstrate the involvement of G proteins in receptor-mediated responses. This was first realized by Pfaffinger et al . , 2 who used whole-cell recording to demonstrate the role of G proteins of the G i family in the muscarinic regulation of potassium channels in heart. Their experiments showed that when precautions were taken to maximize intracellular dialysis by choosing small cells and using low-resistance pipettes, muscarinic responses could only be observed when GTP was added to the intraceUular solution. Previous studies had ruled out all the then-known second messenger systems. Therefore the observation that signaling could be preserved solely by providing GTP lead to the now widely accepted idea that some G proteins can gate ion channels through a "membrane-delimited" pathway, possibly involving a direct interaction of the G protein with the channel itself. This elegant experiment established GTP dependence as one of the critical criteria for demonstrating the involvement of a G protein in transmembrane signaling phenomena.

Historically the importance of adding GTP to the intracellular solution was not always appreciated. This was probably a consequence of the fact that G-protein-mediated responses can often be recorded in the whole- cell mode in the absence of added GTP. The reason for this is that GTP levels have to be severely depressed before any impairment of the G- protein-mediated signaling becomes evident, l Thus, in the absence of extensive intracellular dilution and disruption of GTP metabolism, endogenous mechanisms can synthesize enough GTP to sustain G-protein function. From a practical standpoint this complicates the demonstration of GTP dependence and could result in the erroneous interpretation that a response is GTP-insensitive. Extensive exchange of the cytoplasm with

1 G. E. Breitwieser and G. Szabo, J. Gen. Physiol. 91, 469 (1988). 2 p. j. Pfaffinger, J. M. Martin, D. D. Hunter, N. M. Nathanson, and B. Hille, Nature

(London) 317, 536 (1985).


the pipette recording solution is particularly difficult to obtain in large cells and also in neurons expressing dendritic arborizations. In the case of neurons, it seems unlikely that even the most extensive cell dialysis at the level of the soma and proximal dendrites would extend far into the rest of the dendritic arbor. As a result agonist administrations that cover the whole cell do not necessarily exhibit a dependence on exogenous GTP added to the recording solution. One way around this problem is to use localized agonist applications that restrict receptor activation to the cell soma and proximal dendrites, the areas most likely to be dialyzed by the recording pipette. This approach has been used to demonstrate the GTP dependence for adenosine in neurons in primary culture 3 and for serotonin in hippocampal brain slices. 4 In these experiments responses to iontopho- retic applications of adenosine and serotonin restricted to the proximity of the recording electrode ran down rapidly when recorded in the whole- cell mode in the absence of GTP. Addition of GTP to the recording solution, however, prevented this rundown.

Hydrolysis-Resistant Guanine Nucleotides

A second fruitful strategy for testing the involvement of G proteins in the signaling of electrophysiological responses has been the use of hydrolysis-resistant guanine nucleotides. After intracellular infusion of guanosine 5'-O-(3-thiotriphosphate) (GTPyS) or guanylyl imidodiphosphate (GppNHp), receptor activation, which normally catalyzes the exchange of the GDP bound to the a subunit of the G protein for GTP, results in the exchange of GDP for the nonhydrolyzable guanine nucleotides. This does not impair the dissociation of the a from the fly subunits. However, because termination of the G-protein activation involves the hydrolysis of GTP, the a subunit is caught in a persistently activated state. Thus, at least in principle, the ability of hydrolysis-resistant guanine nucleotides to trap the a subunit in the activated state can be used as a diagnostic criterion for the participation of G proteins in the signaling cascade. The experiment then is very simple and involves the comparison of agonist- induced responses under control conditions and after infusion of the hydrolysis-resistant guanine nucleotide. Under control conditions a G-protein- mediated response should recover to control after termination of agonist administration. In contrast, after infusion of the hydrolysis-resistant guanine nucleotide, the response should persist after removal of the agonist

a L. O. Trussell and M. B. Jackson, J. Neurosci. 7, 3306 (1987). 4 R. Andrade, Drug Dev. Res. 26, 275 (1992).

[29] INTRACELLULAR INFUSION OF GUANINE NUCLEOTIDES 351

GTP3'S

Fro. 1. Effect of GTPyS on cholinergic responses in rat hippocampal pyramidal cells. (Left) Whole-cell recording obtained using 0.5 mM GTP and 2 mM ATP in the intracellular solution. Administration of carbachol (Carb) elicits a membrane depolarization that recovers to control following removal of the agonist from the bath. Cell membrane potential, -70 inV. (Right) Whole-cell recording obtained from a different cell using 100/zM GTP-yS, 0.5 mM GTP, and 2 mM ATP. Administration of carbachol to the cell elicits an irreversible membrane depolarization. Cell membrane potential, -78 inV. Calibration bar: 2 rain and 5 inV.

(Fig. 1). Intracellular infusion of GppNHp (100 /xM) or a GTPyS/GTP mixture (2 and 0.1 raM, respectively) into bullfrog atrial cells to examine muscarinic regulation of potassium currents has been shown to come very close to this ideal. ~.5

A common complication encountered is that most G proteins exhibit some basal turnover that is independent of receptor stimulation. As a result, infusion of GTPTS or GppNHp per se results in the agonist-independent activation of the response (current) being examined. This agonist- independent current can make it difficult to unambiguously demonstrate that the agonist response is rendered irreversible following infusion of the hydrolysis-resistant guanine nucleotide. One strategy to solve this problem is to slow down the agonist-independent activation of the current. Trapping of the G protein in the activated state by the hydrolysis-resistant analog is not dependent on the absolute concentration of GTP3,S or GppNHp but instead on the relative ratio of the intracellular concentrations of the hydrolysis-resistant nucleotide and GTP.~'4 Thus, the time course of G-protein activation depends not only on the basal (agonist-independent) turnover rate of the particular G protein involved, but also on the concentration of hydrolysis-resistant guanine nucleotide used in the electrode and the concentration of GTP included in the patch pipette (and/or still remaining inside the cell). In the limiting case, which requires a high basal G-protein turnover rate and high ratio of hydrolysis-resistant nucleotide

5 G. E. Breitwieser and G. Szabo, Nature (London) 317, 538 (1985).


to GTP, there is a rapid agonist-independent activation of the current of interest. Such spontaneously developing G-protein-mediated responses have been reported using hydrolysis-resistant nucleotide/GTP concentration ratios ranging from 500/zM GTPyS/no added GTP to 100/~M GTPyS/ 500/.,M GTP. 3'4'6'7 In one of these studies it was possible to slow down the agonist-independent turning on of the response by reducing the ratio of hydrolysis-resistant guanine nucleotide to GTP from 1 : 5 to 1 : 50. This produced a more stable baseline and allowed for a clearer demonstration of the effects of the hydrolysis-resistant guanine nucleotide on agonist responses .4

Guanosine 5'-O-(2-thiodiphosphate) (GDP/3S) is a hydrolysis-resistant guanine nucleotide capable of binding the a subunit of G proteins. This compound can support little or no activation of the G protein 8 and therefore can competitively inhibit G proteins. Thus, in a sense, GDP/3S functions as either an antagonist or a weak partial agonist at the level of the G protein. The availability of GDPBS allows for a second avenue to examine the involvement of G proteins in the signaling of electrophysiological responses. In the simplest form the experiment compares the response to an agonist obtained using a control intracellular solution with that obtained with the same solution plus GDPflS. A G-protein-mediated response should be inhibited by intracellular infusion of GDP/3S concentrations capable of inhibiting G-protein activation (Fig. 2). Such inhibitions have been demonstrated in a number of systems using mid-micromolar concentrations of GDPflS, 3,9 a finding that is consistent with the concentration of GDP/3S needed to inhibit G~ in vitro. 8 Although GDP/3S has been reported to inhibit G-protein-mediated responses, some caution in using this compound is advised as biochemical reports indicate that GDP/3S can itself activate G proteins, albeit much less efficiently than GTP or hydrolysis-resistant analogs. 8

In addition to the technical and biological concerns outlined above, some practical steps are taken in the author's laboratory when working with GTP and hydrolysis-resistant analogs. These compounds are unstable at room temperature, and therefore precautions are routinely taken to minimize degradation. The final intracellular solution containing both GTP and ATP is prepared fresh every day. We have found it easiest to prepare the final solution by mixing two separate stocks. The first

6 A. C. Dolphin, J. F. Wootton, R. H. Scott, and D. R. Trentham, Pfluegers Arch. 411, 628 (1988).

7 K. S. Elmsley, W. Zhou, and S. W. Jones, Neuron 5, 75 (1990). 8 F. Eckstein, D. Cassel, H. Levkovitz, M. Lowe, and Z. Selinger, J. Biol. Chem. 254,

9829 (1979). 9 A. C. Dolphin and R. H. Scott, J. Physiol. (London) 386, 1 (1987).


Control 5-HT Baclofen

1 min

GDP/3S 5-HT Baclofen

FIG. 2. Effect of GDP¢tS on 5-HT (5-hydroxytryptamine) and baclofen responses in the rat hippocampus. (Top) Control responses were recorded using sharp microelectrodes filled with 2 M potassium methyl sulfate. Under these conditions both 5-HT- and baclofen-induced large membrane hyperpolarizations. (Bottom) Responses recorded using sharp microelectrodes containing 2 M potassium methyl sulfate and GDPflS (33 raM). Under these conditions the first application of serotonin and baclofen results in greatly reduced hyperpolarizing responses that recover only incompletely (see Andrade et al.14), whereas subsequent agonist applications are without effect.

stock contains all of the required salts, calcium chelators, and buffers (ionic stock). This stock is stable and can be stored at room temperature or in a regular laboratory refrigerator. The concentrations of the salts, calcium chelators, and buffers are such that when 4 ml of the stock is diluted to a final volume of 5 ml they are all present at the desired concentration. The second stock contains the ATP, GTP, and hydrolysis- resistant analogs (nucleotide stock). In this stock all of the nucleotides are dissolved in deionized water at concentrations 5-fold higher than those desired in the final recording solution, and the pH is adjusted to 7.3. This stock solution is unstable at room temperature and therefore is stored frozen at - 40 ° in l-ml aliquots for daily use. Nucleotide stock solutions containing ATP and GTP can be stored for over 2 weeks without apparent loss of activity. The final recording solution is prepared by mixing 4 ml of the ionic stock and 1 ml of freshly thawed nucleotide stock. The final solution is kept on ice and can be used for at least 4 hr.


An alternative to this protocol is to dissolve the nucleotides directly into the final recording solution. However, even in the presence of 10 mM HEPES, the addition of the nucleotides changes the pH of the final recording solution, forcing a second pH adjustment.

Use of GTP and Nonhydrolyzable Guanine Nucleotides in Single-Channel Recording

The use of single-channel recording in inside-out membrane patches brings unprecedented power to the analysis of G-protein regulation of ion channel activity. Under these conditions only intracellular components closely associated with the membrane are retained. Thus, recording under these conditions can be thought of as a refinement of whole-cell recordings optimized to obtain a complete dialysis of the interior of the cell. The classic evidence for a direct gating of potassium channels by G proteins was provided by Kurachi and collaborators using this approach. 10 In these experiments they showed that the single-channel activity observed in the presence of acetylcholine or adenosine in the patch pipette disappeared rapidly after the membrane patch was excised but that channel activity could be recovered by applying 100 lzM GTP to the inside surface of the patch. In addition, they also showed that application of GTP3~S to the cytoplasmic side of the patch could activate channel activity without the need for agonist inside the pipette. These results clearly demonstrated that all of the components necessary for the activation of the channel except GTP resided in close association with the membrane. The extraordi- nary power of this technique, however, goes beyond this and extends to examining the functioning of single channels and also to enabling exchange of the solution bathing tl" • .*oplasmic side of the membrane using solutions of known composition. This can be exploited to examine the identity of the G proteins involved in the gating of the channel as outlined elsewhere in this volume. More pertinent to the scope of this chapter, it has also allowed a preliminary quantitative analysis of the relationship between GTP concentration and the functioning of G proteins.11

Use of Nonhydrolyzable Guanine Nucleotides in Conjunction with Conventional Sharp Microelectrode Recordings

Traditional sharp microelectrode recording techniques are also suitable for intracellular infusion of hydrolysis-resistant guanine nucleotides. Al-

l0 y. Kurachi, T. Nakajima, and T. Sugimoto, Pfluegers Arch. 407, 264 (1986). 11 y. Kurachi, H. Ito, and T. Sugimoto, Pfluegers Arch. 416, 216 (1990).


though the principles involved are essentially identical to those outlined above, there are some important practical differences. Unlike whole-cell recordings, sharp microelectrode work requires that the cell be impaled with a high-resistance microelectrode. As a result, the technique does not work well for small cells, and, even under the best of circumstances, cells are stressed immediately after impalement. On the other hand, the use of high-resistance microelectrodes does not result in the dialysis of the interior of the cell, and therefore responses mediated through complex biochemical cascades can be recorded for prolonged periods of time after impalement.

The drawbacks (and benefits) of infusing hydrolysis-resistant guanine nucleotides using sharp microelectrodes are the direct results of the technical differences outlined above. Because the cell interior is not dialyzed, it is not possible to test directly the dependence of the response on intracellular GTP. Therefore, the technique is confined to the use of hydrolysis- resistant nucleotides. In addition, when infusing hydrolysis-resistant nucleotides, it is not possible to precisely control the intracellular concentration nor the hydrolysis-resistant analog to GTP concentration ratio. Fi- nally, because infusion from sharp microelectrodes is time-dependent and extremely difficult to quantify, the final intracellular concentration of the hydrolysis-resistant nucleotide is not known. Indeed, to ensure delivery of the hydrolysis-resistant guanine nucleotide to the interior of the cell, these are generally dissolved in the microelectrode at concentrations much higher than those used in whole-cell recording. Typical concentrations reported in the literature for GTP,/S and GDP/3S are highly variable, ranging from the mid-micromolar ~2,~3 to mid-millimolar) 4-16 Some of the variability probably reflects differences in the microelectrodes used by different investigators, which would strongly influence the rate of hydrolysis-resistant nucleotide infusion into the cell. Of course the intracellular GTP concentration is also outside investigator control since it is determined by cell metabolism. In spite of these drawbacks sharp microelectrodes offer the only currently available avenue to inject guanine nucleotides without simultaneously dialyzing the cell. It is known that many G- protein-mediated responses run down rapidly while recording in the whole- cell mode even in the presence of added GTP. This is thought to reflect the loss of other essential cytoplasmic constituents. As such rundown

12 K. Sasaki and M. Sato, Nature (London) 325, 259 (1987). ~3 p. j. Williams, B. A. MacVicar, and Q. J. Pittman, J. Neurosci. 10, 757 (1990). 14 R. Andrade, R. C. Malenka, and R. A. Nicoll, Science 234, 1261 (1986). i~ G. K. Aghajanian, Brain Res. 524, 171 (1990). 1~ R. A. North, J. T. Williams, A. Suprenant, and M. J. Christie, Proc. Natl. Aead. Sci.

U.S.A. 84, 5487 (1987).


would not occur when recording with sharp microelectrodes, this technique offers the only currently known avenue for testing the effect of guanine nucleotides on such responses.

Conclusion

The intracellular infusion of GTP and guanine nucleotides has been an important technique for demonstrating the involvement of heterotrimeric G proteins in regulating membrane ion currents. This traditional role, however, has become less critical with the realization that heterotrimeric G proteins are an obligatory component of the signaling mechanism used by receptors of the seven-transmembrane-spanning domain super- family. Nevertheless, it is likely that these molecules will continue to play an important role in the experimental approaches used to understand transmembrane signaling. For example, little is known at present regarding the kinetics of G-protein activation and inactivation by agonists in situ. Such knowledge is essential, however, if we want to understand how such proteins function in synaptic transmission. Inroads into this problem have been made by trapping G protein in the active conformation using GTPyS,1 and the widespread availability of caged GTP analogs promises to greatly facilitate these studies. 17 In addition, the use of hydrolysis-resistant guanine nucleotides has been principally restricted to studies on the physiology of heterotrimeric G proteins. These proteins, however, are not the only class of intracellular effector that uses GTP. A second class of GTP- binding proteins, the small GTP-binding proteins, are also thought to play an important role in cell physiology. Thus, a second area where guanine nucleotide analogs will continue to prove useful is in determining the function of small G proteins in cellular physiology 18 and exploring their role in the regulation of membrane excitability.

Acknowledgments

Work in the author's laboratory is supported by National Institutes of Health Grant MH 43985 and the Alfred P. Sloan Foundation. I thank Dr. Sheryl Beck for helpful suggestions on the manuscript.

17 A. C. Dolphin, J. F. Wootton, R. H. Scott, and D. R. Trentham, Pfluegers Arch. 411, 628 (1988).

18 N. K. Pryer, L. J. Wuestehube, and R. Schekman, Annu. Reo. Biochem. 61, 471 (1992).

[30] U S I N G G - P R O T E I N A N T I S E R A I N E L E C T R O P H Y S I O L O G Y 357

[30] Injection of Antisera into Cells to Study G-Protein Regulation of Channel Function

By I. MCFADZEAN, M. P. CAULFIELD, Y. VALLIS, and D. A. BROWN

Introduction

It is clear that GTP-binding proteins (G proteins) play a crucial role in excitable cells by coupling many types of neurotransmitter receptors to ion channels, whether by interacting directly with the ion channel or by activating an enzyme to liberate a soluble second messenger which interacts with the channel. Over the past few years, a number of electrophysiological experiments have been designed to identify which G proteins are involved in such responses. Two broad approaches have been employed. The first, and probably the more widely used to date, has made use of the fact that in some cases the G proteins are sensitive to pertussis toxin (Ptx). This means that endogenous G proteins can be inactivated by pretreatment of cells with Ptx, and the ability of purified preparations of G proteins or their subunits to reconstitute the response can be tested. The methodology relating to this approach is covered elsewhere in this volume) The main disadvantage of reconstitution studies is that they do not positively identify the normally operant transduction pathway. To do this requires that the pathway be blocked. Antibodies raised against the C-terminal portion of a subunits of various G proteins have been used to this end. 2 In this chapter, we discuss some methods for loading cells with such antibodies, prior to electrophysiological recordings of ion channel currents and their modulation by neurotransmitters.

Checking Adequate Loading of Cells with Antibody

Whichever procedure is adopted, it is important to establish whether there has been successful loading of cells with antibody. Ideally, this should be done for the cells from which recordings of currents were made, but the success rate can be established in separate batches of cells. Antibody loading can be inferred by including dye in the antibody solutions (e.g., Fast Green3), but this presumes that the access of dye to the cell

i A. Yatani, this volume [33]. 2 G. Milligan, this series Vol. 237 [21]. 3 R. M. Harris-Warrick, C. Hammond, D. Paupardin-Tritsch, V. Homburger, B. Rouot,

J. Bockaert, and H. M. Gerschenfeld, Neuron 1, 27 (1988).



interior accurately reflects the access of antibody. We have directly visualized antibody using an immunohistochemical approach to check loading of rabbit anti-G-protein antibodies both in NG108-15 neuroblastoma x glioma hybrid cells 4 and rat cultured sympathetic neurons (M. P. Caulfield and Y. Vallis, unpublished). This uses a second antibody, raised against rabbit immunoglobulins (usually in sheep or pig) and labeled with fluorescein.

Cells are first permeabilized with a 95% ethanol, 5% acetic acid solution (at -20 °) for 15 rain, before being incubated for 30 min at 37 ° with an appropriate dilution in phosphate-buffered saline (PBS) of a fluorescein- conjugated, porcine immunoglobulin raised against rabbit immunoglobulins. We use a 1 : 100 dilution of second antibody, selected after preliminary experiments with dilutions ranging from 1 : 50 to 1 : 200. After being washed with excess phosphate-buffered saline and mounted using Citifluor, the cells are viewed using a fluorescence microscope.

Two points need to be made about these verification procedures. The first is that it is impossible to quantify the amount of antibody that has gotten into the cell. All that can be said is that the fluorescence is significantly above background levels. The second point is that lack of functional effect of the antibody used (given this uncertainty about antibody concentration in the cell) cannot be taken as evidence that the G protein to which the antibody binds is not involved in mediating the neurotransmitter response studied.

C o n t r o l s

The properties of cells loaded with antibodies or antiserum cannot validly be compared to those of unloaded ceils. This raises the question of appropriate controls. A possible control for experiments with antiserum is to use preimmune serum. 4 However, this does not allow verification of successful intracellular penetration of the control treatment with cyto- chemical methods. Heat-treated antibodies have been used as controlsY but this relies on the assumption that the heating protocol has inactivated the antibody, as (usually) there is no independent way of verifying the lack of activity of the heat-treated antibody. An alternative approach is to use another (non-G-protein binding) antibody as a control. In experiments with rat sympathetic neurons, we have successfully used rabbit antiserum raised against rat glial fibrillary acidic protein as a control for

4 I. McFadzean, I. Mullaney, D. A. Brown, and G. Milligan, Neuron 3, 177 (1989). 5 p. M. Lledo, V. Homburger, J. Bockaert, and J. D. Vincent, Neuron 8, 455 (1992).

[30] USING G-PROTEIN ANTISERA IN ELECTROPHYSIOLOGY 359

G-protein antibody-injected cells. This has the advantage that it allows selection for recordings of cells which have been successfully injected.

Methods for Loading Cells with Ant ibody

Adding Antibody to Patch Pipette Filling Solution

One of the advantages of the whole-cell patch-clamp technique over conventional single-electrode recording methods is that the relatively large tip diameter of the micropipettes used (typically 1-2 ~m) can (theoretically) allow the solution contained within the micropipette to dialyze the cell interior. It follows from this that the most straightforward way of getting antibodies into cells would seem to be to add them to the micropipette filling solution and allow them to diffuse into the cell during the time course of an experiment. This technique has been used successfully in rat anterior pituitary cells to study the coupling between D z and both calcium and potassium channels. 5 Affinity-purified polyclonal antibodies, raised against either % purified from bovine brain or synthetic peptides corresponding to the C termini of the a subunits, are dissolved in the micropipette filling solution at a final concentration of 180 mg/ml.

Although the relative simplicity of this approach makes it an attractive option, there are potential problems which might make it unsuitable in some cases. The inclusion of antibody in the micropipette filling solution may make it difficult to form high-resistance gigohm (G~) seals between the pipette and the cell under study. Seal formation is essential in making low-noise patch-clamp recordings, and problems arise when serum proteins find their way onto the pipette tip, especially when using nonpurified antibody preparations. One way of circumventing this problem is to dip the tip of the micropipette in antibody-free solution for a few seconds prior to backfilling with antibody-containing solution. Obviously this will increase the time it takes for the antibody solution to equilibrate within the cell (see Roe et al. 6 for a discussion of the time taken for polyene antibiotics to diffuse within patch pipettes), and this, along with the slow binding of the antibodies, may mean that effective inhibition of the G proteins fails to occur during the course of a typical patch-clamp recording. Our own experience with this approach has been disappointing; inclusion in patch pipette solutions of an antiserum (! : 100 dilution) raised against Gq~ failed to produce detectable immunofluorescence in NG108-15 cells, even after 1 hr (J. Robbins and Y. Vallis, unpublished).

6 j. Rae, K. Cooper, P. Gates, and M. Watsky, J. Neurosci. Methods 37, 15 (1991).


Direct Injection of Antibodies with Microelectrodes

The first report of the successful use of antibodies was the demonstration that purified rabbit polyclonal antibodies against bovine Goa inhibited the reduction of calcium current in snail neurons produced by activation of dopamine receptors. 3 Here, the antibody is pressure-injected from a microelectrode filled with 165 txg/ml antibody, in a solution of 120 mM KCI (plus 200 mM HEPES, pH 7.4). Currents are recorded using a two- electrode voltage clamp. Although this method is suitable for snail neurons, because of their relatively large size, it is usually not possible with smaller cells to impale the cells with two or three micropipettes.

Usually, membrane currents are recorded with the whole-cell patch- clamp technique,7 and antibodies can be loaded into the cell by preinjection from a microelectrode. This approach has been used successfully in both NG 108-15 neuroblastoma x glioma hybrid cells 4 and cultured sympathetic neurons (M. P. Caulfield, unpublished data).

Antibodies are raised in rabbits to synthetic decapeptides corresponding to the C termini of the G-protein o~ subunits. Antiserum is then injected into NG108-15 cells using micropipettes similar to those used to make conventional intracellular recordings (i.e., with dc resistances 50-100 MO when filled with 3 M KCI), but then broken back (e.g., with a piece of tissue paper, or by gently touching the electrode tip onto the bottom of the dish) until they have resistances of 20-60 Mf~ when filled with antiserum. After filling the micropipette with antiserum, the cells are impaled and the antiserum ejected from the tip by applying positive pressure to the back of the micropipette using a patch-pipette holder attached to a syringe. To check that the antiserum is being ejected when the pressure is applied, the tip of the pipette is first submerged in liquid paraffin and viewed under a microscope. The droplet size ejected under a given pressure is measured and used to make approximations as to the final concentration of antiserum within the cell. The injection pipette is connected to a bridge amplifier so that a successful impalement of the cell is seen as a recorded membrane potential. After a cell is injected, the pipette is withdrawn, the position of the cell on the dish marked, and the cell left for at least 1 hr before patching on to make a whole-cell recording.

In separate experiments, it is established that this procedure is 100% successful when injecting NG108-15 cells with antibody? However, with superior cervical ganglion cells, there are several occasions when sucessful antibody penetration (established immunocytochemically) is less than 30%. Given that many of the injected ganglion cells do not survive the

70. P. Hamill, A, Marty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfliigers Arch. 391, 85 (1981).


injection process, we have adopted a routine of histochemically visualizing antibody in cells after recording currents and neurotransmitter responses (see Fig. 1). We have now successfully recorded responses to noradrenaline in 40 cells verified as having been injected with antibodies to various G proteins, or control antibodies (Figs. 2 and 3)

As a footnote, it is interesting that "microinjectors," which use pre- formed micropipettes, are now available commercially. These machines allow rapid injection of many cells in a preparation. We feel that such devices should be used with caution when antibodies are to be injected, and that verification of successful injection of antibody should always be done. This is because repeated pressure injection of cells with a single microelectrode usually results in the electrode becoming blocked after 20

dissociate

~ ° o ° 0

~:~ ~ ~ o o ° '~ o

ganglion replate

Ca-current Fluorescent visualization \

L; Osram \

\

/

J / [ \

m i c r o i n j e c t / /

~ w a i t

FIG. 1. Summary of steps in preparation of rat superior cervical ganglion neurons for antibody injection, recording of voltage-gated calcium currents, and subsequent verification of antibody loading. Ganglia from 7- to 14-day-old rats are enzymatically dissociated (trypsin and collagenase), then plated out on laminin-coated dishes. After 24 hr in culture, the cells are resuspended (by gently drawing up and expelling medium with a flame-polished Pasteur pipette), then replated. This is to remove processes to improve the space clamp during recording of calcium currents. After allowing the cells to attach to the substrate for about 3 hr, antibody is pressure injected into cells from a micropipette, and patch-clamp recordings are made at least 1 hr later. Cells are identified by their position in a grid square marked on the dish. Identification is usually aided by making a scratch mark close to the cell with a needle. After recording, the patch pipette is gently withdrawn (with application of gentle positive pressure), then cells are stained for antibody and visualized under fluorescent illumination.

362 ~ONS AND CHANNELS [30]

FIG. 2. (Top) Phase-contrast photomicrograph of a sympathetic neuron (A) which has been injected with an anti-Gq antibody; subsequently, voltage-gated calcium currents, and their inhibition by az-adrenoceptor activation, were recorded (via a patch pipette), then the presence of antibody verified by immunocytochemical staining with a second anti-rabbit fluorescein-conjugated antibody. (Bottom) The fluorescence is clearly visible. Note the very low level of fluorescence in the adjacent uninjected neuron (B).


Anti-GFAP Anti-G o

I I ,. .....

Fie. 3. Inhibition of the voltage-dependent calcium current by noradrenaline in rat cultured superior ganglion (s.c.g.) neurons is reduced in cells injected with antibody to Go (M. P. Caulfield and Y. Vallis; unpublished). Each trace is the average of three voltage- dependent calcium currents evoked in the absence (bottom traces) or presence (top traces) of noradrenaline (1/.~M). Recordings from two s.c.g, neurons are shown, one injected with antibody to glial fibrillary acidic protein (GFAP; left) and the other, antibody to G O (right). The mean inhibition of the peak calcium current in 9 cells injected with anti-GFAP was 46.4 -+ 3.1%, whereas in 12 anti-Go-injected cells it was 27.0 -+ 3.3%. This difference was significant at the p = 0.0005 level (two-tailed t-test). In 4 cells injected with antibody to Gq the inhibition produced by noradrenaline was 44.8 +- 8%, a value not significantly different from control. All cells were "image-positive" as described in Figs. 1 and 2 and related text. Whole-cell calcium currents were evoked every 60 sec by 50-msec step depolarizations from a holding potential of - 80 mV to a command potential of + 10 mV following a 20-msec prepulse to - 40 mV (to reduce capacitive transients). Recordings were made using a switching amplifier (Axoclamp 2A) operating at a switching frequency of 4-8 kHz. Patch pipettes were filled with a Cs+-based solution which reduced, but did not abolish, voltage-dependent potassium currents evoked by the depolarizing step. Compensation for the residual outward current was achieved by digitally subtracting currents evoked in calcium-free solution containing 2.5 mM cobalt. Scale bars represent 200 pA (vertical axis) and 10 msec (horizontal axis).

or so cells have been injected. This is apparent as an increase in electrode resistance (recorded with the bridge amplifier) and lack of cell swelling when pressure is applied to the back of the pipette.

Scrape Loading

A drawback of the microinjection technique is that only a fraction of the cells under study can be loaded with antibody. An alternative approach which overcomes this limitation has been used by Menon-Johansson and Dolphin s to load antibodies into cultured rat dorsal root ganglion neurons. These workers used a "scrape-loading" technique based on that first described by McNeil et al. 9 The cells are replated after 3 to 4 days in culture by washing them gently from the coverslips to which they are

8 A. S. Menon-Johansson and A. C. Dolphin, J. Physiol. (London) 452, 177P (1992). 9 p. L. McNeil, R. F, Merphy, F. Lanni, and D. L. Taylor, J. Cell Biol. 98, 1556 (1984).


attached using a small volume of culture medium and a fire-polished Pasteur pipette. This process transiently severs the neurites and attachment plaques and allows the antibodies, which have been added to the replating medium, access to the cell interior. McNeil et al. calculated a loading efficiency of around 25% for a 150-kDa molecule, so the antibodies are added to the medium at a concentration four times higher than that required within the cell.

In the case of dorsal root ganglion neurons, simple mechanical dissociation is all that is required to detach the cells; others have added a trypsinization step. This method has been used to load the oncogene protein T 2 4 - r a s into embryonic chick neurons.t° The cells are incubated for 30 to 45 rain at 37 ° with a 0.25% trypsin solution in CaZ+/Mg2+-free phosphate-buffered saline. After washing with excess culture medium the cells are centrifuged (40 g for 5 min) before being resuspended in 25 to 40 ml of medium containing the Ras protein at a concentration of 2.5 to 25 mg/ml. This cell suspension is then passed through a siliconized Pasteur pipette (1.1 mm internal tip diameter) 20 times to facilitate cell loading. We have found, however, that the relatively low success rate of immunocytochemically verifiable antibody loading following scrape-loading of superior cervical ganglion cells (Fig. 3) means that microinjection and postrecording verification remains our method of choice.

Acknowledgments

We thank Anatole Menon-Johansson for providing unpublished details of the scrape- loading technique. Experiments in the authors' laboratory were supported by the Medical Research Council and a Beit Memorial Fellowship to I. McF.

10 G. D. Borasio, J. John, A. Wittinghofer, Y. A. Barde, M. Sendtner, and R. Heumann, Neuron 2, 1087 (1989).

[31] WHOLE-CELL CLAMP ANALYSIS 365

[31] Whole-Cel l C lamp Analysis for G-Pro te in

Regula t ion of Channels

By J. HESCHELER

Introduction

In 1985 Breitwieser and Szabo Z and Pfaffinger et al. 2 simultaneously reported on the involvement of pertussis toxin-dependent G proteins in the muscarinic stimulation of cardiac K ÷ channels. The suggestion that G proteins directly interact with the channels without additional intermediate steps was corroborated by the elegant experiments of Yatani and coworkers, 3 who measured the K ÷ channel activity in inside-out patches and reconstituted G proteins to the inner side of the channel. The following phase of experimental studies on other types of channels including Na +, Ca 2÷, and C1- channels demonstrated the crucial role of G proteins on the one hand but also revealed the high complexity of channel regulationY A good example for such an integrative role of channels for various signaling pathways is the voltage-dependent Ca 2÷ channel which is expressed in most eukaryotic cells and functionally couples the membrane potential to the intracellular Ca 2- concentration (for subunit composition, see Singer et al.5). In pituitary cells we could demonstrate that G proteins of the Go family are involved in the hormonal inhibition of Ca 2÷ channels, G proteins of the Gi family are involved in the stimulation, and in addition the cAMP- dependent protein kinase as well as protein kinase C modulate the activity of the channel. 6-8 The diversity of G-protein a subunits is demonstrated in the diagram of Fig. 1 based on amino acid sequence similarities. There are G proteins with large differences in amino acid sequence (e.g., G

I G. Breitwieser and G. Szabo, Nature (London) 317, 538 (1985). 2 p. Pfaffinger, J. Martin, D. Hunter, N. Nathanson, and B. Hille, Nature (London) 317,

536 (1985). 3 A. Yatani, J. Codina, and A. M. Brown, Science 235, 207 (1987). 4 A. M. Brown and L. Birnbaumer, Annu. Reo. Physiol. 52, 197 (1990). 5 D. Singer, M. Biehl, I. Lotan, V. Flockerzi, F. Hofmann, and N. Dascal, Science 253,

1553 (1991). 6 W. Rosenthal, J. Hescheler, K.-J. Hinsch, K. Spicher, W. Trautwein, and G. Schultz,

EMBO J. 7, 1627 (1988). 7 j. Hescheler, W. Rosenthal, K.-D. Hinsch, M. Wulfern, W. Trautwein, and G. Schultz,

EMBO J. 7, 619 (1988). 8 M. Gollasch, H. Haller, G. Schultz, and J. Hescheler, Proc. Natl. Acad. Sci. U.S.A. 88,

10262 (1991).



,(/

G s family (4 splice variants) Golf transducins (arae)

(Xo2 --1 | G O family

~, a°l .~J

ail 1 ----" ai2 G i family ai3

az

x ~ Gq family ~ _ _ (C~l,a 11 'a14'a15 '°16 )

G12 family (c¢12, a13 ) FIG. 1. The ez subunits of G proteins are grouped by amino acid sequence identity [see

M. Simon, M. Strathmann, and N. Gautam, Science 252, 802 (1992)]. The hatched areas represent the points of interaction to suppress G-protein c~ subunits either with bacteriotoxins or antisense oligonucleotides, ez corn represents an oligonucleotide complementary to a coding sequence conserved in the a subunits of Gs, Gil_3, Goi_2, Gz, and transducin; for other antisense oligonucleotides see text.

proteins of the Gs and Gl2 family) as well as G proteins generated as splicing products which exhibit up to 90% identity (e.g., Gol and Go2). Hence , one of the central questions with respect to G-protein involvement in channel regulation is the determination of specificity of G proteins in these signaling pathways.

The complexi ty of channel regulations made it necessary to combine closely biochemical, molecular biological, cell culturing, and electrophysiological techniques. With regard to the electrophysiological approaches, experiments may be classified into two major categories. (1) During the experiment the function of G proteins is modified and the hormonal response is measured in the same cell before and after modification. This approach includes the intracellular application of GDP/GTP analogs via the patch pipette, the infusion of antibodies, and the reconstitution of


purified G proteins. (2) A large ensemble of cells is divided into a control group and a group of cells previously altered in G-protein function. The cells are measured and statistically compared. The classic example of such an approach is the pretreatment of cells with pertussis toxin, which blocks the function of G i and Go. The most recent experimental approach is the injection of antisense oligonucleotides specific for the respective mRNA encoding the G protein.

Methods

Techniques for Whole-Cell Recordings

Whole-cell membrane currents are recorded by patch pipettes according to the method described by Hamill et al. 9 The pipette is sealed to the surface of the cell, a gigohm (Gf~) seal is obtained and thereafter the small membrane patch under the tip of the pipette is disrupted. The low- resistance access to the cytoplasm allows the voltage clamp of the membrane potential and a diffusional exchange between the pipette solution and the intracellular solution. It is important to mention that the ionic conditions in the external and pipette solutions should be chosen so as to maximize the current of interest and to minimize all others. For example, to measure selectively voltage-dependent Ca 2+ currents we use Ba 2+ as divalent charge carrier (increased amplitude) and Cs + as blocker K + channels. 7

Typically, two basic voltage pulse protocols are used to characterize the action of a G-protein-dependent modulation of whole-cell currents.I°,11 In the first protocol, the holding potential is held constant and the membrane is depolarized during test pulses (ranging from 10 to 1000 msec) to various potentials. Measuring the current during the test pulses versus the respective voltage reveals the current-voltage relation. Assuming that the activation of the current has reached the steady state during the test pulse, clamping back to the holding potential should result in a tail current, that is, a current through the opened channels arising from the driving force at the holding potential. If the amplitude of the tail current is set into correlation with the activating voltage, the so-called steady-state activation curve is obtained. In the second protocol, the holding potential is set to various potentials and the current is measured during a constant

90. P. Hamill, A. Marry, E. Neher, B, Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981).

l0 B. P. Bean, Nature (London) 340, 153 (1989). ~ B. Hille, "Ionic Channels of Excitable Membranes." Sinauer, Sunderland, Massachu-

setts, 1991.


test pulse. This protocol allows one to determine the so-called steady- state inactivation curve (it is assumed that the current is in a steady state during the periods of the holding potential).

Such voltage protocols have revealed the common mechanism for G proteins to shift the activation curve but to leave the inactivation unaltered. For example, the Go-dependent inhibition of voltage-dependent Ca ~+ channels is based on a shift of the activation curve to more positive potentials,~° meaning that strong depolarizations may overcome the block.

lntracellular Perfusion by Patch Pipettes

The whole-cell configuration allows modification ofintracellular signaling pathways by excess infusion of intracellular messengers or enzymes, infusion of pharmacological tools, or reconstitution of exogenous proteins) 2 If the agent to be infused into the cell is added directly to the pipette solution, the diffusion starts right after obtaining the whole-cell configuration. Alternatively, a small polyethylene tube placed in the interior of the patch pipette may be filled with the infusate. 13 If pressure is applied to the inner tube, the infusate is released at a defined time during the course of the experiment. The pipette perfusion also allows the repeated exchange of the pipette solution. ~z

The empirically determined times of diffusion are a function of the molecular weights of the infusate and most critically of the geometry of the pipette.X4,15 This is in good agreement with the theoretical calculations applying Fick's law on a simplified geometrical model for the pipette. 12,~6,~7 The concentration increase in the cell is expressed as an exponential process:

c(t) = c0(l - e -t/y) (1)

where c(t) is the time-dependent intracellular concentration and c o is the concentration in the infusate. The time constant ~- depends on the cell volume (V), the diffusion coefficient (D), and a factor (G) that describes the geometry of the pipette:

r = VG/D (2)

t2 j . Hescheler, M. Kameyama, and R. Speicher, in "Practical Electrophysiological Meth- ods" (H. Kettenmann and R. Grantyn, eds.), p. 241. Wiley-Liss, New York, 1992

13 M. Soejima and A. Noma, Pfluegers Arch. 4110, 421 (1984). 14 M. Pusch and E. Neher, Pfluegers Arch. 411, 204 (1987). 15 j. Hescheler, W. Rosenthal, M. Wulfern, M. Tang, M. Yajima, W. Trautwein, and G.

Schultz, Ado. Second Messenger Phosphoprotein Res. 21, 165 (1988). ~6 C. Oliva, I. Cohen, and R. Mathias, Biophys. J. 54, 791 (1988). 17 R. Mathias, I. Cohen, and C. Oliva, Biophys. J. 58, 759 (1990).


The geometrical factor is calculated according to G = d/[Trro(ro + d tan ~b)] as 1.5 × 10 6 m -1 for the standard patch pipette (assuming a diffusion distance d of 100/xm, a radius of the pipette tip r0 of 1.4/xm, and a half- tip cone angle of the pipette ~b of 8°).

The GTP analogs GTP~/S [guanosine 5'-O-(3-thiotriphosphate), 100 to 500/xM] and GMP-PNP (5'-guanosine imidodiphosphate, 100 to 500 ~M) associate to the a subunits of all known G proteins and keep them in the activated form. GDPflS [guanosine 5'-O-(2-thiodiphosphate), 100 t~M] prevents the receptor-induced activation of G proteins. Typically, the analogs are infused via the patch pipette into the cytoplasm. With molecular weights of about 350 and diffusion coefficients D of about 5 x 10-~0 mZ/sec, the time constant ~- is calculated as 100 sec from Eq. (2). Hence, the time until 90% of the maximum concentration is reached in the cytoplasm is determined to be about 4 min, which is in good agreement with the experimental observations. For example, infusion of GTP~/S into a neuronal cell inhibited the voltage-dependent Ca 2+ current within approximately 6 min. ~5 GDPflS needed to be infused for about 5 min to abolish the receptor dependent modulation of Ca z+ currents in PC-12 cells. 18

The pipette infusion technique has also been used to reconstitute highly purified G proteins into cells with endogenous G proteins previously inactivated by pretreatment with pertussis toxin 15 (Fig. 2). Owing to the higher molecular weights of G-protein a subunits (around 40,000) the diffusion time is longer compared to GTP analogs. Typically full reconstitution of hormonal modulations of channels is obtained within approximately 15 min (see Fig. 2). It should be mentioned that the specificity of G-protein o~ subunits in reconstituting modulatory events is concentration-dependent. At high concentrations (10 to 100 nM) all tested G proteins reconstitute the modulatory events, but at low concentrations (0.1 to 10 nM) only specific G proteins are able to reconstitute. It is also possible to activate purified G proteins in vitro by GTP3,S or cholera toxin prior to infusion into cells. The preactivated G proteins cause a modulation of membrane currents similar to receptors.~5 Infusion into cells of affinity-purified functional antibodies toward G-protein a subunits has been used to knock out the hormonal modulation of channels. However, the experimental use of antibodies is rather limited owing to the high molecular weight (approximately 160,000). Because the full diffusion of antibodies needs more than 40 min [according to Eq. (2)] the recorded current must be very stable, which rarely is the case (run-down phenomenon of currents, see Belles

18 M. Gollasch, J. Hescheler, K. Spicher, F.-J. Klinz, G. Schultz, and W. Rosenthal, Am. J. Physiol, 260, C1282 (1991).

3 7 0 IONS AND CHANNELS [ 3 1 ]

I pertussis J toxin

opioid opioid

receptor receptor I I ,

Qo i

calcium channel calcium channel

( i n h i b i t i o n ) (no i n h i b i t i o n )

I ertussis ] QI/Q° toxin opfoid

rece pt o ~ . ~ - - " reconstituted G proteins

calcium channel

( r econs t i t u t ed i nh i b i t i on )

Gi + Go (15 nM each)

nA 0.(

0.~

0.7

DADLE DADLE DADLE

p; ~ t I ~ I 10 15 20 25 30 min

FIG. 2. Reconstitution of purified G proteins after suppression of endogenous G proteins. (Top) Principle of experiments. (Bottom) The voltage-dependent Ca 2+ channel current was repetitively measured in pertussis toxin-pretreated neuroblastoma × glioma (NG108-CC 15 lga) cells during voltage clamp pulses from -80 to 0 inV. Each point in the time course represents the maximal amplitude of the Ca 2+ current. After disruption of the membrane patch (time 0 min), the diffusion of purified G proteins was started. The reconstitution of G proteins was tested by applications of an opioid receptor agonist (D-AlaZ,D-LeuS-enkephalin, DADLE). After about 30 min of infusion of exogenous G proteins a full recovery of the hormonal inhibition of voltage-dependent Ca 2+ channels was achieved (see Hescheler et al. ~5).

et al.19). The usage of antibodies to determine the role of G proteins in the hormonal modulation of channels is more extensively reviewed in [30] in this volume.

19 B. Belles, J. Hescheler, W. Trautwein, K. Blomgren, and J. O. Karlsson, Pfluegers Arch. 412, 554 (1988).

~9a B. Hamprecht, T. Glaser, G. Reiser, E. Bayer, and F. Probst, this series, Vol. 109.

[31] WhOLe-CELL CLAMP ANALYSIS 371

Usage of Caged Compounds

GTP analogs (GTP~,S, GMP-PNP) can be coupled to a photolabile group [1-(2-nitrophenyl)ethyl-3-phosphate ester derivatives] and infused into the cytoplasm with similar diffusion times as described above. A xenon flash lamp (200 W/sec maximal output) is used to induce a light pulse (wavelength 300-370 nm) which releases about 2% of the caged compound. In a study by Dolphin et al. ,2o caged GTPyS was demonstrated to reduce the amplitude of voltage-dependent Ca z+ current in sensory neurons. Such an effect is expected since many receptors reduce the Ca 2+ current via a G protein.

Statistical Approach to Investigate G-Protein-Dependent Channel Modulations

A group of control cells is compared with another group of cells whose G-protein function or expression has previously been altered. The effect of the G-protein alteration is proved by statistical tests. Of course, the test is rather trivial if the effects are large (e.g., in the case of pertussis toxin pretreatment). If the effect is small and/or the variation of effect within a given population of cells is large, however, the number of measurements must be increased considerably to be of significance. Figure 3 demonstrates a typical distribution of modulatory events in a larger ensemble of PC-12 cells (pheochromocytoma). It shows that even the width of the distribution may vary from hormone to hormone, there being a smaller variability of the inhibition of Ca 2+ current by adrenaline (o~z receptors) than by carbachol (presumably M2 receptors). Although the empirically determined distribution of hormonal effects on currents may be fitted by a Gaussian curve, there is no clear theoretical basis to assume that the effects are normally distributed. Therefore, statistical tests for unpaired values not presuming a Gaussian distribution should be applied, for example, the Mann-Whitney-U-Wilcoxon or the Kolmogorov-Smirnov test.

Pretreatment o f Cells with ADP-Ribosylating Bacterial Toxins

Pertussis toxin 2~ consists of six subunits with the glycohydrolase activity being located on the A protomer, which enters mammalian cells only in the presence of the five other subunits (B oligomer). After entry the activated A protomer ADP-ribosylates G proteins of the G~ and G o families

2o A. C. Dolphin, J. F. Wootton, R. H. Scott, and D. R. Trentham, Pfluegers Arch. 411, 628 (1988).

2~ M. Ui, in "ADP-Ribosylating Toxin and G-Proteins" (J. Moss and M. Vaughan, eds.), p. 45. American Society for Microbiology, Washington, D.C., 1990.

372 IOtaS AND CHANNELS [31J

A 20-

B

_~ 15- o

15 ,_ 10-

e - 5-

0 -1 V"I r-'l 1"-7 o ~o 20 so ~o 5o 6o io so

8 15

E -1 C

calcium current inhibition (% of control)

~o

lO

l- lO 20 30 40 50 60

n ~o ~o

calcium current inhibition (% of control)

FIG. 3. Distribution of hormonal effects in PC-12 cells. The voltage-dependent C a 2+

channel current was measured during voltage clamp pulses from -80 to 0 inV. (A) Adrenaline (1/.~M) and (B) carbachol (10/zM) were applied, and the maximal inhibition(given as percentage of control current) was assessed (V. Degtyar and J. Hescheler, unpublished results, 1993).

as well as transducins (see Fig. 1). ADP-ribosylated G proteins are no longer accessible for activation by receptors. Low concentrat ions of pertussis toxin are usually required to achieve a complete ADP-ribosylation of G proteins in cultured ceils. Typically, cells are incubated with the toxin for 24 hr at concentrat ions ranging from l0 to 100 ng/ml; shorter incubation intervals require higher concentrat ions of the toxin. Cells may differ considerably in sensitivity to the toxin; owing to an apparent lack of acceptor sites, erythrocytes and platelets are not sensitive to the toxin


(for review, see Rosenthal e t al.22). There are many reports on a full abolishment of the hormonal modulation of membrane currents by pertussis toxin, including the receptor-mediated stimulation of K + channels in atrial cardiomyocytes, pituitary cells, and insulin-secreting ce l l s , 4 inhibition of Ca 2+ currents in neuronal and nonneuronal secretory cells, and stimulation of Ca 2+ currents in pituitary and adrenocortical ce l l s . 23

Like pertussis toxin, cholera toxin ADP-ribosylates G proteins of the G~ family and G proteins specific for primary sensory cells (Golf, transducins, see Fig. 1). In contrast to the case of pertussis toxin, a complete and specific ADP-ribosylation of Gs o~ subunits by cholera toxin is difficult to achieve, even under optimal conditions. ADP-ribosylation of Gs proteins stay permanently activated. For electrophysiological experiments cholera toxin has been used only in a few cases. The only model where a Gs function on ionic channels is well established is the cardiomyocyte. Activa- tion of Gs stimulates voltage-dependent Ca 2+ currents (in cooperation with a cAMP-dependent phosphorylation) and inhibits Na ÷ currents. 24'25

Usage of Antisense Oligonucleotides to Knock Out G Proteins Specifically

The theory of action of antisense oligonucleotides is extensively described in a contribution elsewhere in this series, z6 Only a few short sequence regions of G-protein a subunits allow for selective hybridization of antisense oligonucleotides; longer antisense oligonucleotides are likely to lack selectivity. Figure 1 highlights some examples ofantisense oligonucleotides used for investigations on the G-protein-dependent modulation of Ca z+ currents in rat pituitary GH 3 cells. 27 It should be emphasized that oligonucleotides could be designed to knock out either a whole family of G proteins (e.g., anti-icom, anti-ocom) or individual proteins (e.g., anti- 01, anti-02). Antisense oligonucleotides were used not only to suppress a subunits of G proteins but also 13 and 3' subun its.28 There was no differ-

22 W. Rosenthal, C. Kleuss, J. Hescheler, B. Wittig, and G. Schultz, "'Methods of Pharmacol- ogy," 7, 141 (1993).

23 G. Schultz, W. Rosenthal, W. Trautwein, and J. Hescheler, Annu. Rev. Physiol. 52, 275 (1990).

24 A. Yatani, J. Codina, Y. Imoto, J. P. Reeves, L. Birnbaumer, and A. M. Brown, Science 238, 1288 (1987).

25 B. Schubert, A. M. J. VanDongen, G. E. Kirsch, and A. M. Brown, Science 245, 516 (1989). 26 C. Kleuss, G. Schultz, and B. Wittig, this series, Vol. 237 [27]. 27 C. Kleuss, J. Hescheler, C. Ewel, W. Rosenthal, G. Schultz, and B. Wittig, Nature

(London) 353, 43 (1991). 28 C. Kleuss, H. Scherilbl, J. Hescheler, G. Schultz, and B. Wittig, Nature (London) 358,

424 (1992).


ence in oligonucleotides hybridizing to the translated or to the untranslated region of the mRNA.

Apparently, the most efficient method is the direct injection of oligonucleotides into the nucleus. Because the antisense oligonucleotide-injected cells need to be incubated for approximately 48 hr to allow degradation of the endogenous G proteins, it was a major technical problem to find the injected cells precisely. Two approaches have been used successfully. In the first, all cells within a defined area of the glass coverslip are injected (about 1000 cells) and the electrophysiological meaurements are made only on cells within this frame. In the second approach, a fluorescent marker is coinjected and the injected cells are recognized with a fluorescence microscope. The latter technique requires controls demonstrating that the marker is without effect on the membrane currents of measured cells. For statistical comparison of effects of various oligonucleotides, usually 20 to 50 cells for each probe are measured. The large number of cells to be measured is certainly one of the major challenges for electro- physiologists.

Transient suppression of G-protein subunits with antisense oligonucleotides was successfully applied to demonstrate that the inhibition of Ca 2÷ currents in pituitary GH 3 cells is mediated by Go. Furthermore, it could be demonstrated that receptors specify for the two splice variants of the G O o~ subunit, aoi and ao2, as well as for the/3 and 3' subunits: whereas muscarinic agonists inhibit Ca 2+ currents via aoi/3374, somatostatin inhib- itis Ca 2+ currents via C¢o2fll'Y3. The effectiveness of antisense oligonucleotides to knock out G proteins specifically was directly monitored by immunofluorescence microscopy using specific peptide antibodies as probes for G proteins. 27

Trituration Method for Intracellular Application of Proteins

Another method for intracellular application of proteins prior to electrophysiological measurements is based on the ability of cells to take up proteins through microlesions. Cells are concentrated by centrifugation to about 1.5 × 107 cells/ml and incubated in a small volume of Ca>-free buffer supplemented with the respective protein (for ras proteins see Hescheler et al.29). Flushing the cells through a small cannula causes microlesions through which the protein enters the cell. The efficiency of this technique was about 90% as assessed by fluorescence microscopy. After resuspension of cells on glass coverslips they are used for electrophysiological examination.

29 j. Hescheler, F.-J. Klinz, G. Schultz, and A. Wittinghofer, Cell. Signalling 3, 127 (1991).

[32] WHOLE-CELL RECORDING 375

Concluding Remarks

Measuring whole-cell membrane currents provides an important tool for the investigation of G-protein-dependent regulatory processes. In contrast to single-channel measurements, the whole-cell current measurements are more reliable (average currents of 1000 to 10,000 channels) and guarantee that the channel current is determined under the cytosolic environment. The close combination of whole-cell current measurements with molecular biological techniques provides an excellent opportunity to study cellular signaling cascades. Besides suppression experiments with antisense oligonucleotides, the overexpression of signaling proteins may gain importance in the future. From the biological point of view it may be rewarding to investigate the more complex signaling pathways, for example, the modulation of currents by differentiation factors working on tyrosine kinases and in a yet unknown manner via small GTP-binding proteins (e.g., ras proteins, see Hescheler et al.29).

[32] W h o l e - C e l l P a t c h R e c o r d i n g in B r a i n Slices

By ROBERT D. BLITZER and EMMANUEL M. LANDAU

Introduction

Until relatively recently, intracellular electrophysiological research in brain slices has relied on sharp electrode techniques, in which cells are impaled by electrodes with tip diameters typically less than 0.1 ~m. There are some important restrictions associated with this method, such as the difficulty in introducing large molecules into the cell and the amount of background noise contributed by high-resistance electrodes.

The adaptation of the whole-cell patch clamp technique to brain slices is a technical innovation which addresses these and other problems. The method employs pipettes with much larger tip diameters (typically > 1 /zm). Rather than impaling a cell, a high-resistance seal (termed a gigaseal) is formed between the pipette tip and the cell membrane, and the region of the membrane within the pipette tip is then disrupted to establish communication with the interior of the cell. The method may be considered an extension of traditional slice methods, and as such it requires only modest expenditure and training in laboratories presently conducting slice research. Whole-cell methods have been used in slices from a variety of

Copyright © 1994 by Academic Press, Inc. METHODS 1N ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved.


rat brain regions, including hippocampus,1 dentate,2 cerebral cortex,3 locus ceruleus, and cerebellum. 4 With proper attention to the composition of the solution used to fill the pipette (and which dialyzes the cell interior), it is possible to record stable G-protein-mediated responses for 1 hr or more. 5

The whole-cell method in the slice has several advantages over the sharp electrode method. First, access to the cell interior is much greater in the whole-cell technique. Relatively large molecules can enter the cytosol by diffusion from the electrode in whole-cell recording, 6 permitting the design of experiments involving the intracellular injection of proteins and peptides. 7 In the sharp electrode technique, protein injection requires that the tip of the electrodes be broken down to a relatively large diameter, 8 resulting in substantial damage to the cell on impalement. Second, the signal-to-noise ratio is higher in the whole-cell method, because of decreased electrode noise associated with the lower access resistance as well as higher leak resistance. This advantage permits the accurate measurement of relatively small signals, such as miniature excitatory postsyn- aptic currents (EPSCs). 9-11 Third, the low-resistance pipettes used in whole-cell recording are capable of passing larger currents than are sharp electrodes, especially depolarizing currents.

Variants o f Whole-Cell Method: Thin and Thick Slices

The whole-cell slice technique has been developed in two variants: thin slice and thick slice (or "blind"). In the thin slice variant, 4 individual cells are visualized and selected. This permits recording from identifiable cell types, ~2 including those which are sparsely distributed, and even particular cell regions, such as soma or proximal dendrites. This variant is more expensive to set up and technically more demanding than the blind variant, 3 for which no special optics are required and the slice is

I T. A. Pitier and B. E. Alger, J. Neurosci. 12, 4122 (1992). 2 B. U. Keller, A. Konnerth, and Y. Taari, J. Physiol. (London) 435, 275 (1991). 3 M. G. Blanton, J. J. LoTurco, and A. R. Kriegstein, J. Neurosci. Methods 30, 203 (1989). 4 F. A. Edwards, A. Konnerth, B. Sakmann, and T. Takahashi, Pfluegers Arch. 414,

600 (1989). 5 L. Zhang, J. L. Weiner, and P. L. Carlen, J. Neurosci. 12, 4510 (1992). 6 M. Pusch and E. Neher, Pfluegers Arch. 411, 204 (1988). 7 M. Alveja and G. K. Aghajanian, Neurosci. Lett. 134, 113 (1991). 8 G.-Y. Hu, O. Hvalby, S. I, Walaas, K. A. Albert, P. Skelflo, P. Andersen, and P.

Greengard, Nature (London) 328, 426 (1987). 9 C. R. Lupica, W. R. Proctor, and T. V. Dunwiddie, J. Neurosci. 12, 3753 (1992).

~0 R. Malinow and R. W. Tsien, Nature (London) 346, 177 (1990). 11 j . M. Bekkers, G. B. Richardson, and C. F. Stevens, Proc. Natl. Acad. Sci. U.S,A. 87,

5359 (1990). 12 p. Sah, S. Hestrin, and R. A. Nicoll, J. Physiol. (London) 430) 605 (1990).


not cleaned prior to the placement of the recording pipette. In this section the variants are briefly described; the use of the blind variant for recording from rat brain slices is presented in greater detail in the next section.

Thin Slice Technique. In the thin slice variant, slices 100-200/xm in thickness are prepared on a Vibratome (Model 1000, Lancer, St. Louis, MO). Prior to recording, the slice is placed in the tissue chamber and observed on an upright microscope with differential interference contrast (or Nomarski) optics. The surface of the slice is teased apart with jets of bath solution from a blunt pipette in order to expose a relatively clean area of membrane on the cell of interest. A recording pipette can then be brought against the membrane in preparation for recording.

Blind Technique. The procedure for the blind method is similar to that used for conventional slice work. Slices 400-500 /xm in thickness are made on a Vibratome or tissue chopper (TC-2, SorvaU, Wilmington, DE). In the recording chamber, the slices are observed with a dissecting microscope, and the recording pipette is brought to the surface of the slice. As the pipette is advanced through the slice, contacts with membranes are signaled by increases in apparent pipette resistance.

Major Equipment Required for Blind Technique

Amplifier: A patch clamp amplifier or more general-purpose voltage clamp amplifier can be used. We use an Axoclamp 2A (Axon Instru- ments, Foster City, CA), which permits clamping in both continuous and discontinuous ("switch clamp") modes.

Micromanipulators: The pipette is advanced into the slice using a motor drive (860A, Newport, Fountain Valley, CA) or a piezoelec- tric stepper (CE-2000, Burleigh, Fishers, NY) held by a manipulator. The essential requirement in the manipulator is that it can support the weight of the drive or stepper without drifting. Inte- grated systems designed specifically for patch clamping are available, though expensive. An economical and adequate alternative (suggested by R. Andrade, St. Louis University School of Medi- cine) is to remove the fine X axis micrometer from a Stoelting MM33 manipulator and replace it with a Newport motor drive.

Oscilloscopes: Digital or storage oscilloscopes are particularly useful for monitoring changes in access resistance as membrane contacts are made while advancing through the slice. If discontinuous voltage clamping will be used, a second oscilloscope is needed. We use a Nicolet 3091, Madison, WI and a Tektronix 5113, Beaverton, OR.

Data acquisition and storage devices: Voltage clamp commands can be issued by a software package with an analog-digital convertor (we use pClamp and TL-1, Axon Instruments) or a programmable


controller (Master-8, AMPI, Jerusalem, Israel). To store relatively slow responses, such as agonist-induced currents, we use a chart recorder (2400S, Gould, Valley View, OH). Three channels are useful: low and high gain current channels and a voltage channel. For fast events, such as voltage-gated currents, and for quantitative analysis of data, acquisition software such as pClamp (with an analog-to-digital converter; TL-100, Axon Instruments) is preferable.

Other major equipment: This includes an air table (preferably with tapped holes), a zoom dissecting microscope on a boom stand, and a computer with a math coprocessor for any acquisition software.

Age of Rats. The method can be used in adult animals; however, recordings are somewhat easier to obtain in immature animals, since gigaseals form more readily than in adults. The data shown in this chapter were obtained using animals 21 to 40 days old.

Preparation and Maintenance of Brain Slices. The methods for preparing and maintaining slices are the same as for conventional recording. Briefly, the animal is anesthetized and the brain rapidly removed and cooled in ice-cold Ringer's solution for l rain. The hippocampus is then dissected out in a cold petri dish, and positioned on the chilled stage of a tissue chopper (alternatively, a Vibratome may be used, and indeed the Vibratome is preferable in the case of small brain nuclei). The slices are transferred to an interface chamber, where they are maintained at the interface between Ringer's solution and a humidified atmosphere of 95% 02/5% CO2 at room temperature. Slices can be kept in this manner for more than 12 hr.

Pipette Fabrication. Pipettes are pulled from 1.5 mm outer diameter nonfilamented thin wall blanks (WPI, Sarasota, FL) using a programmable horizontal puller (P86C, Sutter, San Francisco, CA). The tip outside diameter is 2-3 ~m, resulting when filled in an electrode resistance of 2-4 Mfl. The taper of the electrode is considerably more gradual than that of a classic patch pipette; rather than a bullet shape in which the pipette walls curve in at the tip, the pipette shape is nearly conical from the shoulder to the tip. Each pipette is examined microscopically to ensure that the tip is flat and of the correct diameter. The tips are not fire-polished or treated in any other way after pulling.

Solutions Used to Fill Pipettes. All pipette solutions are filtered with a 0.2-t~m syringe filter. The osmolarity of the solutions is measured and adjusted, because osmolarity influences two crucial properties of the pipettes: ease ofgigaseal formation and propensity to reseal (i.e., to undergo an increase in access resistance during recording). Resealing is a more common problem in whole-cell recording from slices than from cultured or dissociated cells. In general, low ionic strength favors both gigaseal


formation and resealing. Conversely, high ionic strength solutions make gigaseal formation difficult but also discourage resealing. A useful approach to this dilemma is to tipfill with a solution which is hyposmotic (by 10-20 mosM) to the Ringer's, and to backfill with a slightly hyperosmotic solution. Typical values in our laboratory are (in mosM) tipfill (280), backfill (310), and Ringer's solution (295). The composition of the tipfill solution is (in mM) potassium gluconate (128), HEPES (40), MgCI2 (2), and EGTA (0.6), pH 7.30-7.35; no Ca 2+ is added. The backfill solution additionally includes ATP (4), GTP (0.3), phosphocreatine (5), and creatine phosphokinase (50 U/ml).

Recording Procedure. The hippocampal slice is immobilized in a submersion recording chamber between two meshes 13 and constantly superfused with Ringer's solution. The pipettes solutions are kept on ice in the dark, and the pipette is filled immediately before use. The tip is filled by pressure with a syringe needle terminating in a short length of polyethylene tubing (PE 10). The volume of tip filling can be chosen in order to speed or slow the diffusion of back-filled substances to the tip. As the osmolarity at the tip rises owing to diffusion from the high-osmolarity backfill solution, gigaseal formation becomes difficult; filling to a point halfway between the tip and the shoulder of the pipettes permits reliable gigaseal formation for a period of 5-10 rain after pipette filling.

The pipette is mounted in a patch pipette holder (Warner, Hamden, CT), with the suction port attached to a 5-ml glass syringe by polyethylene or silicon tubing. Contact between the headstage input and the pipette solution is made by an AgCl-coated wire which reaches to the shoulder of the pipette; this arrangement allows for minimal pipette filling volumes, helping to limit pipette capacitance. The pipette is advanced into the solution above the slice while gentle pressure is applied with the syringe. With the amplifier in current clamp mode, a periodic current pulse is set up, the amplifier bridge is balanced, and the pipette is advanced toward the surface of the slice. Contact with the surface of the slice is detected by an increase in resistance (conveniently monitored with an audio amplifier). The increased resistance may be due to contact with a cell membrane, but recordings of superficial cells are generally less stable than deeper recordings. In addition, superficial cells are more likely to have been damaged during slice preparation. Therefore, no attempts are made to obtain gigaseals until the pipette has been advanced 10-20/~m deeper.

Before progressing into the slice, the pipette is cleared with a pressure pulse, and the pipette is voltage clamped at 0 inV. An iterative command of +2 mV is given (Fig. la), and the pipette is slowly advanced through

13 R. A. Nicoll and B. E. Alger, J. Neurosci. Methods 4, 153 (1981).

380 Iot~s AND CHANNELS [32]

a b

J u I .......

C

- - ~ J b ° .

J FIo. 1. Steps in the acquisition of a neuron in CAl. (a) Current response to a +2 mV

command before contact with membrane. (b) Response to same command after membrane contact. (c) Current response to a -20 mV command after establishment of gigaseal. (d) Response to same command after breakthrough. Calibrations: (a) and (b), 400 pA/20 msec; (c) and (d), 200 pA/5 msec.

the slice. Apparent membrane contacts are observed as decreases in the current response to the command pulses by about 50 to 75% (Fig. lb). Each putative contact is challenged by a brief, gentle pressure pulse. If the conductance is seen to increase transiently during the pressure pulse and then to decrease again after the pulse, it is likely that contact with a membrane has been made, and an attempt to form a gigaseal is made.

Gigaseals are established by applying suction with the syringe. Good results can be obtained by either a gradual application of gentle suction or a brief pulse of stronger suction. The rapidity with which gigaseals form is variable; while most seals form within a few seconds after the application of suction, adequate seals in some cases require minutes. As a rule, the highest resistance seals are those which form rapidly. The seal resistances obtained range from 3 to 10 GO. In the case of a failure to obtain a gigaseal, additional at tempts may be made with the same pipette, but with a diminished likelihood of success.

As the gigaseal forms (Fig. lc), the command voltage is changed to a value near the expected resting membrane potential (such as - 6 0 mV).


The holding current indicates the seal resistance (e.g., at -60 mV, a current of 60 pA is passed when the seal resistance is 1 Gf~). To rupture the membrane within the pipette tip ("breakthrough"), the 5-ml syringe is replaced by a section of a 1-ml plastic syringe barrel. The holding potential is set at -60 mV, and suction is then applied by mouth while observing the current response to an iterative voltage step. An effective technique is to apply the suction in brief pulses, at first gently and progressing to stronger suction. On breakthrough, there is a dramatic increase in the size and duration of the capacitance currents along with an increase in conductance (Fig. ld).

Problems and Suggested Solutions

Adequacy of Access and Resealing Problem. The completeness of the breakthrough can be quickly determined by switching to current clamp mode and balancing the pipette resistance with the bridge. Generally, full access to the interior of the cell is not obtained on breakthrough (i.e., the breakthrough is partial). The obstruction may be caused by membrane fragments or intracellular constituents. Access can often be improved by applying gentle pressure or suction to the pipette. In some cases, after the obstruction is cleared by a pressure or suction pulse, access remains stable with the pipette vented to atmosphere. However, it is not uncom- mon for access to worsen repeatedly (owing to either resealing or plugging), requiring the frequent (and risky) application of pressure and/or suction. A more reliable solution to the resealing problem, in addition to the use of a slightly hyperosmotic backfill solution (see above), is to apply and occasionally adjust a continuous pressure or suction (in the range of ---1-5 mm Hg). This can be accomplished conveniently with a pressure generator/transducer (DPM-IB, Biotek, Winooski, VT). Even with these measures to improve access resistance, full access is difficult to achieve and maintain. Stable access resistances typically range from 8 to 15 MI).

Series Resistance. The access resistance is in series with the membrane, and any current passing through the pipette induces a voltage across this series resistance. An error in the voltage clamp is thus introduced as current is passed. The contribution of the access resistance to total series resistance can introduce substantial error when whole-cell recording in the slice. This is so because access resistance can be relatively high (in comparison to values obtained in isolated cells) and membrane resistance relatively low (probably reflecting the large membrane surface area associated with extensive neuronal processes). The membrane resistance of a CA1 pyramidal cell immediately after breakthrough is 120-160 MI) and starts to decline soon after breakthrough. After 1 hr of recording, mere-


brane resistance can be as low as 80 MI), so that the error in the voltage clamp associated with uncompensated series resistance might be l0 to 20%.

Two solutions to the series resistance problem are (1) resistance compensation and (2) discontinuous single-electrode voltage clamping (dSEVC). The first approach requires that the command voltage be adjusted to offset the series resistance error14; however, this method is limited to about 80% compensation, above which the clamp circuit is likely to oscillate. Most patch clamp amplifiers incorporate series resistance compensation controls.

Series resistance errors can be eliminated by using dSEVC. In this method, the headstage passes current and senses voltage at different points in a cycle. The voltage is measured a certain time after the end of the current-passing phase, when the voltage across the access resistance (which has a relatively short time constant) has completely discharged but before the membrane voltage has changed substantially. An advantage of dSEVC over the traditional patch method is its relative insensitivity to changes in access resistance because series errors are avoided as long as the pipette voltage fully settles before the voltage is measured. However, it is essential to monitor this settling constantly throughout the experiment. If the time constant of the pipette increases due to worsening access, one has the option of either attempting to improve access or increasing the cycle duration of the dSEVC (lowering the cycle frequency).

The temporal resolution of the clamp in dSEVC is limited by the frequency of the cycling between current-passing and voltage-sensing modes. The shorter the time constant of the pipette, the more quickly the voltage across the access resistance drops, and the more rapidly the circuit can be cycled. Rapid cycling also limits the error associated with the discharge of membrane voltage while waiting for the voltage to discharge across the pipette. Using patch pipettes, dSEVC frequencies of over 5 kHz can be routinely employed. Techniques to limit the time constant of the pipette include using the lowest possible pipette resistance consistent with reliable gigaseal formation (increasing the angle of pipette tip convergence can help), coating the pipette with an insulator, such as Slygard (Dow Corning, Midland, MI), and limiting the submersion of the pipette in the bath by using the lowest possible bath level and advancing the pipette vertically.

Response Rundown. The rundown, or gradual loss, of responses in the whole-cell configuration is well-documented in isolated cells. It may

14 F. J. Sigworth, in "Single-Channel Recording" (B. Sakmann and E. Neher, eds.), p. 29. Plenum, New York and London, 1983.


A

5-HT BACLO

FIG. 2. Whole-cell recordings of agonist-induced responses in CA1 neurons. (A)Outward currents induced by 10/xM serotonin (5-HT) and 10/~M baclofen (BACLO). Holding potential, -60 mV; agonists applied in the bath. (B) Inhibition of 1AHp by norepinephrine. Norepi- nephrine was applied ionophoretically in CA1 (0.2 M in 2 M NaCI, +10 nA for 0.5 sec). The left trace was recorded before norepinephrine application, middle trace 60 sec after norepinephrine, and right trace 20 min after norepinephrine. Holding potential, -50 mV; IaHp was evoked by a command to + 100 mV for 200 msec (trace truncated in the records). Calibrations: (A) 100 pA/l min; (B) 100 pA/l sec.

reflect the loss through dialysis of energy sources, second messenger components, or cofactors necessary to normal physiology. Thus, to prevent or at least slow the process of rundown, substances thought to play these roles have commonly been added to the pipette solution. In our experience, the inclusion of GTP 15 and an ATP-regenerating system are crucial in obtaining stable second messenger-mediated responses. Figure 2 illustrates such responses in pyramidal cells of rat hippocampus. Both the serotonin (5HTI~)- and 3,-aminobutyric acid (GABAb)-mediated outward current (Fig. 2A) and the inhibition of the CaZ+-dependent K ÷ current (IAH P) by norepinephrine (Fig. 2B) require intracellular Ca z+ and the activation of a G protein. These responses are stable for at least 1 hr under the conditions employed.

Junction Potentials. When the patch pipette is immersed in the bath solution, an electrical potential develops as a result of the differences in the compositions of the pipette solution and the bath solution, z6 This junction potential can be different after breakthrough than when the pipette is immersed in the bath. Because the pipette potential is zeroed in the bath, a change in junction potential on breakthrough will produce erroneous

~5 L. O. Trussel and M. B. Jackson, J. Neurosci. 7, 3306 (1987). 16 p. H. Barry and J. W. Lynch, J. Membr. Biol. 121, 101 (1991).


membrane potential measurements. In addition, the junction potential may change with time as the cell interior is dialyzed.~7

To get an independent indication of the true membrane potential, one can use voltage-sensitive membrane properties as an assay. For example, Iq is an anomalous rectifier which activates negative to about -90 mV. To estimate the true membrane potential, one can apply increasingly negative current commands from the resting potential until 1Q is just activated. The membrane potential produced by this command is considered to be -90 mV, and the amplifier offset is then adjusted as appropriate. Other possible membrane characteristics that may be used as indicators of membrane potential are spike amplitude, firing threshold, and the reversal potential of an agonist-induced K + current.

Inclusion of Proteins in Pipette. For studies of signal transduction, the ability to inject proteins into neurons is a great advantage of the whole- cell technique. However, the presence of proteins in the pipette tends to discourage gigaseal formation. Therefore, proteins (including the phosphocreatine kinase routinely used) should be added only in the backfill solution. Lipophilic proteins, such as holo-G proteins and fly subunits, pose a separate problem: the necessity of including a detergent in the solution. If the detergent concentration is low enough, no obvious adverse effect on membrane characteristics are seen (e.g., 0.01% cholate seems to be tolerated). However, higher detergent concentrations can produce a rapid and profound drop in membrane resistance.

Acknowledgments

This work was supported by Veterans Administration Merit Grant 7805-003 and a NARSAD Young Investigator award to R.D.B.

17 A. Marry and E. Neher, in "Single-Channel Recording" (B. Sakmann and E. Neher, eds.), p. 115. Plenum, New York and London, 1983.

[ 3 3 ] RECONSTITUTING CHANNELS IN MEMBRANE PATCHES 385

[33] Recons t i tu t ion of R e c e p t o r - R e g u l a t e d Ion Channels in Isolated Pa tch M e m b r a n e

By ATSUKO YATAN1

Introduction

The involvement of G proteins in the coupling process of membrane receptors to ion channels such as the cardiac muscarinic receptor-activated K ÷ channel (K ÷ [ACh]) and the Ca 2 ÷ channels has been reported.l-4 Most of the experiments describing such regulation have employed the whole-cell configuration of the patch clamp technique 5 to test the effects ofintracellularly applied GTP or GTP analogs that alter G-protein function. Ion channel activity can be modified by direct interaction of the channel with an activated G protein or with a cytoplasmic second messenger formed by interaction of an activated G protein with membrane-associated enzymes. To identify which G protein(s) mediates the muscarinic response and to examine how a G protein interacts with an ion channel, we have measured single-channel currents that give less ambiguous results than whole-cell currents. 6

The basic approach used to assay G-protein effects is the cell-free, inside-out patch recording. In this configuration, G proteins are applied to the cytoplasmic side of the membrane. Such cell-free systems can be used to demonstrate the direct effects of the G protein on channels. One major problem associated with inside-out patch recording is that vesicle formation at the pipette tip 5 causes false-negative effects. Thus, in addition to the standard patch clamp procedure, it is important to test GTP effects before applying the test proteins and again at the end of experiments to test the GTP or GTPTS effects to exclude sealed or partially sealed patches. G-protein effects can be tested on all types of ion channels; however, ion channels that produce spontaneous changes in activity after formation of

1 p. j. Pfaffinger, J. M. Martin, D. D. Hunter, N. M. Nathanson, and B. Hille, Nature (London) 317, 536 (1985),

2 G. E. Breitwieser and G. Szabo, Nature (London) 317, 538 (1985). 3 y . Kurachi, T. Nakajima, and T. Sugimoto, Pfluegers Arch. 407, 264 (1986). 4 L. Birnbaumer, J. Codina, R. Mattera, A. Yatani, N. Scherer, M. J. Toro, and A. M.

Brown, Kidney Int. 32, S-14 (1987). O. P. Hamill, A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981).

6 A. Yatani, J. Codina, A. M. Brown, and L. Birnbaumer, Science 235, 207 (1987).

Copyright © 1994 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 238 All rights of reproduction in any form reserved,


an inside-out patch, that is, cardiac L-type C a 2+ channels, 7,8 are more difficult to study. However, this is not a problem for K+[ACh], and there is practically no change in channel activity during inside-out recordings. Techniques to measure the activation of K + [ACh] currents by G proteins are described in this chapter.

Methods

Preparation of Cells

Clean plasma membrane, free of connective tissue, is a prerequisite for the formation of a gigaseal between the patch pipette and the cell membrane. Many types of primary cultured cells can be used for these assays. In our laboratory, we mainly use acutely dissociated adult guinea pig atrial cells isolated by a modified procedure originally described by Taniguchi et al. 9

An adult guinea pig (400-500 g) is anesthetized with pentobarbital (50 mg/kg) and the heart is removed, attached to the base of a Langendorff column, and perfused with Tyrode's solution followed by nominally Ca z +- free Krebs solution by retrograde coronary perfusion. For enzymatic digestion we use 0.04% w/v collagenase type I (-200 units/mg; Worthington, Freehold, N J) and I0 mg/ml bovine serum albumin (BSA, essentially fatty acid free; Sigma, St. Louis, MO) added to the Ca2+-free Krebs solution. After 25-30 min of digestion, the enzyme solution is washed with high K+/low CI- solution. 1° The cells are kept at room temperature and are used the same day they are isolated. All solutions are oxygenated and prewarmed to 37 °.

Solutions for Cell Preparation

Tyrode's solution (mM): NaC1, 135; KCI, 5.4; CaCI2, 1.8; MgC12, 1.0; HEPES, 5; and glucose, 1.8 (pH 7.4 with NaOH)

Ca2+-free Krebs solution (raM): NaC1, 110; KC1, 2.6; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3,25; glucose, 11.1 (pH 7.4 with a mixture of v/v 95% 02 and 5% COz)

High K+/low CI + storage solution (mM): KOH, 70; e-glutamic acid, 50; KCI, 40; taurine, 20; KHzPO4, 20; MgCI2, 3; glucose, I0; HEPES, 10; EGTA, 0.5 (pH 7.4 with KOH)

7 A. Cavalie, R. Ochi, D. Pelzer, and W. Trautwein, Pfluegers Arch. 398, 284 (1983). 8 A, Yatani, J. Codina, Y. Imoto, J, P. Reeves, L. Birnbaumer, and A. M. Brown, Science

238, 1288 (1987). 9 j. Taniguchi, S. Kokubun, A. Noma, and H. Irisawa, Jpn. J. Physiol. 31, 547 (1981). 10 K. Yazawa, M. Kaibara, M. Ohara, and M. Kameyama, Jpn. J. Physiol. 40, 157 (1990).


Electrophysiological Recordings

Single-channel current measurements are made with the gigaseal patch clamp method involving cell-attached and inside-out configurations as described by Hamill et al.,5 with a List EPC7 patch clamp amplifier (Medi- cal System Co., Greenvale, NY). All experiments are carried out at room temperature (20°-22°).

Pipette Glass. Glass patch pipettes are fabricated from either thick- walled, hard glass capillaries (Type 7052), or thin-walled, soft glass capillaries (Type 8161) from Garner Glass (Claremont, CA). A hard glass pipette has less background noise from capacitance across the glass wall, but the success rate of obtaining gigohm seals is 20-30% less than that of soft glass pipettes. Results obtained with either type of glass are indistin- guishable. The background noise can be reduced by coating the pipette with Sylgard 182 (Dow Corning Co., Midland, MI) up to a distance about 200/xm from the tip.

Pipettes are pulled with a two-stage microelectrode puller (Model pp- 83, Narishige, Japan) and are fire-polished to a final tip diameter of 0.5-1.0 /xm by using a microforge (Model MF-83, Narishige). When filled with pipette solutions, the pipettes have resistances of 5-I0 Mfl. Gigohm seals between the pipette and cells are obtained by applying gentle suction, and seal resistances range between 20 and 100 GO.

Solutions. The standard patch solution contains the following (in mM): KCI, 140; MgCI 2 , 2; EGTA, 5; HEPES, 5 (pH 7.3 with Tris base). Musca- rinic agonists, carbachol (1-10/xM) or acetylcholine (0.1-1/zM), can be added to the pipette solution to detect the presence of K+[ACh] in the patch. Bath solution (internal solution for inside-out patch recording), which is the same as patch solution, is used to depolarize the cell membrane potential to approximately 0 mV. For Mg 2÷ experiments, EGTA is replaced by EDTA, and divalent cation concentrations are calculated with disassociation constant for EDTA. All chemicals are analytical grade. Nucleotides are from Boehringer Mannheim (Indianapolis, IN).

Data Acquisition and Analysis

Single-channel currents are stored on video cassette tape (PCM- 1 Medi- cal System Co.) for subsequent analysis. The currents are displayed on a paper recorder for direct analysis or low-pass filtered with a four-pole Bessel filter at 1-2 KHz and digitized between 5 and 10 KHz on an IBM PC/AT.

Transitions between closed and open levels are detected using an interactive threshold detection program, which finds the half-maximum amplitude of single unit openings, and are converted to idealized events.


To quantify the effects of test agents, we average the probability of the channel being open, P, for N channels in a membrane patch, NP, for a period of generally 20-30 sec after channel activity reaches a steady level or at a fixed time after applying the test agents. The relative activities are normalized to NP obtained by GTP or GTPyS. The rates at which the agents produce the effects are estimated from the time course of NP or by integrating NP continuously to give cumulative NP and measuring the slope as described previously. 1~-~3

Assay for G-Protein Effects

Application Method

Test agents including G proteins can be applied either by perfusion into the recording chamber or by direct addition by a micropipette to test stimulatory effects on quiescent channels or inhibitory effects on GTP- or GTP analog-activated channels. However, with the second procedure, the effects are limited by the diffusion of the agents in the bath solution. To examine the precise time course of the effects, and since the supply of G proteins are generally limited, we have used the concentration-clamp method TM with which rapid solution exchange can be produced in small volumes (200-400/~1) as described previously. 14,15

Briefly, a tapered polyethylene tube serves as the host chamber for different solutions. The tip of the patch pipette, containing an excised patch of membrane, is inserted into the host chamber through a circular hole approximately 1 mm in diameter. The lower end of the host chamber has an effective volume of 200-400/A and is exposed to different test solutions by rotating the stage on which other chambers containing different test solutions are located. The solutions are exchanged when a negative pressure of about - 15 cm Hg is applied to the upper end of the host chamber via a miniature Teflon solenoid valve (General Valve, series 2), driven for controlled durations by a stimulator (Type $44, Grass Instru- ment Co., Quincy, MA).

Figure 1A shows the speed of solution exchange estimated from the time required for the change in holding current at 0 mV under inside-out patch recording, during exchange of intracellular K + concentration from

11 A. Yatani, K. Okabe, L. Birnbaumer, and A. M. Brown, Am. J. Physiol. 258, H1507 (1990). 12 K. Okabe, A. Yatani, T. Evans, Y. K. Ho, J. Codina, L. Birnbaumer, and A. M. Brown,

J. Biol. Chem. 265, 12854 (1990). 13 K. Okabe, A. Yatani, and A. M. Brown, J. Gen. Physiol. 97, 1279 (1991). 14 N. Akaike, M. Inoue, and O. A. Krishtal, J. Physiol. (London) 379, 171 (1986). 15 A. Yatani and A. M. Brown, Science 245, 71 (1989).

[33] RECONSTITUTING CHANNELS IN MEMBRANE PATCHES 389

A Valve open Valve open

KCI ~1 KCI // ; I ~ KCI 1 4 0 m M ~ ~ 140raM 28raM

200msec

B vGTP W FGTP ..

/i I OpA /// ~----GTPIOpM " ' - - . . 20see

200msee FIG. 1. Concentration-clamp method for rapid exchange of solutions. (A) System response

time measured by the shift of the current at a holding potential of 0 mV during inside-out patch recordings on guinea pig atrial cells. The current shifted 50 msec after a change in the intracellular K + concentration from 140 to 28 mM (system dead time) to a new level within 10 msec (system response time). (B) Time course of GTP activation of guinea pig atrial K+[ACh] currents recorded in the inside-out patch configuration. A representative current record of the GTP (10/zM) activation and deactivation time course after washing (W) was made. The patch solution contained carbachol at a concentration of 10 p,M. The recording potential was - 80 mV, and downward deflections denote channel openings. Single- channel currents at fast time resolution are also shown at bottom.

140 to 28 raM. After a delay of about 50 msec (time required for solution moved as a bolus), the holding current changes within 10 msec. Because G-protein and GTP-analog effects have a much slower time course, these measurements should reflect the true times for the effects to occur. Using this method, in every experiment, we examine GTP effects to evaluate access of bath-applied agents to the patch before applying the test agents.

Figure 1B shows a typical example of the effect of GTP on K+[ACh] currents. In the presence of 10 /xM carbachol, the rate and extent of activation are dependent on the concentration of GTP, and removal of GTP from the bath inactivates K+[ACh] within 20 sec; thereafter the patch should be completely silent. Magnesium ion is an important cofactor for GTP activation, and half-maximum activation (Kd) by Mg 2+ is 3 /xM) 1 This test for GTP dependency of channel activation can be repeated without change in response) 3 Generally, we accept for evaluation those


patches in which the activation by GTP has a half-time (tl/2) of less than 3 sec and the tt/2 of deactivation is less than 20 sec.

Figure 2 shows an example of the effects of different concentrations of GTP analogs (GTPyS, GMP-PNP, and GMP-PCP) on the rate of activation measured by this method. The muscarinic agonist carbachol (10/zM) was present throughout experiments. At lower concentrations of GTP analogs, K + [ACh] began to open after an initial delay and reached a steady-state level over the next several minutes. The rate of activation was concentration-dependent, and GTPyS was most effective among the three analogs tested. In all cases Mg 2÷ is an absolute requirement for channel activation as in the case of GTP. However, in contrast to the channel activation by GTP, GTP-analog activation persists after removal ofMg 2 +. The maximum rate of activation obtained with GTP analogs, which produce irreversible reaction, was slower than that obtained by GTP of about 50/min.13 To test how receptors regulate K + [ACh] activation rates, similar experiments

Aa ~ GTP7 s

lOOnM

4 ~'P~I ] ~ ±ROan I I I I I'll, T ,'r~'1~',~rr~'~v~r!F~,~r,,~l~ll~lp, II~,

~ IO01JM

- G M P - P N P 5IJM ~ 501JM

" ~ 'lq' I q rl n ql~'"~l, ",'1-.-T-.r T ~ ii.t ,-rr,'-p.~.lTr,,~-~ t T 5pA

2 0 s e c

B

6

"-,= 2

< o

0 .001 i i i i

o .01 o.1 1 lO l o o

GTP analog concentrations (~M)

FIG. 2. Effects of different concentrations of GTP analogs on rate of K+[ACh] activation. (A) Example of activation by GTPyS (a) and GMP-PNP (b). Single-channel currents at - 8 0 mV were recorded in the inside-out configuration. Carbachol (10 tzM) was present. (B) Concentratlon-dependent activation rates. Data are means of 4-8 experiments. 0 , GTPyS, ©, GMP-PNP; /x, GMP-PCP.


were repeated in the absence of carbachol. Without receptor activation, the concentration-dependent activation curves for GTP analogs shifted to higher concentrations by about 10-fold (not shown). The observations indicate that activated receptors promote the GTP-GDP exchange rate of G proteins and produce faster activation of K+[ACh] as seen in other signal-transduction pathways.

Effects o f Buffer Solutions on Receptor-Activated Potassium Channel

As control experiments, we have tested the effects of reagents commonly used for G-protein buffers, individually or as buffer solution, on K+[ACh]. The G-protein buffers used in the following experiments (Gk, Yatani et al.6; Gs, Codina et ai.16; c~ k, Codina et al.17; recombinant (ak, Mattera et al.18) when diluted 100-fold in bath solution (assuming G-protein stock concentration is 100/xg/ml and G-protein concentration is in the 20 nM range), had no effects on the channel. A G-protein concentration higher than 200 nM often caused patch breakdown.

The nonionic detergent Lubrol PX inhibited K + [ACh] currents activated by GTP, in either the presence or absence of carbachol in the pipette solution, at concentrations higher than 5/zM. 12 On the other hand, in our experiments the zwitterionic detergent CHAPS activated K+[ACh] currents at a concentration as low as 1 t~M, and the effects were concentration-dependent. 11 At a concentration of 100/xM, the value of activity was about 50% of that obtained by GTPyS. Concentrations higher than 300 /zM often caused patch breakdown. Other reagents individually tested included ethanol (concentrations higher than 0.2% caused inhibitory effects); bovine serum albumin (up to 0.02%), dithiothreitol (DTT, up to 1 mM), and glycerol (up to 0,4%) had no effects on K + [ACh] currents.

Uncoupling of Endogenous G Protein by Pertussis Toxin and Activation of Potassium Channel by Activated G Proteins

Figure 3A shows that in the presence of NAD + (1 raM), activated pertussis toxin (PTX), which prevents receptor interactions with particular G proteins, blocked K + [ACh] currents. The PTX effect was concentration dependent between 0.4 and 40/zg/ml. Generally complete block was obtained within 20 rain after application of PTX plus NAD + . The PTX- induced block was due to reduced frequency of channel opening; neither

t6 j. Codnia, J. D. Hildebrandt, R. D. Sekura, M. Birnbaumer, J. Bryan, C. R. Manclark, R. Iyengar, and L. Birnbaumer, J. Biol. Chem. 259, 5871 (1984).

17 j. Codina, A. Yatani, D. Grenet, A. M. Brown, and L. Birnbaumer, Science 236, 442 (1987). 18 R. Mattera, A. Yatani, G. E. Kirsch, R. Graf, K. Okabe, J. Olate, J. Codina, A. M.

Brown, and L. Birnbaumer, J. Biol. Chem. 264, 465 (1989).


Carb FGTP w r GTP W r

A IOOpM fPTX+NAD ~ IOONM l ,mM ] GTP'/S

C-A I-o

IOarb i ~ ; GTP'~mM " 2rim GTP PTX + NAD r-G1 ( 100~M 5" 5' ~'3" 1 5 A' ~5 pA

l O s

C C-A I-O f GK* 50,.2 pM 25,PM 203,PM 50pM 2' ~ ' " ' '~ ~ . . . . . ' - ~ 'm,, ,r ,r~l,~,~' ~

;5pM 5pM 50pM 5' 20" D

F 'mrl~" - ~ ~P"~"'rn~ll'nl~ 11~'""l'l'"r~,/,/ ~ r "~t L~i"~cr~m

r Carb r GTP- free ctrl r ec°mbct*'-3

FIG. 3. Reactivation of K+[ACh] by GTP-yS (A) and unactivated Gk (B) in the presence of 1 n ~ GTP, after uncoupling of endogenous 13 protein by activated PTX plus NAD ÷ at concentrations of 10 ~gtml and 1 raM, respectively. Activatiun of K÷[ACh] currents by hRBC Gk (C), hRBC ~ (D), and recombinant ~ (E). In (12), (b), and (E), G proteins were preactivated with GTPTS, as denoted by asterisks. Single K + channel currents were recorded in the cell-attached (C-A) and inside-out (I-O) patch configurations as noted. Carbachol (10 v.M) was present in the patch solution throughout in (A), (B), and (E). The recording potential was -80 mV. Numbers above records denote time elapsed in minutes (') or seconds (") between solution change and the beginning of the record.

the current amplitude nor open times were altered. After PTX treatment GTP, even at a concentration of 1 raM, could not activate the channel, but GTPyS was able to activate the channel. The PTX block was also overcome by nonactivated PTX-sensitive human erythrocyte (hRBC) G i (because of its effects on K+[ACh], we refer to this protein as G~) at concentrations of 2 nM, in the presence of 1 mM GTP 19 (Fig. 3B).

To test if exogenous purified G protein mimics muscarinic receptor effects on K+[ACh], we have tested G proteins preactivated by GTPTS (denoted by asterisk). Figure 3C shows that hRBC Gk* activated K + [ACh]

r9 A. Yatani, J. Codina, and A. M. Brown, in "G Proteins" (R. Iyengar and L. Birnbaumer, eds.), p. 241. Academic Press, San Diego, 1990.

[33] RECONSTITUTING CHANNELS IN MEMBRANE PATCHES 393

in the absence of agonist. Gk* was effective at picomolar concentrations, and maximum effects were obtained with 50-100 pM. Preactivated t~ subunit of Gk, Otk*, was as effective as holo-Gk ~7 (Fig. 3D). For exogenous G-protein effects Mg 2+ was not required, and the activation was irreversible. The Gk*- or ~k*-activated current had channel properties (i.e., open time and conductance) identical to those of muscarinic agonist plus GTP- activated channels. The reconstitution experiments suggest that the receptor-G protein-K+[ACh] complex is not continuously associated but loosely coupled, since exogenous G protein could replace the natural endogenous coupling G protein. Neither preactivated Gs* nor its ct subunit as* was effective in K+[ACh] activation. Activated bovine brain Go* showed weak activation. 6

Amino acid sequence analysis showed that hRBC o~ k encoded the oq3 gene, and the recombinant ai3 was expressed in Escherichia coli cells using plasmid pT7-7 and tested for activityJ 6 As shown in Fig. 3E, even though the recombinant protein had about 50-fold lower potency than purified G protein, it was effective in activating K + [ACh] currents. Subse- quently, we found that purified native bovine brain Otil and hRBC oq2 as well as recombinant ~il and oq2 are equally active as ai3 j9 In contrast,/33, dimers purified from several sources including human erythrocytes, human placenta, bovine brain, and bovine rod transducin inhibited K+[ACh] currents produced by GTP or carbachol plus GTP in a concentration- dependent mannerJ 2

Based on our data we have proposed that o~ subunits of G proteins are the physiological activator of K + [ACh], and the mechanism of interaction was termed direct, resembling adenylate cyclase and rod cell phosphodiesterase regulation. However, the possibility that membrane-delimited lipid- soluble local second messenger or an intermediary membrane protein modulates the channel activation has not been critically tested.

Conclus ion

Inside-out patch recording is a useful assay method to test the effects of G proteins in receptor-mediated ion channel function. As described above, studies from our and other laboratories have shown that several G proteins, such as Gil, Gi2, and Gi3 (possibly Go) activate K+[ACh] currents. 2°,z~ It is possible that various G proteins interact with a single effector and have little specificity in effector regulation; however, the

z0 A. Yatani, R. Mattera, J. Codina, R. Graf, K. Okabe, E. Padrell, R. lyengar, A. M. Brown, and L. Birnbaumer, Nature (London) 336, 680 (1988).

2E D. E. Logothetis, Y. Kurachi, J. Galper, E. J. Neer, and D. E. Clapham, Proc. Natl. Acad. Sci. U.S.A. 85, 5814 (1988).


assay bypasses interactions between receptors and G proteins. Our preliminary data show that affinity-purified antibodies to COOH-terminal decapeptides specific to ai3 (Gai3 antibody) blocked muscarinic receptor-mediated activation more effectively than all- and ai2-specific antibodies. 22 Further studies on the coupling processes between receptors and G pro- reins 23,24 need to be made to differentiate functional specificity in physiological cellular responses to external signals.

Acknowledgments

I am grateful to Drs. L. Birnbaumer and J. Codina for introducing us to the G-protein- coupled receptor projects, and for the gift of G proteins. I thank Dr. R. Iyenger for helpful comments.

22 W. F. Simond, P. K. Goldsmith, J. Codina, C. G. Unson, and A. M. Spiegel, Proc. Natl. Acad. Sci. U.S.A. 86, 7809 (1989).

23 C. Kleuss, J. Hescheler, C. Ewel, W. Rosenthal, G. Schultz, and B. Wittig, Nature (London) 353, 43 (1991).

~4 R. Taussig, S. Sanchez, M. Rifo, G. Gilman, and F. Belardetti, Neuron 8, 799 (1992).

[34] R e g u l a t i o n o f P o t a s s i u m C h a n n e l s b y G - P r o t e i n

S u b u n i t s a n d A r a c h i d o n i c Acid M e t a b o l i t e s

By MITSUHIKO YAMADA, ANDRE TERZIC, and YOSHIHISA KURACHI

G-Protein Activation of Cardiac Muscarinic Potassium Channel

GTP-binding (G) proteins transmit the signal from receptors containing seven transmembrane segments to intracellular effectors. Agonist-bound receptors activate heterotrimeric (aflT) G proteins by catalyzing the replacement by GTP of GDP bound to the a subunit, resulting in the functional dissociation of G proteins into GTP-bound a (Ga-GTP) and fly(G/3y) subunits. 1-3 Both subunits, Ga and G/3y, could potentially activate effectors. Because of the smaller molecular heterogeneity of Gfly in various G proteins, it has been thought that only Ga-GTP mediates signals to effectors. However, specific roles of G97, other than the binding to Ga-GDP,

I L. Stryer and H. R. Bourne, Annu. Rev. Cell Biol. 2, 39l (1986). 2 A. G. Gilman, Annu. Rev. Biochem. 56, 615 (1987). 3 L. Birnbaumer, J. Abramowitz, and A. M. Brown, Biochem. Biophys. Acta 1031, 163

(1990).


[34] G PROTEIN, ARACHIDONIC ACID, AND K ÷ CHANNEL 395

have been demonstrated in various systems and include modulation of channels, phospholipase A2 (PLA2), adenylyl cyclase, pheromone action in yeast, and/3-adrenergic receptor kinase. 4-1°

Several plasma membrane ion channels can be directly regulated by G proteins without the mandatory participation of cytosolic second messengers. This type of G-protein regulation of ion channels has been defined as "membrane-delimited." The archetype in this class of channels is a specific, inwardly rectifying K+-selective channel named the muscarinic K + (KAch) channel. The KACh channel is present in cardiac atrial and nodal cells and is coupled to the m2-muscarinic and Al-adenosine receptors through pertussis toxin (PTX)-sensitive G proteins (GK).

To indicate an involvement ofa G protein it is necessary to demonstrate first the GTP dependence of the agonist action. Indeed, acetylcholine (ACh) induces an inwardly rectifying K + current in whole-ceU voltage- clamped atrial cells only where GTP or nonhydrolyzable GTP analogs were included in the patch pipette, iL~2 To establish the concept that G proteins directly (i.e., without the required participation of a cytosolic component) activate the KACh channel, it was necessary to demonstrate that the channel can be activated in cell-free inside-out patches of atrial cell membranes by GTP (in the presence of agonists in the patch pipette), T M

nonhydrolyzable analogs 14,~5 or purified or recombinant G protein subunits (in the absence of agonists ) . 4'16-19

4 D. E. Logothetis, Y. Kurachi, J. Galper, E. J. Neer, and D. E. Clapham, Nature (London) 325, 321 (1987).

5 C. L. Jelsema and J. Axelrod, Proc. Natl. Acad. Sci. U.S.A. 84, 3623 (1987). 6 T. Katada, K. Kusakabe, M. Oinuma, and M. Ui, J. Biol. Chem. 262, 11897 (1987). 7 M. Whiteway, L. Hougan, D. Dignard, D. Y. Thomas, L. Bell, G. C. Saari, F, J. Grant,

P. O'Hara, and V. L. MacKay, Cell (Cambridge, Mass.) 56, 467 (1989). 8 W.-J. Tang and A. G. Gilman, Science 254, 1500 (1991). 9 A. D. Federman, B. R. Conklin, K. A. Schrader, R. R. Reed, and H. R. Bourne, Nature

(London) 356, 159 (1992). to j. A. Pitcher, J. Inglese, J. B. Higgins, J. L. Arriza, P. J. Casey, C. Kim, J. L. Benovic,

M. M. Kwatra, M. G. Caron, and R. J. Lefkowitz, Science 257, 1264 (1992). it p. j. Pfaffinger, J. M. Martin, D. D. Hunter, N. M. Nathanson, and B. Hille, Nature

(London) 317, 536 (1985). 12 G. E. Breitwieser and G. Szabo, Nature (London) 317, 538 (1985). 13 y . Kurachi, T. Nakajima, and T. Sugimoto, Am. J. PhysioL 251, H681 (1986). 14 y . Kurachi, T. Nakajima, and T. Sugimoto, Pfluegers Arch. 407, 264 (1986). 15 y . Kurachi, T. Nakajima, and T. Sugimoto, Pfluegers Arch. 407, 572 (1986). 16 A. Yatani, J. Codina, A. M. Brown, and L. Birnbaumer, Science 235, 207 (1987). ~7 A. Yatani, R. Mattera, J. Codina, R. Graf, K. Okabe, E. Padrell, R. Iyengar, A. M.

Brown, and L. Birnbaumer, Nature (London) 336, 680 (1988). 18 A. Yatani, this volume [33]. 19 y, Kurachi, H. Ito, T. Sugimoto, T. Katada, and M. Ui, Pfluegers Arch. 413, 325 (1989).


A number of other receptors have been reported to be coupled to various ion channels directly by a G protein, z°,21 implying that direct G-protein regulation of ion channel function is a general cell signaling mechanism. It should be noted that in this context "direct" is used for "membrane-delimited," since it is possible that additional membrane components are interposed between the G protein subunits and the ionic channel.

Because both Ga and Gfly could conceivably be the active G-protein subunits responsible for the regulation of an effector, the question emerges as to how to establish experimentally which specific subunit(s) activates a channel. This is the topic of the first part of this chapter. Thus far, the strategy primarily employed to identify the G-protein subunit involved in the regulation of ion channel function has been to apply to inside-out cell membrane patches (or to lipid bilayers containing channels) purified and recombinant G-protein subunits. Other approaches have also been employed and include the transfection of antisense oligonucleotides into intact cells in order to suppress the expression of specific G-protein subunits. 22 It has been reported that activated Ga subunits regulate all the channels so far investigated with the notable exception of the KAc h channel, which could be regulated by both Ga and G[3y subunits.

Methods Used to Study Regulation of Cardiac Muscarinic Potassium Channel by G-Protein Subunits

Preparation of Cardiac Cells and Perfusion System

Single atrial and ventricular cells of guinea pig hearts can be obtained by an enzymatic dissociation method. 23'24 In guinea pigs (200-300 g), anesthetized with pentobarbital and artificially ventilated, the aorta is rapidly cannulated, and the heart is retrogradely perfused through the coronary arteries at a constant temperature (37°). The heart is perfused with the following sequence of solutions (for composition, see below): first, control bathing solution (Tyrode's) for 5-10 min, then a nominally Ca2+-free Tyrode's solution for 5 min, followed by a nominally Ca 2+- free solution containing collagenase (0.04%, w/v, Sigma type I; Sigma Chemical Co., St. Louis, MO) for 25 min, and finally a high-K + , low-Cl-

20 A. R. North, J. T. Williams, A. Suprenant, and M. J. Christie, Proc. Natl. Acad. Sci. U.S.A. 114, 5487 (1987).

21 A. M. Brown and L. Birnbaumer, Annu. Rev. Physiol. 52, 197 (1990). 22 K.-L. Laugwitz, K. Spicher, G. Schultz, and S. Offermanns, this series, Vol. 237 [22]. 23 G. Isenberg and U. Klockner, Pfluegers Arch. 395, 6 (1982). 24 y. Kurachi, Pfluegers Arch. 394, 264 (1982).

[34] G PROTEIN, ARACHIDONIC ACID, AND K + CHANNEL 397

solution for 5 min. The heart is then stored in the latter solution at 4 ° for same-day use. A small piece of cardiac tissue is dissected and gently agitated in a narrow recording chamber.

Myocytes are permitted to adhere to the glass floor of the chamber and then superfused at a constant flow rate ( -5 -7 ml/min). To prevent surface absorption of hydrophobic substances all glass surface in the perfusion systems should be siliconized, and the tubing should be made of Teflon. Although Teflon is the material of choice, polyethylene tubings can be also used. In addition, to further prevent binding of hydrophobic substances to the glass walls or tubing, a solution of fatty-acid free albumin (0.3% w/v) may be perfused through the system before the experiment. Coating of the glass floor of the experimental chamber with silicone usually makes it difficult for isolated cells to adhere to the bottom of the chamber. Hence, cells should be seeded on uncoated coverslips beforehand and then transferred to the assay chamber on the coverslip just prior to the experiment. The bath temperature is maintained at room temperature (22 ° ) or at 37 ° (to within 2°). Quiescent relaxed atrial or ventricular cells with a smooth surface and clear striations are used for experiments.

Electrophysiological Recordings and Quantification of Channel Activity

The patch clamp technique, and more specifically its inside-out variant (which allows free access to the cytosolic side of the plasma membrane), is employed for electrophysiological recordings ?5 A patch clamp amplifier (e.g., EPC-7, List Medical, Darmstadt, Germany; Axopatch-Ic, Axon Instruments, Foster City, CA) is used to measure single-channel currents, which can be monitored on-line on a high-gain digital storage oscilloscope (e.g., VC-6025, Hitachi, Tokyo, Japan). Patch electrodes are made by pulling, in two steps, capillary borosilicate glass tubes (e.g., 1.5- 1.8 × 100 mm, Kimax-51, Kimble Products, Toledo, OH) with the aid of a vertical puller (L-M-3P-A, List Medical). The tip of the electrode is coated with Sylgard (Dow Corning, Midland, MI) to reduce random noise and fire-polished.

The resistance of the patch electrodes can be determined by repetitively applying a voltage step (e.g., 5 or 10 mV) to the pipette once it is immersed in the recording chamber filled with the bath solution. A bath electrode is used to ground the bathing solution and can be made of Ag/ AgC1 incorporated in an agar salt bridge. Usually, patch electrodes with a resistance in the range of 5-7 M~ (which corresponds to a tip diameter of around 1 ~m) are used to make a seal. Once a gigohm seal is formed

2~ O. P. Hamill, A. Marry, E. Neher, B. Sakmann, and F. J. Sigworth, Pfluegers Arch. 391, 85 (1981).


between the pipette glass and the membrane of a cell, the electrically isolated patch of the membrane is excised by retreating the pipette from the cell. The resulting patch is inside-out with the cytoplasmic side facing the bathing solution.

Data are stored on tape using a PCM converter system (e.g., VR10 or VR10B, Instrutech, New York, NY), reproduced and low-pass filtered at 1.5-2 kHz ( - 3 dB) by a Bessel filter (e.g., Frequency Devices, Haverhill, MA), sampled at 5 kHz, and analyzed off-line with the aid of a personal computer and standard software. In biological membranes channels open and close stochastically in an all-or-none manner. For single-channel analysis, the threshold for judging the open state is set at half of the single- channel amplitude. 26

Solutions and Chemicals

The control bathing solution contains (in mM) 136.5 NaCI, 5.4 KC1, 1.8 CaCI2, 0.53 MgC12, 0.33 NaH2PO4, 5.5 glucose, and 5.5 HEPES-NaOH buffer (pH 7.4). The composition of the hig-K + , low-C1- solution is (in raM) l0 taurine, 10 oxalic acid, 70 glutamic acid, 25 KCI, I0 KH2PO 4, 11 glucose, 0.5 EGTA, and 10 HEPES-KOH buffer (pH 7.3-7.4). The composition of the pipette solution (in mM) is 140 KCI, 1 CaC12, I MgC12, and 5 HEPES-KOH (pH 7.4). In the inside-out patch clamp experiments the bath is perfused with "internal" solution containing (in mM) 140 KCI, 0.5-MgCI2, 5 EGTA-KOH, 5 HEPES-KOH buffer (pH 7.3). In Mg2+-free internal solution, MgCI 2 is omitted and EGTA is replaced with equimolar EDTA.

GTP (Na + salt), 5'-guanylimidodiphosphate (GppNHp), ATP (Na + or K + salt), and acetylcholine (ACh) can be purchased from Sigma, whereas guanosine 5'-O-(2-thiodiphosphate) (GDPflS) and guanosine 5'-O-(3-thiotriphosphate) (GTPyS) are available from Boehringer Mannheim (Mann- heim, Germany). All nucleotides should be stored at - 20 °.

GTPyS-bound Ga (Ga*) and G/3y of pertussis toxin G proteins are purified from brain (see below). Alternative sources of G-protein subunits can also be used (e.g., placenta, retina). Gila* (3.7 to 6.5 ~M), Gi2a* (3.0 or 6.2/zM), Gi3ot* (1.2/xM), Goa* (12.8/zM), Goa-GDP (1/xM), and Gfly (6.5 to 20 tzM) are stored at -80 ° in solutions containing 50 mM Na HEPES (pH 7.4), 0.1 mM EGTA, and 0.7% (w/v) CHAPS or 0.5% Lubrol PX. The subunits are dissolved in internal solution at a concentration of I00 nM and stored at 4 ° (stock preparation). The stock preparations (used within 5 days) are further diluted in the internal solution to the desired final concentrations just before use. To prevent the surface absorption of

26 D. Colquhoun and F. J. Sigworth, in "Single-Channel Recording" (B. Sackmann and E. Neher, eds.), p. 191. Plenum, New York, 1983.


lipophilic substance, diluted assay solutions should be stored in polypropylene tubes instead of glass containers.

Preparation of G-Protein Subunits

A detailed account of how to purify and characterize different G-protein subunits from bovine brain membranes has been previously described. 27 All purified a subunits interact with/37 subunits and serve as a substrate for pertussis toxin-catalyzed ADP-ribosylation. The identity of the a subunits (i.e., all, oti2, 0~i3, OLo, O~o2) is confirmed by immunoblot analyses using specific antibodies raised against various a subunits of G proteins, z8 The functionality of the purified G-protein subunits should be tested in a known system before being tested for putative effects on ionic channels. For example, the active forms of the Ga subunits purified from brain, GTP3,S-bound Ga, inhibit the adenylyl cyclase activity of murine lymphoma $49 cyc- membranes which have been reconstituted with GTPTS-treated Gs .27

Transducin is extracted from photolyzed rod outer segment membranes with GTP and purified by hexylagarose column chromatography. 29 The a and /3~/ subunits of transducin (Ta and T/37) are purified from transducin by ~o-aminooctyl-agarose column chromatography. 3° For functional assays and characterization of transducin the reader is referred to another chapter in this volume. 31

Diffusion of G-Protein Subunits and Other Limitations of Inside-Out Patch Methodology

The patch clamp methodology allows the investigator to monitor in situ and in real time the behavior of channels, and it has been widely accepted as a powerful approach to evaluate the regulation of ion channels by G-protein subunits. Usually the density of channels in an excised patch is small owing to the limited size of the patch surface. This often allows a clear identification of the studied channel with little "contamination" by other channels. However, to perform meaningful population studies a large number of patches must be studied.

27 I. Kobayashi, H. Shibasaki, K. Takahashi, K. Tohyama, Y. Kurachi, H. Ito, M. Ui, and T. Katada, Eur. J. Biochem. 191, 499 (1990).

28 T. Katada, K, Kontani, A. Inanobe, I. Kobayashi, Y. Ohoka, H. Nishima, and K. Taka- hashi, this series, Vol. 237 [10].

29 B. K.-K. Fung, J. Biol. Chem. 258, 10495 (1983). 30 Y.-K. Ho and B. K.-K. Fung, J. Biol. Chem. 259, 6694 (1984). 3~ A. Tar, T. D. Ting, and Y.-K. Ho, this volume [1].

400 ruNS AND CHANNELS [34]

Although the inside-out configuration allows access to either side of the patch, it should be kept in mind that excised membranes are removed from their natural environment and that segments of signaling cascades may be disrupted. Also, patch membranes contain, to an unknown extent, proteins and other molecules which may be functional to various degrees, which should be taken into consideration in interpreting the findings. During an experiment resealing of an inside-out patch can occur which will prevent the access of substances to the internal side of the patch. Another major limitation of the inside-out methodology is the possible rundown of some channels in a cell-free milieu.

The ability of various G-protein subunits to access the membrane, to incorporate into the bilayer, and to reach the side of action is unknown. Therefore, the concentrations of subunits applied to the internal side of the patch membrane may not represent the actual concentrations of specific subunits once they have been incorporated into the lipid bilayer of the membrane and have diffused to the effector sites. The applied concentrations should only be viewed as apparent and may not be used as a valid indicator of which subunit(s) activates the channel in vivo.

The investigator should establish for each assay system whether it represents a valid reconstitution system. In other words, it may not be sufficient to determine the functionality of G-protein subunits by biochemical methods; rather, their abilities to reach a plasmalemmal channel (or other target) should also be tested.

Criteria for Defining Active G-Protein Subunits

The following criteria can be used, in patch clamp experiments, to identify which are the active subunits responsible for the activation through a "membrane-delimited" mechanism of the KACh or other channels.

1. The subunit- and GTP-activated channels should possess identical conductance and kinetic properties (e.g., reversal potential, unit conductance, rectification, mean open time), as well as voltage dependency (usually expressed as the relative open probability of the channel at different membrane potentials). The stimulation by active subunits of the studied channel should be highly reproducible (Fig. 1).

2. The intracellular GTP-induced activation of a channel (with agonists in the pipette) should show a characteristic concentration dependency. In this regard, the G-protein subunit(s) which regulates a channel in vivo can also be expected to exhibit a concentration-dependent pattern with regard to the ability to activate a channel. However, owing to the limita-


A GTPIO#M GTPYS-bound a id 300pM /3Y 10nM

B GTP10gM

GTPYS-bound ai-3 3nM flY 3nM GTPYS 10/lM

C GTP 10aM GTPYS-bound ao 10nM fly 10nM

lmin

F~G. 1. G-protein/3y but not a subunits consistently and fully activate muscarinic KACh channels. Recordings of the KACh channels were made in the inside-out configuration from guinea pig atrial myocytes. The pipette solution contained 0.5 gM ACh. (A) GTP (10 ttM) maximally activated KACh channels and induced an inward current at a holding potential of -60 inV. After washing out GTP, channel activity disappeared. Application for 7 rain of the GTPyS-bound form of ai~ (300 pM) did not activate the channel significantly. The lack of Ga effect was observed in 68% of patches analyzed. Subsequent application of 10 nM G~, to the same patch almost fully activated the KAC, channels. Gl3y activation was seen in 98.5% of the cases (132 of 134 patches). (B) In the inside-out patch, 3 nM of GTPyS- bound ai3 partially stimulated KACh channels following a lag period of more than 3 rain. This is an example of extreme responsiveness to a subunits, which was very rarely observed. Subsequent application of 3 nM of G/3y rapidly and fully activated the KACh channels. Further application of GTPyS did not produce an additional increase in channel activity beyond the level already attained by G/3y. The holding potential of the patch was - 80 mY. (C) Marginal activation of KACh channels by a high concentration of the GTPyS-bound form of ao (10 riM). This type of response to Ga subunits was obscrved in 32% of patches studied (40 of 124 patches) and did not exceed 20% of the maximal channel activity. In the same patch, G/3y (10 nM) maximally activated the channel. The holding potential was -80 inV.

t ions o f the a s s a y s y s t e m (e .g . , i n c o r p o r a t i o n o f e x o g e n o u s l y a pp l i e d subun i t s in to the m e m b r a n e l ip id b i l a y e r and o t h e r d i f fus ion s t eps o c c u r be fo re the subun i t s ac tua l l y in t e rac t wi th the ta rge t ) , th is c o n c e n t r a t i o n d e p e n d e n c y m a y no t n e c e s s a r i l y re f lec t the in t r ins ic s t o i c h i o m e t r y tha t g o v e r n s the i n t e r a c t i o n o f the subuni t ( s ) wi th the e f fec tor . A s a rgued a b o v e , the r e l a t i v e p o t e n c y o f d i f fe ren t subun i t s in ac t iva t ing a specif ic c hanne l d o e s no t p e r se def ine w h i c h o f the subun i t s a c t i v a t e the c ha nne l in vivo.

402 iONS AND CHANNELS [34]

3. The buffer solution on its own, that is, without the G-protein subunits, should not activate the channel to a significant degree, whereas the buffer solution containing the active subunits should markedly activate the channel.

4. Boiled preparations should not activate the channel since denatured G-protein subunits are biologically inactive. Note, however, that peptides corresponding to the active portion of certain G-protein subunits have been found to regulate effectively a specific effector. 32

5. The Ga subunits should be tested in both the GDP-bound and GTPyS-bound forms. If Ga is the subunit directly responsible for the G-protein activation of a channel, then the GDP-bound Ga should not activate the channel. Also, as it is known that at least some a-GTPyS complexes are unstable in Mg 2+-free solution, preincubation of a-GTPyS complexes in Mg2+-free internal solution, containing GDP or GDPflS, should inactivate Ga subunits.

6. Gfly-induced activation of a channel should be abolished by preincubation of G/3y with an excess of GDP-bound Ga.

Application of Criteria

What follows is a description of the practical use of the above-defined criteria to determine which subunit(s) may be responsible for channel activation. As a didactic example, the activation of the extensively studied cardiac KACh channel by G-protein subunits was chosen. A comparison between G-protein subunit activation of the KACh and the KATp channels is also presented, as an illustration of distinct regulations of cardiac K + channels by pertussis toxin-sensitive G proteins.

Gfly Subunits Mimic GTP in Activating Muscarinic Potassium Channel

Gfly subunits (10 riM) applied from the internal side of guinea pig atrial cell membrane patches induce persistent openings of a K + channel which has identical conductance and kinetic properties as the KAc h channel. 33 Indeed, as is the case with the GTP-activated channel (in the presence of ACh), the Gfly-activated channel has a unit conductance of approximately 40-45 pS when 150 mM K + bathes both the external and internal sides of the membrane and shows pronounced inward rectification in the presence of 2 mM Mg 2+ in the solution bathing the internal side of the patch

3z H. M. Rarick, N. O. Artemyev, and H. E. Harem, Science 256, 1031 (1992). 33 H. Ito, R. T. Tung, T. Sugimoto, I. Kobayashi, K. Takahashi, T. Katada, M. Ui, and Y.

Kurachi, J. Gen. Physiol. 99, 961 0992).


A xfg

GTI' 100 ~M /3Y I0 nM

B G*I'P /3Y with Mg 2+ ~3Y withol~t Mg2+

0 - - , - - . . . . . ,

membrane D~tential C _ .ll][l -50 0 ~ d . . + 5 0 mV

i'T .e-

L: /

09g ms

0 g nls 0 5ms

E

I[I I

80 -40 (I +411 +[411 mV

membrane potential

FIG. 2. Activation of KACh channels by GTP and G-protein/33, subunits. (A) After forming an inside-out patch in internal solution containing 2 mM MgCI2, 100/~M GTP activated the KACh channel with 0.3 /xM ACh in the pipette solution. After washing out GTP, channel activity disappeared. Subsequent application of G/3T activated the KACh channel irreversibly. The patch was held at - 80 mV. (B) Expanded recordings of the Kach channel at various holding potentials induced by GTP or G/3 T with or without 2 mM Mg 2+ (indicated above each column). (C) Current-voltage if~V) relationship of Kac h channel. Filled triangles, open squares, and filled squares represent the I/V relationship induced by GTP with Mg 2 + (2 mM), G/3T with Mg 2+ (2 mM), and G/3T without Mg 2. , respectively. Strong inward rectification was noted using GTP with Mg 2+ and G/3T with Mg 2+. (D) Open-time histograms of the KAC~ channel currents induced by GTP and G/3 T at - 8 0 mV. (E) Voltage-dependent channel activity in the absence of Mg 2+ induced by G/3T (filled circles) and GppNHp (open circles). The relative NPo was obtained in reference to the NPo induced by 10 nM G~r or 10/~M GppNHp at - 8 0 mV. The results were expressed as means +- S.D. (n = 3 each). (From lto et al) 3)

(Fig. 2A-D). The current-voltage relationships of the GTP- and G137- activated channels are superimposable (Fig. 2C). Like that induced by GTP, the open-time histogram of the GilT-activated channel can fit a single exponential curve with a time constant of approximately 1 m s e c . 4'33

It should be pointed out that the GTP-dependent channel activation by ACh requires intracellular Mg z+ 15,34 whereas G/37-mediated activation of the KAC h channel does not. This difference does not discredit G/3T

34 y . Kurachi, T. Nakajima, and T. Sugimoto, Pfluegers Arch. 410, 227 (1987).


as the active subunits since it is in harmony with the Mg2+-dependent dissociation of trimeric G proteins into active Ga and GilT. 2 Because Gfly subunits applied exogenously are already separated from the Ga subunits, they do not need Mg 2+ to become active. Actually, this feature can be exploited to evaluate the effect of the Gfly subunits on a specific channel in the absence of Mg 2+. In the absence of Mg 2+ in the internal solution, the Gfly-activated KA¢ h channel exhibits no inward rectification (Fig. 2C).

In addition, the channel activity decreases in a voltage-dependent manner as the holding potential is depolarized to more positive values than the equilibrium potential for K + (EK ~ 0 mV in the symmetrical 150 mM K ÷; see Fig. 2E). This voltage-dependent activation of the KACh channel is also observed with GppNHp or GTP3JS in Mg2+-free solutions. 33 Thus, the exogenous Gfly-activated KACh channel exhibits the same conductance and kinetic properties and shows the same voltage-dependence as the channel activated by GTP and GTP analogs.

High Reproducibility of Gfly-Induced Activation of Muscarinic Potassium Channel Not Shared by Got

G/3y subunits (10 riM) activate, with no significant lag time (<5 sec), the KAC h channel in 132 of 134 guinea pig atrial cell membrane patches so tested? 3 Hence, the frequency of activation of KACh by G/3y is highly reproducible, with a success rate equal to 98.5%. The magnitude of the channel activation by Gfly is equivalent to the activation produced by GTP (in the presence of ACh) or GTPyS (Fig. 1). Neither the reproducibility nor the effectiveness of KACh channel activation by G/3y are shared by different subtypes of Ga subunits (e.g., Gil.2.3a purified from the brain). Indeed, Ga (10 pM to 10 nM), preactivated with GTPyS, inconsistently (in 40 of 124 patches) activates the KACh channel after a lag time of 2 to 4 rain (Fig. 1). This activation is, at best, only partial since it amounts to approximately 20% of the GTP3,S-induced maximal channel activity. Hence, exogenously applied Ga and Gfly affect the KAC h channel in a different manner.

Concentration Dependence of Gfly-Induced Activation of Muscarinic Potassium Channel

Gfly subunits (10 pM to 100 riM), purified from brain and sequentially applied to the internal side of guinea pig atrial cell membrane patches, activate the KACh channel in a concentration-dependent manner. The minimal concentration of bovine brain G/3y required to activate the channel is approximately 300 pM, and the ECs0 is about 6 riM. The relationship between the concentration of G/3y versus relative NPo (an index of channel


activation, where N is the number of channels in the patch and Po is the probability of each channel to be open) can be fitted using the Hill equation:

y = Vmax/{1 + (Kd/[Gfly]) n}

where y is relative NPo, Vm,x is the maximal NPo, K a is the GBy concentration required for the half-maximal activation of the channel, and H is the Hill coefficient. The relative NPo for each G/3y concentration is obtained in reference to the NP o induced in the same patch by 100 ~M GTP (in the presence of 1/xM ACh) or 10-100/xM GTPyS (i.e., the maximal NPo). The Hill coefficient is calculated to be 3.12. 33 Although this value is very similar to that reported for the relationship between the concentration of GTP versus channel activation 3s'36 it does not necessarily mean that exogenously applied subunits simply mimic the effects of intracellular GTP in the stoichiometry of channel activation. Indeed, in interpreting these results the investigator should take into account the complicating element that exogenously applied subunits first have to be incorporated, and then diffuse into the membrane, before interacting with the effector. In the case of the KACh channel, however, a similar Hill coefficient of around 3 has also been reported in the case of the GTPyS activation of the channel, 37 which, as is the case with the action of brain/3,/subunits, is for all practical purposes irreversible. Thus, the steep concentration dependency may be a genuine property of the G-protein regulation of the KAC h channel function and could be explained by the presence of a still unidentified (amplifying) step.

G/3y subunits from nonbrain sources also activate the KACh channel in a concentration-dependent manner. This is the case, for example, with transducin /33' (see next section). It is not known whether retinal and nonretinal G-protein subunits share a common pathway in activating the KACh channel.

G/3y Activation o f Muscarinic Potassium Channel Not Dependent on Detergents or Buffer Constituents

To prevent aggregation, hydrophobic G/3y subunits of many G proteins must be suspended in a detergent, such as CHAPS or Lubrol PX. Cholic acid is not used as a detergent in electrophysiological experiments because it disintegrates the cell membrane. Because at least in principle detergents

35 y. Kurachi, H. Ito, and T. Sugirnoto, Pfluegers Arch. 416, 216 (1990). 36 H. Ito, T. Sugimoto, I. Kobayashi, K. Takahashi, T. Katada, M. Ui, and Y. Kurachi, J.

Gen. Physiol. 98, 517 (1991). 37 T. Nakajima, T. Sugimoto, and Y. Kurachi, J, Gen, Physiol. 99, 665 (1992).


could on their own activate a channel, 38,39 detergents used to suspend protein subunits should be directly tested on the channel of interest to exclude whether they have a significant effect by themselves.

In the case of the KACh channel, low concentrations (10-200/zM and up to 500 /~M) of the zwitterionic detergent CHAPS (purchased from Dotite, Kumamoto, Japan, or Sigma) added to the intracellular side of an inside-out patch do not activate the channel, whereas subsequent application in the same patch of Gfly (I0 nM) suspended in 5.7 to 17.5/zM of CHAPS activates the KAch channel. 33 This indicates that (1) CHAPS on its own does not activate the KACh channel and (2) the lack of effect by CHAPS is not due to vesicle formation or disintegration of the patch. It should be pointed out that higher concentrations of CHAPS (in the millimolar range) could cause a breakdown of the patch. Therefore, Gfy subunits should be suspended in CHAPS concentrations not exceeding 200/zM.

To rule out the remote possibility that the activation of a channel by G-protein subunits is due to a unique combination of the detergent (i.e., CHAPS) with the subunits, it is also desirable to suspend the G-protein subunits in a detergent with physicochemical properties different from those of the original one. In the example of the KACh channel, instead of CHAPS, the Gfly subunits are suspended in a nonionic detergent, Lubrol PX. The Knch channel is also activated in a concentration-dependent manner by Gfy suspended in Lubrol PX. 33 At 10 nM Gfy, the concentration of Lubrol PX is 0.00025%. At this concentration, Lubrol PX on its own has no effect, whereas at 0.001% this detergent inhibits even the ACh-mediated, GTP-induced activation of the KACh channel. Hence, independent of whether the detergent is zwitterionic or nonionic Gfy subunits activate the KAC h channel.

To exclude altogether any implication of a detergent, in some cases advantage can be taken of the property of rare hydrophilic Gfy subunits which do not need to be suspended in a detergent. This is the case with Try. A preparation of hydrophilic Try (100 nM-3 /zM), not containing any detergent, when applied to the internal side of the patch membrane, promptly and reversibly activates KAc h in a concentration- dependent fashion with an ECs0 of approximately 1/zM. 4° These experiments strongly indicate that the activation of the KAc h channel is indeed due to the Ely subunits with no participation of the detergents whatsoever.

3s G. E. Kirsch, A. Yatani, J. Codina, L. Birnbaumer, and A. M. Brown, Am. J. Physiol. 254, H1200 (1988).

39 A. Yatani, K. Okabe, L. Birnbaumer, and A. M. Brown, Am. J. Physiol. 258, H1507 (1990). 40 M. Yamada, Y. K. Ho, R. H. Lee, K. Kontani, K. Takahashi, T. Katada, and Y. Kurachi,

Biochem. Biophys. Res. Commun. in press (1994).


In additional control experiments it has also been determined that the various G/33" buffer solutions employed cannot on their own (in the absence of the/3,/subunits) activate the KACh channel. 33

Inactive Preparations of G/33" Cannot Activate Muscarinic Potassium Channel

A conventional approach to inactivating G-protein subunits is to denature them by boiling at 100 ° for 5 min. A preparation of boiled G/3y subunits does not, whereas native G/33' does, activate the KAC h channel in the same patch, indicating that a heat-labile substance (like a protein), but not a heat-stable substance (like the detergent CHAPS), is responsible for the activation of the KAC h channel.19'33

Contrary to the case for/33, subunits, it has been reported that a - GTPyS complexes are not stable in Mg2+-free solutions containing GDP or GDP/3S. 41 This difference in behavior can be exploited to rule out whether a contamination of a/33' preparation with preactivated (or GTPTS- bound) Ga subunits is responsible for the G-protein subunit-mediated activation of a specific channel. In the case of the KAC h channel, preparations of G/33, subunits are preincubated in EDTA-Mg2+-free solution containing 2-10/xM GDP or GDP/3S for 24-48 hr at 4 °. G/33' subunits, incubated with Mg2÷-free EDTA, do not lose the ability to activate the KAc ~ channel. Following identical preincubation in Mg 2+-free soultions neither Gilot, Gi2ot, nor Goa (up to 10 nM) could activate the KAC h channel. ~9,33 These experiments point out that (I) the activation of the KAC h channel by G/33' is not due to contamination of the G/33" preparation by Got and (2) the participation of a G-protein subunit genuine to the cardiac membrane in the activation of the KAC h channel by exogenously applied G/33' subunits appears unlikely since Mg 2+ and GTP are necessary for activation of G proteins, ~4 and GDP/3S is expected to block the activation of the native G proteins by binding to Got. 2

A more "physiological" way of inactivating G/3y, rather than boiling the subunits, is to preincubate a G/33" preparation with an excessive concentration of Got-GDP. During preincubation G/33' may bind to Ga-GDP to form an inactive heterotrimer. 2 This approach has been employed to further confirm the specific effect of G/3y on the KAC h channel. G/3y (5 nM) is preincubated with an excessive amount of Goot-GDP (10 riM) for 5 rain at 35 ° and then perfused to the internal side of the membrane. G/33' preincubated with Goa-GDP does not activate the KAC h channel, whereas further application of G/3y alone does activate the KAC h channel in the same

41 j. Codina, J. D. Hildebrandt, and L. Birnbaumer, J. Biol. Chem. 259 11408 (1984).

408 IONS AND CHANNELS [34l

patch. 33 Boiled Goa-GDP does not prevent Grit activation. Ga-GDP reverses the activation of the KAC h channel in a patch pretreated with GrT. 42 These types of observations can be used to suggest that the Gry is the activating arm of the GK that regulates the atrial KAC h channel.

Ga but Not Gry ATP-Sensitive Potassium Channel

It is important to demonstrate the "functionality" of the G-protein subunits not only by conventional biochemical assays (see Preparation of G-Protein Subunits), but also in an experimental setting that is identical to the one in which the channel of interest is studied. For example, in some instances it is possible to test the functionality of the subunits by electrophysiological means on another channel that is coexpressed with the channel of interest in the same or similar preparation.

When applied to inside-out patches, Gila and Gizot (purified from bovine brain) activate the KAT P channel in guinea pig ventricular cell membrane, where the KAC h channel is not expressed. 33 The internal solution contains 100/xM ATP and 0.5 mM MgCl2 to keep the KAT P channel in its phosphorylated state. 43 When the patch pipette contains adenosine (10 /zM) the KAT P channel activity increases during the application of GTP (100/xM), probably owing to the activation of endogenous G proteins already present in the patch membrane.44 In contrast to the case of the atrial KAC h channel, further application of l0 nM Gry inhibits the GTP-induced increase of KAT P channel activity. By itself, Gry does not affect the background KATP channel activity. 33 Thus, both Ga and Gry can be incorporated into the lipid bilayer and/or reach specific plasmalemmal targets, but only Ga can activate the KAT P channel (Fig. 3).

In atrial cell membranes, both KAT P and KAC h channels are expressed. When Gila (300 pM) is applied to the internal side of inside-out patches, in the internal solution containing 100/zM MgATP, burst openings of the 90 pS KAT P channel are induced with no significant effect on KAC h (Fig. 3). Subsequent application to the same patch of Gry (10 nM) dramatically increases the openings of the 40-45 pS KAC h channel. 33 This approach rules out any artifact arising from incomplete incorporation of subunits into the membrane or diffusion limitations since in the same patch a specific subunit activates one type of channel but not the other.

42 D. E. Logothetis, D. Kim, J. K. Northup, E. J. Neer, and D. E. Clapham, Proc. Natl. Acad. Sci. U.S.A. 85, 5814 (1988).

43 R. T. Tung and Y. Kurachi, J. Physiol. (London) 437, 239 (1991). 44 G. E. Kirsch, J. Codina, L. Birnbaumer, and A. M. Brown, Am. J. Physiol. 259, H820

(1990).


Modulation of G-Protein Regulation of Potassium Channels by Arachidonic Acid Metabolites

It has been demonstrated that various fatty acids modulate the function of certain types of ion channels. Although most of the fatty acids affect channel function without being metabolized by the cells, 45-47 arachidonic acid (AA) regulates some K + channels through intracellular conversion to metabolites. 48-5~

In various cell types, AA is metabolized to a number of biologically active metabolites (see Fig. 4), such as prostaglandins by cyclooxygenase and leukotrienes (LT) by 5-1ipoxygenase (5-LO). 5z This metabolism can occur either when AA is exogenously applied to the cells or when AA is released from the esterified stores in membrane phospholipids by the action of phospholipases on chemical (e.g., neurohormonal) and physical (e.g., ischemia) stimuli. The AA metabolites are known to serve as important intra- and intercellular biological signals in various physiological and pathophysiological conditions.

The lipoxygenase metabolites of AA have been proposed to be a novel class of intracellular second messengers in various agonist-dependent regulations of ion channels.48-5°,53 The metabolites appear to regulate channel function by interacting with the K ÷ channel itself or with some associated site(s), such as G proteins, within the cell membrane. Although the site of action of the metabolites is not unequivocally identified as yet, it has been demonstrated that certain eicosanoids modulate the function of the cardiac KACh channel through its regulatory G protein, G~ .49,53 The following describes how to deal with arachidonic acid metabolites and how to evaluate electrophysiologically the effects of AA metabolites on channels and G proteins with specific reference to the GK-gated KACh channel.

45 R. W. Ordway, J. V. Walsh, and J. J. Singer, Science 244, 1176 (1989). 46 D. Kim and R. A. Duff, Circ. Res. 67, 1040 (1990). 47 M. A. Wallert, M. J. Ackerman, D. Kim, and D. E. Clapham, J. Gen. Physiol. 98,

921 (1991). 48 D. Piomelli, A. Volterra, N. Dale, S. A. Siegelbaum, E. R. Kandel, J. H. Schwartz, and

F. Belardetti, Nature (London) 328, 38 (1987). 49 y. Kurachi, H. Ito, T. Sugimoto, T. Shimizu, I. Miki, and M. Ui, Nature (London) 337,

555 (1989). 50 D. Kim, D. L. Lewis, L. Graziadei, E. J. Neer, D. Bar-Sagi, and D. E. Clapham, Nature

(London) 337, 557 (1989). 51 p. Schweitzer, S. Madamba, and G. R. Siggins, Nature (London) 346, 464 (1990). 52 p. Needleman, J. Turk, B. A. Jakschik, A. R. Morison, and J, B. Lefkowith, Annu. Rev.

Biochem. 55, 69 (1986). 53 T. Nakajima, T. Sugimoto, and Y. Kurachi, FEBS Lett. 289, 239 (1991).

A CiA I/(3 ~ i i-~,oo,,----- ~- a *-i -GTPYS 300 pM pY I0 nM

l 2 "14

3

n t - ~ ~ i ~ ~i--~rt~,~, "~ " " T 1"~1# "lw

B C ~ 1500~ K^T p channe l

[ k ~ = l . 4 m s d 50o[ "~

. . z o l 0 +40 mV 0 2 4 6 ms -2

10001- ~l KACh c h a n n e l

~ = I . I ms

0 l] 2 4 6 ms

D

K ATpchannel

~-.+® GTP

ACh

Adenosine

M / ~ ~ K channel ACh

[34 ] G PROTEIN, ARACHIDONIC ACID, AND K + CHANNEL 411

Preparation of Reagents

Arachidonic acid and metabolites are lipophilic and usually unstable. Thus, special precaution is necessary to handle these substances while working with intact cells or cell membranes during patch clamp experiments.

Dissolution of Arachidonic Acid and Eicosanoids into Aqueous Solu- tion. Make concentrated stock solutions with benzene or ethanol, and keep them in the dark at less than - 2 0 ° under argon or nitrogen. The product of AA with greater than 99% purity provided by Nu Chek Prep, Inc. (Elysian, MN) is a reliable reagent. Other commercially available products and samples used long after breaking the seal should be repurified before usage.

To dissolve AA and eicosanoids into aqueous solutions, place an appropriate amount of an aliquot in a Pyrex glass test tube and remove the

FIG. 3. Distinct G-protein subunits activate KAT P and KACh channels in cardiac cell membranes. (A) Effects of GTPyS-bound Gila and G/3~, on the KAT P and KAC h channels in the atrial cell membrane. The pipette solution contained 1 ~M ACh. The inside-out patch was formed at the arrow above the current trace in the internal solution containing 100/zM ATP and 0.5 mM MgCI2. GTPyS-bound Gila (300 pM) was first applied to the internal side of the patch, which clearly induced openings of the KATP channel ( -90 pS) (trace 2) without affecting background activity of the KAC h channel. Subsequently, G/3y (10 nM) was applied to the patch, which caused a dramatic increase of 45 pS KAC h channel openings in the same patch (traces 3 and 4). Numbers above the current trace indicate the location of each expanded current trace below. In the expanded current traces, the dashed line is the first level of the KAC h channel and the continuous line is that of the KAT P channel. The zrrowhead at each trace is the zero current level. (B) Current-voltage relation of KAT P channel induced by GTP-/S-bound Giic~ in ventricular (open squares) and in atrial (open circles) cell membranes, and G/3y-induced KAC h channel in the atrial cell membrane (filled circles). (C) Open time histograms of KAT P channel induced by GTP3~S-bound Gila (in ventricle) and G/3"y- induced KAC h channel (in atrium) at - 80 inV. (D) Proposed mechanism of the PTX-sensitive G-protein subunit activation of the KATp and KAC h channels in cardiac cell membranes. On stimulation of the receptors by adenosine or ACh, PTX-sensitive G proteins may he functionally dissociated into Gtx-GTP and G/3y. Ga-GTP may activate the KAT P channel, whereas Gfl), activates the KAC h channel. This scheme does not represent any quantitative relationship between each component and does not take into account possible intermediate steps between components. The former mechanism exists in both ventricular and atrial cells, whereas the latter may exist in atrial but not in ventricular ceils. Because cardiac myocytes contain miUimolar concentrations of intracellular ATP, the G-protein activation of the KAT p channel system may not be operative under physiological conditions. However, the system might play a significant role in the ischemia-induced shortening of cardiac action potentials. Although we cannot completely exclude the possibility that the Gia-GTP pathway may in part contribute to the GK activation of the KAC h channel, this pathway cannot be the major regulatory mechanism for the KAC h channel. (From Ito eta/ . 33)


Membrane ~ Arachidonic Phospholipids ~ Acid w

~ [ PGD2 ~ 11-epi-PGF2c t

PGE 2

F PGG2.~ 3 PGH2 ~ PG 2c~ .~ PGI 2 .......... 6-keto-PGFlo ~

~ . T X A 2 ........... TXB 2

~ , . HHT

5-HPETE

15

1G

12-HPETE

5-HETE~lo LTB4 LTA 4

LTC 4 ~ L T D 4 ~ LTE 4

12-HETE 17 Hepoxilin A3.--.~ Trioxilin A 3 17 Hepoxilin B3...--=. Trioxilin B3

11, 12-LTA4..~,. 11, 12-diHETE

12-KETE

15-HETE 18 15-HPETE Lipoxins

EETs ~ DHETs

- ~ 19-hydroxy-eicosatetraenoicacid, 19-oxo-eicosatetraenoic acid

20-hydroxy-eicosatetraenoic acid, eicosatetraen-1,20-dioic acid

FIG. 4. Arachidonic acid metabolism and the enzymes participating. Solid lines indicate enzymatic processes and dashed lines are either nonenzymatic or unknown processes. Enzymes: 1, Phospholipase A2, or phospholipase C and diacylglycerol lipase; 2, prostaglandin (PG) endoperoxide synthase (cyclooxygenase); 3, PG endoperoxide synthase (hydroper- oxidase); 4, PGD synthase; 5, PGE s ynthase; 6, PGF synthase; 7, PGI synthase (cytochrome P-450-1ike enzyme?); 8, thromboxane (TX) synthase (cytochrome P-450-1ike enzyme?); 9, 5-1ipoxygenase; 10, LTA4 hydrolase; 11, LTC4 synthase; 12, y-glutamyltranspeptidase; 13, dipeptidase; 14, 12-1ipoxygenase; 15, glutathione peroxidase; 16, cytochrome P-450-1ike enzyme?; 17, epoxide hydrolase; 18, 15-1ipoxygenase; 19, cytochrome P-450. Metabolites: PG, Prostaglandin; TX, thromboxane; HHT, 12-hydroxyheptadecatrienoic acid; 5-HPETE, 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid; 5-HETE, 5( S)-hydroxy-6-trans- 8,11,14-cis-eicosatetraenoic acid; LT, leukotriene; 12-HPETE, 12(S)-hydroperoxy-5,8,14- cis-lO-trans-eicosatetraenoic acid; 12-HETE, 12(S)-hydroxy-5,8,14-cis-lO-trans-eicosatetraenoic acid; I I,12-diHETE, l l,12-dihydroxyeicosatetraenoic acid; 12-KETE, 12-keto- 5,8,14-cis-lO-trans-eicosatetraenoic acid; 15-HPETE, 15(S)-hydroperoxy-5,8,11-cis-13- trans-eicosatetraenoic acid; 15-HETE, 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid; EET, epoxyeicosatrienoic acid; DHET, dihydroxyeicosatrienoic acid.

solvent under a stream of argon or nitrogen immediately before use. The thin layer of lipid coating the glass is usually redissolved in ethanol or dimethyl sulfoxide or directly dispersed in the aqueous solution by sonication or vortex mixing. However, it should be noted that organic solvents


and detergents, on their own, often have various nonspecific effects on ion channels. Therefore, appropriate control studies must be always conducted when these substances are employed as solvents. In addition, AA and metabolites are more or less oxidized during sonication or vigorous mixing. Thus, it is important to use these substances in a range of concentrations at which they can be rather easily dissolved into aqueous solutions.

The substances stay as monomers in aqueous solutions when.they are at less than the critical micellar concentration. Above this concentration, the amphipathic structures form micelles and tend to cause various nonspecific effects on biological membranes and ion channels. The pKa value of these substances should be also taken into consideration. For example, the pK~ of AA is around 4. Thus, alkaline solutions can dissolve more AA than acidic solutions. Using this property, it is also possible to dissolve AA first in 0.1 M NaHCO3/NaOH at pH 9.3 and then dilute it to the desired concentration with 50 mM HEPES-buffered solution at neutral pH. When the final active metabolite of AA is identified, one may be able to design experiments with this product at low concentration.

Albumin. Albumin has specific binding sites for various hydrophobic substances and makes practically insoluble substances apparently soluble in aqueous solutions. 54,55 With this property, albumin prevents the adsorption of AA in aqueous solutions to glass and plastic surfaces of the tissue baths and test tubes. 56 Albumin also represses the tendency of nonspecific solubilization of amphipathic substances and inclusion in the cell membrane. 57 The protein also stabilizes some eicosanoids such as LTA4 in solution at neutral pH by keeping the acid-sensitive structure in the hydrophobic, alkaline environment of its own molecule. 58 Thus, albumin is useful in dissolving such lipophilic substances as AA and metabolites in a desired solution.

However, the equilibration of albumin with fatty acids is rather complex. 54 The apparent equilibration constant of binding of the substances to albumin is a function of the molar ratio of ligands to albumin, pH, and the presence of additional substances which can bind albumin. 57 In addition, some eicosanoids are chemically modified by the actions of

54 A. A. Spector, J. Lipid Res. 16, 165 (1975). 55 U. Kragh-Hansen, Pharmacol. Rev. 33, 17 (1981). 56 D. R. Samples, E. A. Sprague, M. J. K. Harper, and J. T. Herlihy, Am. J. Physiol. 257,

C 1166 (1989). 57 A. D. Purdon and A. K. Rao, Prostaglandins, Leukotrienes Essent. Fatty Acids 35,

213 (1989). 58 F. A. Fitzpatrick, D. R. Morton, and M. A. Wynalda, J. Biol. Chem. 257, 4680 (1982).


albumin: 9,6° Cellular metabolism of AA is also affected by albumin in extracellular solutions. 61 It is also reported that commercially available albumin products contain a varying amount of fatty acids 62 and also exhibit PLA z activity, probably owing to contamination. 63 Thus, when albumin is employed, these issues need to be taken into consideration.

Inhibitors of Arachidonic Acid Metabolism. As described above, AA is metabolized to numerous biologically active eicosanoids through sequential enzymatic conversions (Fig. 4). Therefore, in attempts to elucidate the mode of action of AA on ion channels, it is necessary to define the metabolic pathway and final product which mediate the AA action. For this purpose, various inhibitors of AA metabolism are useful. Com- monly used inhibitors are listed in Table I. However, the listed agents are neither the sole inhibitors of AA metabolism, nor are they deprived of nonspecific effects. It should be pointed out that, in general, the known inhibitors of AA metabolism exert to some degree nonselective effects on various biological systems. Therefore, it is always necessary to examine the specificity in individual experimental conditions. It goes without saying that logical consistency in the data must be obtained with various inhibitors and eicosanoids.

It has been demonstrated that some eicosanoids exert the biological effects through specific receptors. Various antagonists of these receptors have been developed. For example, LTD4/E4 and LTB4 receptor antagonists are now available. 64 These tools may also be useful in studying the mode of action of AA metabolites.

Electrophysiological Techniques

Perfusion System. To minimize surface absorption of AA and metabolites by various parts of the assay system, the guidelines stated above should be followed. It is also important to diminish the surface area of the tubing in the perfusion system and of the experimental chamber. In this regard, the application of solutions by pressure ejection from micropipettes placed in the vicinity of cells could be advantageous. 65 However, Samples et al. 56 reported that the concentration of AA added to the aque-

59 j. Maclouf, H. Kindahl, E. Granstrom, and B. Samuelsson, Eur. J. Biochem. 109, 561 (1980).

60 F. A. Fitzpatrick and M. A. Wynalda, Biochemistry 20, 6129 (1981). 6z E. Dratewka-Kos, B. Kindl, and D. O. Tinker, Can. J. Biochem. CellBiol. 63, 792 (1985). 62 D. J. Birkett, S. P. Myers, and G. Sudlow, Clin. Chim. Acta 85, 253 (1978). 63 p. Elsbach and P. Pettis, Biochem. Biophys. Acta 296, 89 (1973). 64 D. W. Snyder and J. H. Fleisch, Annu. Rev. Pharrnacol. Toxicol. 29, 123 (1989). 65 S. M. Sims, J. J. Singer, and J. V. Walsh, J. Physiol. (London) 367, 503 (1985).


ous solution in tissue baths and in glass tubes declines rapidly and progressively with time. Therefore, ideally, the concentrated stock solutions containing AA and metabolites should be added directly to the assay chamber instead of being applied through the perfusion system. If this approach is impractical, however, the concentration of the substances applied through the perfusion system should be measured in the assay chamber. Inclusion of albumin in the solution prevents the surface absorption of AA or metabolites, 56 but as described above other precautions are necessary when albumin is used.

Arachidonic acid and metabolites are light-sensitive. Therefore, when the substances are perfused in the assay system for a relatively long period, the entire assay system should be shielded from light. Because these substances are also heat-unstable, it is recommended to conduct the experiment at low temperature. Finally, after each experiment, the perfusion system must be extensively rinsed with ethanol and subsequently with a sufficient amount of distilled water.

Application of Patch Clamp Method. Despite the inherent difficulty in handling lipophilic substances when used on intact cells or cell membranes in an electrophysiological setup, the appropriate combination of the advantages of the individual patch-clamp configurations facilitate the investigation of AA and metabolites on ion channels. 25

Whole-Cell Configuration. In the whole-cell configuration, the following solution could be used for the pipette solution (in mM): 140 KCI, 1 MgCIz, 3 Na2ATP, 5 EGTA-KOH, and 5 HEPES-KOH buffer at pH 7.2-7.4. GTP and analogs, such as GTPyS and GDPflS, should also be added at the desired concentration depending on the aim of the experiments.

This technique is the most appropriate in screening for a putative effect of a substance on ion channels. However, it is essential to identify the channel affected by the substance properly. It should also be kept in mind that by applying the lipophilic substances to the bathing solution the cell may efficiently incorporate them through the entire whole membrane surface. In this case, the channels under observation are exposed to the amphipathic substances in the bath solution. Thus, unless appropriate precautions are taken, direct nonspecific effects on the channels may cause artifacts and make interpretation difficult. The activity of various enzymes participating in AA metabolism may also be artificially modified by the ingredients of the pipette solution. To minimize such modification of the intracellular milieu under tight-seal whole-cell clamp conditions, the nystatin method of whole-cell recording can be u s e f u l . 66

R. Horn and A. Marry, J. Gen. Physiol. 92, 145 (1988).

4 1 6 IONS A N D C H A N N E L S [ 3 4 ]

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Cell-attached configuration. The main advantage of the cell-attached configuration is the preservation of an intact cell, which allows the conversion of AA to eicosanoids through natural metabolic processes. As is the case with the whole-cell clamp technique, it is possible to apply AA and metabolites to the bath solution. In the cell-attached patch, the lipophilic substance could presumably reach ion channels in the patch membrane most likely via intracellular pathways whether or not it is metabolized. Contrary to the whole-cell configuration case, the channel under observation is not directly exposed to the amphipathic structures, at least not at the concentration applied to the bath. From our experience in cardiocytes, AA, LTA 4, and LTC4 added to the bath effectively activate the KAC h channel in the cell-attached configuration. However, the accessibility of substances to the patch membrane may differ among cell types and substances. Therefore, if the bath application of some substance does not lead to channel activation, confirmation of the result should be made by employing different configurations.

A substance could also be added to the pipette solution. If the substance affects the channel in a membrane-delimited manner from the external side of the membrane, the channel function will be affected in these experimental conditions. To elucidate the mode of action of a substance on a channel, a first step may be to compare the effects of the substance on the channel in the cell-attached form following bath versus pipette application. 67,68

Inside-out configuration. The advantages and limitations of the inside- out configuration when used with hydrophobic substances have been described earlier in this chapter. However, it should be restated here that the various cytosolic enzymes and cofactors required for the natural metabolic process are lost, to a unknown degree, in this configuration. In other words, if a substance modulates the function of a channel in the cell- attached or whole-cell configurations, the same substance may produce a negative result in the inside-out patch unless the substance can activate the channel only through a membrane-delimited pathway, including the membrane-associated metabolism pathway. The substrates essential for membrane-bound functional proteins should be added to the internal solution in this configuration (e.g., GTP for G proteins).

Outside-out configuration. In the outside-out configuration, the pipette could be filled with the same solution that is used in the whole-cell configuration with GTP or an analog. In this case, it is possible to apply AA and metabolites to the extracellular surface of the isolated patch membrane.

67 B. Sakmann, A. Noma, and W. Trautwein, Nature (London) 303, 250 (1983). 68 M. Soejima and A. Noma, Pfluegers Arch. 400, 424 (1984).


However, because the various cytosolic factors are lost or diluted into the pipette solution, only the membrane-delimited response can be observed in this configuration. Ordway et al. 45 employed this method to investigate the direct activation by fatty acids of the K + channels in smooth muscle cells. As a variant of this configuration, Levitan and Kramer 69 reported a new patch clamp configuration, the perforated vesicle using nystatin. With this form, it is possible to investigate single-channel function while preserving the intracellular milieu to a certain degree. Actually, these authors showed that the vesicle maintains the function of some intracellular signal transduction systems residing in the cytosol. Thus, this configuration may be useful for the investigation of the effect of AA and metabolites on ion channel function. To our knowledge, however, this configuration has not yet been used in this field.

Assay of Action of Arachidonic Acid Metabolites on G-protein Function Using Patch Clamp Technique

To elucidate whether AA metabolites stimulate the channel itself or through modulation of the function of G proteins, the following points should be taken into account. The mode of action of AA metabolites on the GK-gated KAC h channel is used as an example.

General Principles

1. The effect of AA and metabolites on the channels must be observed at less than the respective critical miceUar concentration. In addition, the vehicle of the substances should have no significant effect on the channel function.

2. In terms of reversal potential, conductance, and kinetic properties, the current induced by AA and metabolites must be clearly distinguished from nonspecific leakage currents caused, for example, by the disintegration of the phospholipid membrane by the amphipathic structures.

3. If the action of AA on the G-protein regulation of channel function is mediated through a specific metabolite following the metabolic conversion of AA, then the effect of AA should be mimicked by the eicosanoid (and precursors) that should be directly responsible for the modulation of the KACh channel. Also, inhibitors of the production of that eicosanoid should block the action of AA. Nonspecific action of the inhibitors should be evaluated under each assay condition.

69 E. S. Levitan and R. H. Kramer, Nature (London) 348, 545 (1990).


Criteria for Direct Activation of Muscarinic Potassium Channel by Arachidonic Acid Metabolites

1. If AA and/or metabolites activate the KAC h channel itself without the involvement of G proteins, then the KACh channel should be activated in a way independent of intracellular GTP. Intracellular GDP/3S should not alter the metabolite effect on the KAC h channel.

2. Arachidonic acid should activate the KAC h channel independently of the patch clamp configuration provided that the proper metabolism is maintained. The final substance should activate the KAC h channel in a membrane-delimited way in the absence of GTP.

Criteria for lndirect Activation of Muscarinic Potassium Channel through Modulation of Gx Function

1. If AA and/or metabolites activate the KACh channel through the modulation of G proteins, then the activation should be GTP-dependent. Intracellular GDPflS should antagonize the metabolite-activation of the KACh channel.

2. To elucidate further the mode of action of the substances, the kinetic analysis of the GTP-induced activation of the KACh channel by the AA metabolites may be essential. The GTP-dependent activation of the KAC h channel can be enhanced either by an increase of the activated G proteins in the patch membrane or by a facilitation of the interaction between GK subunits and the KACh channel.

If arachidonic acid metabolites somehow modulate the function of GK and enhance the KAC h channel activity, the steady-state relationship between the KACh channel activity and intracellular GTP should be altered. In general, the steady-state activity of G protein is the function of the rate of GDP/GTP exchange (the turn-on reaction) and GTP hydrolysis (the turn-off reaction). Therefore, to clarify the mechanism by which the substance modulates the apparent steady-state G-protein function, the effect of the substance should be evaluated on the respective reaction rate. As long as one can assume that the substance does not affect the kinetic properties of a given G-protein-effector system other than the two rates, each of the rates can be estimated in terms of the turn-on and turn- off rate of the channel current on instantaneous application and removal of GTP in the inside-out configuration) 7 It is also possible to estimate the values by keeping the concentration of GTP at a constant value (i.e., concentration-clamp) in the whole-cell and outside-out configurations and by abruptly changing the concentration of agonists on the extracellular


side of the cell membrane. 7° For this type of kinetic analysis, a special device such as the oil-gap 71 or concentration-clamp system n is usually a powerful tool. However, as described above, it may be necessary to modify the apparatus to prevent surface absorption and deterioration of AA metabolites.

When the interaction between G K subunits and the KAc h channel is facilitated by AA metabolites, maximal activation of the channel activity induced by intracellular GTP and analogs, and exogenous active G-protein subunits, may be enhanced.

Reported Effects of Arachidonic Acid and Metabolites on Cardiac Muscarinic Potassium Channel

In atrial myocytes, AA (50/zM) in the bath solution activates the KAC h

channel in the cell-attached configuration in the presence of m2-muscarinic or Al-adenosine receptor antagonists. Ethanol (up to 0.5%), the vehicle of AA, has no effect on the channel. The effect of AA is mimicked by LTC 4 (1 .6 /xM) and specifically blocked by an inhibitor of 5-LO, AA861 (3/.tM). Thus, LTC 4 and/or metabolites are likely to mediate the effect of AA on the KACh.

The action of the AA metabolites on the cardiac KACh channel has been proposed not to be mediated by G proteins but rather to be due to a direct activation of the channel. 5° Inconsistent with such a hypothesis, the current induced by AA or metabolites in the cell-attached configuration suddenly disappears on formation of the inside-out configuration .49 Subse- quent application of GTP to the cytosolic side of the patch membrane restores the current. GDPflS antagonizes the effect of GTP. Therefore, the action of the AA metabolites on the modulation of KACh channel function is probably mediated by a G protein.

Generally, in the absence of agonists, the maximum effective concentration of GTP induces in the C1- internal solution only about 20% of the maximum activation of KAc h channel observed in the presence of agonists. 36 However, in the inside-out patch excised from a cell pretreated with the AA metabolites, GTP alone can induce the full activation of the channel in the absence of agonists. In principle, this could be due to the enhancement of the turn-on rate and/or the inhibition of the turn-off rate of G-protein function.

7o A. S. Otero, Y. Li, and G. Szabo, Pfluegers Arch. 417, 543 (1991). 71 D. Qin, M. Takano, and A. Noma, Am. J. Physiol. 257, H1624 (1989). 72 A. Yatani and A. M. Brown, Science 245, 71 0989).


In relation to this observation, Scherer and Breitwieser demonstrated that 5-LO metabolites increased the turn-on rate of the KACh current in the absence of agonists. 73 Therefore, LTC4 and/or metabolites may stimulate the GDP/GTP exchange of GK in the absence of agonists. Be- cause these authors also reported that the AA metabolites inhibited the turn-on reaction of the channel current in the presence of agonists, the interaction between the receptors and GK may be modulated by the metabolites. However, as PTX pretreatment of the cell does not affect the action of AA and metabolites in the absence of the agonists, the AA metabolites may not affect the GK-coupled receptors. In addition, LTA 4 and LTC4 applied to the pipette solution do not activate the KACh channel in the cell-attached configuration. Therefore, the site of action of the AA metabolites is likely to be downstream of the receptors. Furthermore, activation of the KACh channel by bovine brain G/3y is not inhibited by an LO inhibitor, nordihydroguaiaretic acid, nor a specific 5-LO inhibitor, AA861, 33 indicating that the LO metabolites also have no effect downstream of GK. From these observations, the most likely site of action of the AA metabolites could be GK and not the KACh channel itself.

It has also been reported that Gfly activated the KACh channel by stimulating PLAz and inducing the proportion of the 5-LO metabolites, whereas Ga directly activated the channel. 5° However, as mentioned above, the KACh channel currents induced by 10 nM G/3y are not blocked by LO inhibitors. As it was reported that the effect of 5-LO metabolites on the KACh channel was 6 to 16 times smaller than the Gfly effect on the channel, it follows that the activation of the KAC h channel by Gfly cannot be explained in terms of the activation of PLA z. In addition, platelet- activating factor activates the KACh channel by stimulating PLA2 through PTX-sensitive G protein in the cell-attached configuration if applied to the bath solution. This response is probably mediated by the activation of GK by 5-LO metabolites of AA. However, platelet-activating factor, if applied to the pipette solution, cannot activate the channel. These data indicate that GK differs from the G protein which regulates PLA2.53 There- fore, GKfly directly regulates the KACh channel, whereas the 5-LO pathway is likely to modulate G~: function in an indirect way by modifying the turn-on reaction of G K .

73 R. W. Scherer and G. E. Breitwieser, J. Gen. Physiol. 96, 735 (1990).

AUTHOR INDEX 423

Author Index

Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A

Aaronson, S. A., 280, 294 Abramowitz, J., 394 Ackerman, M. J., 409 Adams, J. M., 294 Adams, P. R., 307 Adler, E. M., 336 Afendis, S., 243 Aghajanian, G. K., 355, 376 Ahlijanian, M. K., 72, 73(3), 78 Ahn, N., 259 Akaike, N., 388 Akiyama, T., 256, 260 Aktories, K., 124 Albert, K. A., 376 Aldape, R. A., 182, 186(4), 246 Aldrich, R. W., 147 Alessandrini, A., 259 Alger, B. E., 376, 379 Alien, L., 86 Allen, R. A., 72 Allende, J., 321 Almers, W., 146 Alvarez, R., 31, 34, 44, 45(38, 39), 46(38),

50(38, 39), 52(38, 39), 92, 111 Alveja, M., 376 Amakawa, R., 228 Amundson, J., 321, 325 Andersen, P., 376 Anderson, N. G., 258 Anderson, R. E., 227-228 Anderson, W. B., 45 Andrade, R., 348, 350, 351(4), 352(4),

353(14), 355 Annamalai, A. E., 67 Aoyama, A., 271 App, H., 260 Applebury, M. L., 3, 19, 183

Apud, J. A., 86(q), 87 Aragay, A. M., 246 Arai, H., 146 Arai, Y., 416(6), 417 Aramori, I., 146 Armand, J., 280 Arriza, J. L., 395 Artemyev, N. O., 13-14, 19, 19(6), 20(6),

21(6), 23(6), 24(7), 25(7), 26(7), 27(7), 402

Aruch, J., 260 Aruffo, A., 88 Asai, D. J., 126 Ashida, Y., 416(7), 417 Ashman, C. R., 89 Atkinson, J. P., 417 Auger, K. R., 294 Augustine, G. J., 336 Ausubel, F. M., 280, 289(16), 292(16),

293(16) Avruch, J., 256 Awad, J. A., 37 Axel, R., 89, 146, 280 Axelrod, J., 395 Ayres, M. D., 98

B

Bach, A., 86 Bach, M. K., 416(5), 417 Baehr, W., 19, 183 Bahk, Y. Y., 248 Bakalyar, H. A., 57, 95, 96(5-7), 97(5), 117,

118(2), 119(2), 125(2-4), 126(3) Batdassare, J. J., 208 Ball, A., 154, 167, 169, 173(6), 174(6),

179(7), 180(7) Balla, T., 217

424 AUTHOR INDEX

Banerjee, P., 260 Barbacid, M., 294 Barde, Y. A., 364 Barish, M. E., 143 Barouki, R., 117, 125(12, 13) Barr, M., 256 Barry, P. H., 383 Bar-Sagi, D., 255, 409, 421(50), 422(50) Bartfai, T., 190 Barth, L. G., 153 Barth, L. J., 153 Bassnett, S., 298, 308 Bast, A., 416(9), 417 Batzer, A., 255 Batzer, A. G., 255 Baukal, A. J., 217 Bayley, H., 61, 68 Beacham, L. M. III, 57, 58(9) Beam, K. G., 336 Bean, B. P., 335-336, 367, 368(10) Becker, E. L., 416(1), 417 Beckingham, K., 102 Beebe, D. C., 298, 308 Beer, M. S., 86 Beiderman, B., 14, 83 Bekkers, J. M., 376 Belardetti, F., 394, 409 Bell, L., 395 Bell, R. M., 131 Belles, B., 370 Bender, J., 32 Benovic, J. L., 395 Bensadoun, A., 262 Berg, P., 288 Berridge, M. J., 133, 145, 181,188, 195,207,

219, 220(1), 227, 237, 303 Berridge, W. J., 147 Berstein, G., 155, 182 Bertrand, L., 86 Bhakta, S., 147 Bianchi, R., 72 Biehl, M., 365 Bigay, J., 6, 12(10), 13, 183 Binetruy, B., 271,273(9) Bio-Rad Ion Exchange Manual, 190 Birkett, D. J., 414 Birnbaumer, L., 37, 73, 124, 183, 187(27),

321,337,365,373,373(4), 385-386,388, 391,391(6, 11, 12), 393, 393(6, 12, 16), 394-396, 406-408,408(41)

Birnbaumer, M., 391,393(16) Bishop, J. M., 277-278 Bishop, W. R., 131 Bitensky, M. W., 4 Blank, J. L., 155, 182, 183(22), 237-238,

238(7), 239(7), 241(13), 242(7), 243 Blanton, M. G., 376 Blazynski, C., 228 Bliton, C., 331,333(16) Blitzer, R. D., 140, 145, 147, 147(16),

151(16), 246, 375 Bloch, D. B., 83 Block, B., 343 Blomgren, K., 370 Bloomquist, B. T., 228, 246 Blumer, K. J., 260, 270(13) Bockaert, J., 357-358, 358(3), 359(5), 360(3) Bohme, E., 118 Bokoch, G. M., 417 Bominaar, A. A., 207, 215 Bonventre, J. V., 83 Borasio, G. D., 364 Borgeat, P., 416(3), 417 Borleis, J., 117, I18(17), 120(17) Bosse, E., 336 Botchan, M. R., 89 Boton, R., 144, 152, 153(50) Boulikas, T., 106 Boulton, T. G., 258, 260 Bourne, H. R., 14, 82-83, 83(I, 2, 4), 86(1-

3, 16, o), 87, 87(1-3), 91(1, 2), 94(1), 126, 276,294, 394-395

Bouvier, C., 86 Bouvier, M., 126 Bowtell, D., 255 Boyajian, C. L., 72, 73(10), 74(10), 75(10),

76, 79(10), 80(10) Boyer, J. L., 182, 183(18), 196-197, 197(9),

202(14), 206(9, 15), 207(9, 15), 237 Bradford, M. M., 6, 275 Bradham, L. S., 72 Bragt, P. C., 416(9), 417 Braquet, P., 416(3), 417 Brashler, J. R., 416(5), 417 Brattain, K. A., 182, 183(22), 237, 238(7),

239(7), 242(7) Brautigan, D. L., 260 Breckenridge, B. M., 72 Breitwieser, G. E., 349, 351,351(1), 356(1),

365, 385, 395,422

AUTHOR INDEX 425

Brendel, S., 19, 184 Brent, R., 280,289(16), 292(16), 293(16) Brodie, B. B., 41 Brooker, G., 44, 50(36), 72, 80(9), 147,

154(43) Brooks, M. W., 255 Brostrom, C. O., 37, 72 Brostrom, M. A., 37 Brown, A. M., 183, 337, 365, 373, 373(4),

385-386, 388, 391, 391(6, 11, 12), 392- 393, 393(6, 12, 19), 394-396, 406, 408, 421

Brown, A.M.C., 83, 85(11) Brown, B. L., 72 Brown, D. A., 357-358, 360(4) Brown, J. E., 227-228 Brown, K. D., 147 Brown, K. O., 145, 182 Brown, R. L., 23 Browning, M. D., 79, 80(20) Bruder, J., 273,274(12) Bruder, J. T., 256 Bryan, J., 391, 393(16) Buck, K., 86 Buckingham, L., 158 Buday, L., 255 Buku, A., 145,275 Bunzow, J. R., 86 Burgoyne, R. D., 303 Burke, M., 331,333(17) Bushfield, M., 56, 57(4) Buss, J. E., 290 Buttimore, C., 85

C

Cahalan, M. D., 320 Caldwell, K. K., 76-77 Calos, M. P., 89 Campbell, C. R., 88, 110 Campbell, J. S., 259 Campbell, K. P., 335-336, 339, 340(18), 341,

342(20), 343,343(20), 344, 344(20) Camps, M., 132, 181-182, 183(19, 20),

188(21), 193(19), 195(19), 196,237,245, 331

Cantley, L. C., 294 Capdevila, J., 416(4), 417 Capon, D. J., 285

Carcagne, J., 280 Carlen, P. L., 376 Caron, M., 395 Caron, M. G., 86, 126, 145, 147(16), 151(16) Carozzi, A., 132, 182, 183(20), 188(21), 208,

237, 245-246 Carpenter, C., 294 Carter, H. R., 137 Carty, D. J., 94, 141, 145, 145(5), 151.

151(5), 275 Casey, P. J., 395 Cassel, D., 352 Catanzariti, L., 86(0), 87 Catt, K. J., 217 Caulfield, M. P., 357, 363 Cavalie, A., 386 Cepko, C. L., 283 Cerione, R. A., 13, 21(5) Chabre, M., 6, 12(10), 13, 183 Chabre, O., 82, 86(3, o), 87, 87(3) Chae, H. Z., 132, 145, 182 Challiss, R.A.J., 34 Chan, H. W., 90 Chan, P. S., 41 Chang, J., 417 Chardin, P., 255 Charlesworth, A., 86 Charlton, M. P., 336 Cheek, T. R., 303 Chen, C., 89, 275,280 Chen, E. Y., 285 Chen, J., 95, 96(12), 108-109, 113(2), 115(2,

4), 116(4), 117, 118(15), 125(8), 126(8), 135, 145

Chen, L., 95, 96(9, 10), 117, 125(7, 9) Chen, S., 61 Cheng, H.-C., 261 Cheng, V. C., 89 Cheung, W. Y., 72 Ching, N. S., 126 Cho, K. S., 219, 220(5-7), 229 Choi, K. D., 154, 155(1), 168, 181, 195,

196(4), 219, 220(2), 237, 243(3) Choi, K.-J., 131 Choi, W. C., 229 Christie, M. J., 355, 396 Civelli, O., 86 Clapham, D., 147, 148(40), 154(40, 41), 307,

321-322, 322(3), 324(4-6), 325, 331, 333(6, 16, 17), 335(4-6)

426 A U T H O R I N D E X

Clapham, D. E., 330, 393,395,403(4), 408- 409, 421(50), 422(50)

Clark, O. H., 294 Claro, E., 137 Cleland, W. W., 32 Cobb, M. H., 258,260 Cockcroft, S., 137, 154-155, 155(2), 159(3),

167, 167(3), 168-169, 172(5), 173(6), 174(6), 179(7), 180(7)

Codina, J., 124, 135, 183, 187(27), 275, 321, 337, 365, 373,385-386, 388, 391,391(6, 12), 392-393, 393(6, 12, 16, 19), 394- 395,406-408, 408(41)

Cohen, A. I., 228 Cohen, I., 368 Cohn, E. J., 17 Collins, S. J., 184 Colman, A., 330 Colman, M. S., 271,273(6) Colman, R. F., 58, 60(13), 67, 67(11) Colquhoun, D., 398 Colvin, R. A., 72 Cone, R., 86 Cone, R. D., 86 Conklin, B. R., 82, 83(2), 86(2, 3, 16, o), 87,

87(2, 3), 91(2), 126, 395 Cooke, A. M., 147 Cooper, A. G., 417 Cooper, D.M.F., 37, 56, 71-72, 73(3, 10),

74(10), 75(10), 76-79, 79(10), 80(1, 20), 95, 96(11), 117, 120(6), 124(14), 125(6, 14), 126(6), 127(14)

Cooper, G. M., 277, 283 Cooper, K., 359 Coote, P. R., 134 Copeland, T. O., 268 Coronado, R., 343 Correia, J. J., 67 Corson, D. W., 227 Cory, S., 294 Costa, E., 86(q), 87 Coteccia, S., 145, 147(16), 151(16) Counis, R., 44, 50(37) Coussen, F., 57, 95, 96(5), 97(5), 117, 118(2),

119(2), 125(2) Cox, A. D., 271-272, 277, 290 Crews, C. M., 259 Croll, D., 147, 154(43) Cross, M., 294 Crowley, W. F., Jr., 86

Cui, Z., 83 Cullen, B. R., 88 Cunnick, J. M., 15 Cunningham, E., 137, 154, 167-169, 173(6),

174(6), 179(7), 180(7)

D

D'Alayer, J., 95 Dale, N., 409 D'Alonzo, J. S., 14 Daly, R., 255 Daly, R. J., 255 D'Angelo, G., 67 Daniels, D. V., 44, 45(38, 39), 46(38), 50(38,

39), 52(38, 39) Danielsen, M., 90 Dascal, N., 140-141, 142(2), 143(2), 144,

145(4, 14), 146(2, 3), 148, 152, 153(50), 365

Dash, P., 151 Davidson, A., 151 Davidson, N., 141, 144, 145(14), 328, 336 Davidson, R. L., 89 Davis, L. J., 257 Davis, R. L., 117 DeBernardi, M. A., 72, 80(9) De Erausqin, E., 86(q), 87 Degtyar, V., 372 Dekker, A., 200 Delcour, A. H., 336 DeLisle, S., 147 Demaurex, N., 308, 317, 319 Denning, G., 147 DePhinho, R. A., 258 Der, C. J., 271-272, 273(10), 277, 281,283,

285, 290, 290(17) Deretic, D., 14 Deterre, P., 13 De Vivo, M., 131, 135, 145 DeVivo, M., 275 Devlin, M. J., 19 Devreotes, P. N., 117, 118(17), 120(17) De Waard, M., 335, 341, 342(20), 343(20),

344, 344(20) de Weerth, A., 147 Dexter, T. M., 294 Dhanaseharan, N., 14 Didsbury, J. R., 86(m, n), 87

AUTHOR INDEX 427

Dietzel, C., 102 Dignard, D., 395 Dingus, J., 183 Dinsart, C., 118 Dodson, G. S., 255 Dolecki, G. J., 126 Dolphin, A. C., 352, 356, 363,371 Domino, S. E., 45 Dong, K., 147, 151(33) Downes, C. P., 133, 195-197, 199(12),

206(12, 18), 207(12, 18) Downward, J., 255-256 Draijer, R., 215 Dratewka-Kos, E., 414 Dreher, R.-M., 95 Dreyer, W. J., 18, 183 Drummond, A. H., 195 Dubendorff, J. W., 266 Duckworth, B., 294 Duerson, K., 321,322(3), 324(3), 335(3) Duff, R. A., 409 Duh, Q.-Y., 294 Dumont, J. E., 118 Dumont, J. N., 141, 153 Duncan, J. L., 158 Dunn, J. J., 266 Dunn, P. M., 134 Dunwiddie, T. V., 376 Dush, M. K., 126 Dyer, E. L., 32

E

Eckstein, F., 352 Edwards, E., 228 Edwards, F. A., 376 Egan, S. E., 255 Eisen, D., 416(2), 417 Eisfelder, B. J., 83 Elledge, S. J., 256 Ellis, C., 219 EI-Maghrabi, M. R., 67 Elmsley, K. S., 352 Elsbach, P., 414 Emori, Y., 219, 220(11, 12) Endo, M., 256 Ennulat, D., 321,322(3), 324(3), 335(3) Erickson, A. K., 261-262 Erickson, R. L., 259

Ericsson, L., 259 Erlanger, B. F., 417 Erneux, C., 215 Europe-Finner, G. N., 207 Eva, C., 86 Evanczuk, A. T., 19 Evans, T., 82, 83(1), 86(1), 87(1), 91(1),

94(1), 197, 388, 391(12), 393(12) Ewel, C., 373, 374(27), 394 Exton, J. H., 132, 145, 155, 182, 183(22),

237-238,238(7), 239(7), 241(13), 242(7), 243

F

Fain, G. L., 228 Fain, J. N., 137, 208 Falardeau, P., 86 Falck, J. R., 416(4), 417 Farr, A. L., 338 Federman, A., 82, 83(1), 86(1), 87(1), 91(1),

94(1) Federman, A. D., 82, 86(3, 16), 87, 87(3),

126, 395 Feichtinger, H., 294 Fein, A., 227 Feinstein, P. G., 57, 95, 96(6), 117, 118(2),

119(2), 125(2, 3), 126(3) Feigner, D. L., 90 Fensome, A., 167-169, 179(7), 180(7) Fernley, R., 255 Ferreira, P. A., 248 Finkel, T., 283 Firestone, J. A., 79, 80(20) Fisher, G. J., 208 Fisher, S. C., 228 Fitzpatrick, F. A., 413-414, 416(5), 417 Flanagan, C. A., 147, 151(33) Fleisch, J. H., 414 Fleurdelys, B., 257 Flockerzi, V., 336, 365 Flores, P., 271,273(4) Florio, V. A., 37 Fordis, C. M., 89 Forquet, F., 13 Frankenfeld, C. D., 117, 120(11), 124(11),

125(11), 126(11) Franson, R. C., 416(2), 417 Franza, B. R., 286

428 AUTHOR INDEX

Franzini-Armstrong, C., 341, 342(20), 343, 343(20), 344(20)

Frazier, A. L., 86 Frech, G. C., 147 Freedman, V. H., 277, 287(1), 290(1), 294(1) Freifelder, D., 24 Fremeau, R. T., 86 French, M., 72, 80(7) Friedrich, T., 336 Fritsch, E. F., 110, 121, 123(22), 124(22),

186, 249, 250(23), 251(23), 266, 273, 274(13), 280, 289(15), 292(15), 293(15)

Frohman, M. A., 126 Fugiwara, T., 147 Fujisaki, J., 147 Fukuda, K., 147 Fung, B.K.-K., 3-4, 399 Furth, M. E., 257 Furuichi, T., 336

G

Gadek, T. R., 90 Gage, F. H., 280, 288(12) Gale, N. W., 255 Gallagher, C., 246 Gallagher, R. E., 184 Gallego, C., 83,271,272(5), 273(5), 275(5) Gallo, R. C., 184 Galper, J., 393,395,403(4) Gannage, M.-H., 117, 125(12, 13) Gao, B., 95, 96(8), 117, 125(5), 126(5) Garbers, D. L., 32, 45 Garbers, E. L., 35 Gardner, A. M., 258, 260, 262(11), 265(11),

270(13) Garrels, J. I., 286 Garritsen, A., 72, 73(10), 74(10), 75(10), 79,

79(10), 80(10, 20) Gates, P., 359 Gautam, N., 94, 145, 182-183,196, 237,246,

366 Gehm, B., 228 Gelfand, D. H., 122, 249 Geny, B., 155, 159(3), 167, 167(3), 169,

172(5) Gerard, C., 86(n), 87 Gerard, N., 86(n), 87 Gerschenfeld, H. M., 357, 358(3), 360(3)

Gershengorn, M. C., 134, 147 Gettys, T. W., 86(m), 87 Ghalayini, A. J., 227-228 Giannatasio, G., 72 Gibbons, B. H., 126 Gibbons, I. R., 126 Gibbs, J. B., 14 Giddings, B. W., 255 Gierschik, P., 132, 181-183, 183(19, 20),

187(27), 188(21), 193(19), 195(19), 196, 237, 245, 331

Gil, L., 416(4), 417 Gilbert, M., 336 Gilbert, M. M., 336 Gilbert, T. L., 146 Gillo, B., 140, 145, 146(3), 147, 151,151(33),

152, 153(50) Gilman, A. G., 14, 57, 79, 87, 90, 91(31), 95-

96, 96(8), 97(13), 100(13), 101, 105(t5), 106(15), 107(15), 117, 118(2), 119(2), 124, 125(2, 3, 5), 126, 126(3, 5), 196, 245, 394-395, 407(2)

Gingrich, J. A., 86 Girard, S., 147, 148(40), 154(40, 41), 307,

322, 324(4, 5), 325, 335(4, 5) Girardot, J. M., 78 Gispen, W. H., 200 Gluzman, Y., 186 Godinot, N., 86 Goeddel, D. V., 285 Goelet, P., 151 Goetzl, E. J., 147 Goldhammer, A. R., 76 Goldsmith, P. K., 394 Gollasch, M., 365, 369 Gomori, G., 188 Gomperts, B. D., 157, 320 Gonzalez-Ouva, C., 13 Goping, G., 336 Gorman, C., 84 Gospodarowicz, D., 45 Gotoh, Y., 260 Gout, I., 167 Graf, R., 391,393,395 Graham, F. L., 280, 282(14) Grandy, D. K., 86 Granstrom, E., 414 Grant, F. J., 395 Grant, G. A., 17 Graves, L., 259

AUTHOR INDEX 429

Grayson, D. R., 86(q), 87 Graziadei, L., 409, 421(50), 422(50) Graziani, A., 294 Greengard, P., 376 Grenet, D., 391 Grinstein, S., 319 Grissmer, S., 320 Griswold-Prenner, I., 261 Grodberg, J., 266 Grunewarld, K., 294 Grynkiewicz, G., 297, 298(2), 308, 325,

334(7) Guillemette, G., 217 Guillory, R. J., 61 Gundersen, C. B., 141 Gupta, S. K., 83,271,272(5), 273(5), 275(5) Gustafson, M., 303 Gutman, A., 271,273(4)

H

Ha, K. T., 280 Haga, K., 147 Haga, T., 147 Hagen, F. S., 146 Haiech, J., 95 Haldeman, B. A., 146 Hailer, H., 365 Halnon, N. J., 95, 96(9), 117, 125(7, 9) Hamill, O. P., 308, 360, 367, 385, 387(5),

397,415(25) Hamilton, S. L., 337 Hamm, H. E., 13-14, 18-19, 19(6, 20),

20(6), 21(6), 23(6), 24(7), 25(7), 26(7), 27(7), 402

Hammond, C., 357, 358(3), 360(3) Han, P.-L., 117 Hancock, R., 106 Hanley, M. R., 133 Hanoune, J., 117, 125(12, 13) Harada, Y., 147 Harafuji, H., 140 Harden, T. K., 136, 182, 183(18), 195-197,

197(9), 199(12), 202(14), 206(9, 12, 15, 18, 19), 207(9, 12, 15, 18, 19), 237

Hardman, J. G., 32 Hargrave, P. A., 3 Harkins, R., 147 Harkness, J., 13

Harootunian, A. T., 303 Harper, M.J.K., 413,415(56) Harrison, J. K., 259 Harris-Warrick, R. M., 357, 358(3), 360(3) Harrylock, M., 259 Harsh, G., 294 Hartig, P. H., 141 Harvey, M. S., 171 Harwood, A. E., 255 Harwood, J. P., 37 Haugland, R. P., 299 Hauser, C. A., 271 Hawkins, P. T., 196 Hawthorne, J. N., 228 Hayashi, F., 228 Hayashi, M., 416(6), 417 Hayashi, Y., 147 Haystead, C., 259 Haystead, T.A.J., 259 Healy, E. C., 246 Heasley, L. E., 260, 271, 272(5), 273(5),

275(5) Heidecker, G., 273,274(12) Heideman, W., 83, 95 Heldin, C.-H., 219 Hellevuo, K., 117, 124(14), 125(14), 127(14) Hellmiss, R., 321, 322(3), 324(3), 335(3) Helmkamp, G. M., Jr., 171 Hemmings, B. A., 86(o), 87 Hepler, J. R., 145, 182, 245 Herberg, J. T., 32 Herlihy, J. T., 413,415(56) Hernandez-Cruz, A., 307 Herzberg, M., 89 Hescheler, J., 330, 365, 368-369, 369(15),

370, 372-374, 374(27), 375(29), 394 Hess, P., 336 Hestrin, S., 376 Heumann, R., 364 Higashijima, T., 155, 182 Higgins, J. B., 395 Hildebrandt, J. D., 32, 124, 151, 183, 391,

393(16), 407, 408(41) Hiles, I., 167 Hill, S. J., 134 Hille, B., 349, 365,367, 385,395 Hingorani, V. N., 3 Hinsch, K.-J., 365 Hirata, M., 147 Hirose, T., 147

430 AUTHOR INDEX

Hisaka, M. M., 285, 290 Ho, Y.-K., 3-4, 388, 391(12), 393(12), 399,

406 Hoffman, B. J., 141 Hoffman, P. L., 117, 124(14), 125(14),

127(14) Hoffman, R., 309 Hofmann, F., 336, 365 Hogenkamp, H.P.C., 57, 58(10) Hokin-Neaverson, M., 63 Holm, M., 90 Holmes, W. E., 86(1), 87 Holter, W., 89 Homburger, V., 357-358, 358(3), 359(5),

360(3) Homcy, C. J., 95, 96(9), 117, 125(7, 9) Homma, Y., 219, 220(11, 12) Honda, Z., 147 Hopkins, R., 144, 145(14) Hori, S., 146 Horn, R., 418 Hotta, Y., 227 Hou, C., 182, 183(19), 193(19), 195(19), 196,

237, 331 Houamed, K. M., 146 Hougan, L., 395 Housey, G. M., 83 Howard, B. H., 89 Hsuan, J. J., 167-169, 179(7), 180(7) Hu, G.-Y., 376 Huang, K. N., 146 Huang, Y.-C., 72 Hughes, K. T., 211 Hunt, D. F., 259, 261-262 Hunter, D. D., 349, 365, 385, 395 Hunter, T., 285 Hurley, J. B., 4 Hvalby, O., 376 Hwang, P. M., 117

Ichiyama, A., 147 Ifune, C., 144, 145(14) Iguchi, S., 416(6), 417 Imagawa, T., 339, 343 Imai, A., 134 Imler, J. L., 271,273(3)

Imoto, K., 336 Imoto, Y., 337, 373, 386 Inanobe, A., 399 Ingebritsen, T., 261 Inglese, J., 395 Innes, M. A., 122, 249 Inoue, H., 227 Inoue, M., 388 Ip, N. Y., 258 Irisawa, H., 386 Irvine, R. F., 207, 227 Isenberg, G., 396 Ishikawa, Y., 95, 96(9), 117, 125(7, 9) Itaya, K., 199 Ito, H., 354, 395,399, 402, 403(33), 404(33),

405, 405(33), 406(33), 407(19, 33), 408(33), 409, 409(33), 411(33, 49), 421(36, 49), 422(33)

Ito, K., 147 Iversen, L. L., 86 Ives, H. E., 86(o), 87 Iyengar, R., 32, 95, 96(12), 108-109, 113(2),

115(2, 4), 116(4), 117, 118(15), 124, 124(10), 125(8, 10), 126(8, 10), 135, 141, 145, 145(5), 147(16), 151, 151(5, 16), 244, 246, 275, 391,393, 393(16), 395

J

Jackson, M. B., 350, 352(3), 383 Jacobowitz, O., 108-109, 117, 118(15),

124(10), 125(10), 126(10) Jaconi, M., 308, 319 Jafri, M. S., 147 Jainchill, J. L., 280 Jakobs, K. H., 32, 37, 41, 45(34), 56, 57(3),

124, 182, 183(19), 193(19), 195(19), 196, 237, 331

Jakschik, B. A., 409 Jared, D. W., 153 Jarnagin, K., 147 Jarvie, K. R., 86 Jelsema, C. L., 395 Jeng, S. J., 61 Jensen, A. M., 259 Jensen, R. T., 147 Jesse, R., 416(2), 417 Jessel, T. M., 146

AUTHOR INDEX 431

Jhon, D.-Y., 155, 182, 183(24), 186(6), 219, 220(3, 4, 13), 221(13), 229, 237, 243- 244, 244(18), 245-246, 246(4)

Jiang, H., 244 John, J., 364 Johnson, F., 61, 62(17), 63(17), 68, 69(25),

70(25), 71(25) Johnson, G. L., 14, 83, 258, 260, 262(11),

265(11), 270(13), 271, 272(5), 273(5), 275(5)

Johnson, M. D., 83 Johnson, R. A., 31-32, 34-35, 37, 40, 43(2),

45(2), 56-57, 57(3, 4), 58(6), 60(6), 61, 62(17), 63(17), 65(6), 66(6), 67(6), 68, 69(25), 70(25), 71(25), 86, 92, 111

Joho, R. H., 147 Jolles, J., 200 Jones, S. W., 352 Jorquera, H., 321 The Journal of Clinical Investigation, 319 Julius, D., 146

K

Kageyama, R., 147 Kahl, S. D., 335, 341, 342(20), 343(20),

344(20) Kahn, M., 63 Kaibara, M., 386 Kakizuka, A., 147 Kamata, T., 256 Kameyama, M., 368, 386 Kandel, E. R., 409 Kandell, E. R., 151 Kanevshy, M., 336 Kangawa, K., 147 Kao, M., 117, 124(14), 125(14), 127(14) Kapeller, R., 294 Kaplan, D., 256 Kaplan, S., 255 Karin, M., 271,273(9) Karlsson, J. O., 370 Karr, D. B., 49, 54(43) Katada, T., 395, 399, 402,403(33), 404(33),

405, 405(33), 406(33), 407(19, 33), 408(33), 409(33), 411(33), 421(36), 422(33)

Katan, M., 182, 186(4), 219, 220(10), 232, 246

Katsushika, S., 95, 96(9, 10), 117, 125(7, 9) Katz, A., 182, 183(23), 237, 244-245 Kaufman, R. J., 83, 186 Kawabe, J., 95, 96(9) Kawabe, J.-I., 95, 96(10), 117, 125(7, 9) Kawasaki, E., 294 Kaziro, Y., 256 Keller, B. U., 376 Kemp, B. E., 261 Kempf, J., 78 Kendall, D. A., 134 Keown, W. A., 88, 110 Khosravi-Far, R., 285 Kim, C. G., 248 Kim, D., 330,408-409, 421(50), 422(50) Kim, M., 336 Kim, M. J., 248 Kindahl, H., 414 Kindl, B., 414 Kingston, R. E., 280, 289(16), 292(16),

293(16) Kirsch, G. E., 373,391,406, 408 Kirschmeier, P. T., 83 Kitts, P. A., 98 Klapper, D. G., 197, 206(19), 207(19) Klee, C. B., 37, 102 Klemenz, R., 271 Kleuss, C., 19, 184, 330, 373,374(27), 394 Klinz, F.-J., 369, 374, 375(29) Klockner, U., 396 Knapp, M., 151 Knopf, J., 219, 220(13), 221(13), 229, 243,

246 Knopf, J. L., 182, 186(4, 6), 246 Knowles, J. R., 61, 68 Kobayashi, I., 399, 402, 403(33), 404(33),

405, 405(33), 406(33), 407(33), 408(33), 409(33), 411(33), 421(36), 422(33)

Kobilka, B. K., 86 Koesling, D., 118 Kohler, M., 86 Kohnken, R., 183 Kojima, M., 147 Kokubun, S., 386 Konishi, Y., 416(6), 417 Konnerth, A., 376 Konno, M., 416(6), 417

432 A U T H O R I N D E X

Kontani, K., 399 Kosako, H., 260 Koutz, C. A., 228 Kozasa, T., 245 Kragh-Hansen, U., 413 Kraicer, J., 72, 80(7) Kramer, R. H., 419 Krans, H.M.J., 37 Krause, J., 86 Krause, K., 147 Krause, K.-H., 308, 317, 319 Krebs, E. G., 259 Kriegler, M., 288 Kriegstein, A. R., 376 Krinks, M. H., 37 Krishna, G., 41 Krishtal, O. A., 388 Kriz, R., 219, 220(13), 221(13), 229, 243,246 Kriz, R. W., 182, 186(4, 6), 230(21), 232, 246 Kroll, S., 13, 21(5) Kroll, S. D., 145, 151,275 Krupinski, J., 57, 95-96, 96(5), 97(5, 13),

100(13), 117, 118(2), 119(2), 120(11), 124(11), 125(2, 3, 11), 126, 126(3, 11)

Kuang, W. J., 86(1), 87 Kubo, T., 147 Kucherlapati, R. S., 88, 110 Kfihn, H., 13, 183 Kuijper, J. L., 146 Kung, H.-F., 256 Kuno, M., 147 Kuntson, J. C., 90 Kurachi, Y., 354, 385, 393-396, 399, 402-

403, 403(4, 15, 33), 404(33), 405, 405(33), 406, 406(33), 407(19, 33), 408, 408(33), 409,409(33), 411(33, 53), 417, 421(36, 49), 422(33, 53)

Kurjan, J., 102 Kusakabe, K., 395 Kusano, K., 140, 142, 143(10), 153, 326,

327(10) Kushner, J. A., 86 Kwatra, M. M., 395 Kyriakis, J. M., 256, 260

L

Lagarde, A., 134 L'Allemain, G., 258, 259(3)

Lamb, T. D., 228 Lammers, R., 255 Land, H., 277, 285(5) Landau, E. M., 140-141,142(2), 143(2), 145,

145(5), 146(2), 147, 147(16), 148, 151, 151(5, 16), 246, 375

Landis, C. A., 276, 294 Lange-Carter, C. A., 258, 260, 270(13) Langer, S. J., 271,273(6) Lanni, C., 416(1, 2), 417 Lanni, F., 363 Lass, Y., 140, 142(2), 143(2), 145, 146(2, 3),

152, 153(50) Latorre, R., 343 Laugwitz, K.-L., 396 Laurie, D. J., 86 Laursen, R. A., 15 Lebkowski, J. S., 89 Lechleiter, J., 147, 148(40), 154(40, 41), 307,

321-322, 322(3), 324(3-6), 331, 333(6, 16), 335(3-6)

Lecocq, R., 215 Lee, C. H., 90, 182, 245 Lee, C.-W., 182, 183(24), 219,220(3, 4), 227,

229, 237, 243-244, 244(18), 245, 246(4), 248

Lee, D. Y., 211 Lee, E., 14, 101 Lee, H.-H., 182, 219, 220(3), 229, 243,

244(18), 245, 246(4) Lee, J., 86(1), 87 Lee, K.-H., 182, 183(24), 219, 220(3, 4), 227,

229, 237, 243, 244(18), 245,246(4), 248 Lee, K.-Y., 219, 220(5-7), 229 Lee, L. S., 89 Lee, S.-J., 248 Lee, S. Y., 195, 207 Lefkowith, J. B., 409 Lefkowitz, J., 395 Lefkowitz, R. J., 126, 145, 147(16), 151(16) Lefort, A., 118 Lehman, T. C., 117, 120(11), 124(11),

125(11), 126(11) Lending, C. R., 246 Leonard, J. P., 336 Lester, H., 328 Lester, H. A., 141, 144, 145(14), 336 Leung, A. T., 336, 339, 343 Levin, L. R., 117 Levine, A. J., 141

AUTHOR INDEX 433

Levinson, A. D., 281,285 Levis, R. A., 312 Levitan, E. S., 419 Levitzki, A., 73 Levkovitz, H., 352 Lew, D. P., 317, 319 Lew, P. D., 308 Lewin, B., 294 Lewis, D. L., 409, 421(50), 422(50) Lewis, R. S., 320 Li, B.-Q., 256 Li, N., 255 Li, W., 255 Li, Y., 421 Libert, F., 118 Liebman, P., 13 Liebman, P. A., 9, 19 Lira, C., 395 Lin, H. Y., 86(o), 87 Lin, K. C., 117, 118(17), 120(17) Lin, L.-L., 219 Lin, M. C., 37, 41 Lin, Y., 72 Lindau, M., 320 Linder, M. E., 101 L'Italien, J. J., 15 Litosch, I., 208 Livelli, T. J., 146 Lively, M. O., 67 Lledo, P. M., 358, 359(5) Lodish, H. F., 86(o), 87 Logothetis, D. E., 330, 393,395,403(4), 408 Londos, C., 32, 37, 43, 45(35), 46(35), 56,

74, 90, 92(30) Long, R. A., 57, 58(10) Loomis, C. R., 131 Lopez, N. G., 14 Lotan, I., 151, 365 LoTurco, J. J., 376 L6w, H., 37 Lowe, M., 352 Lowenstein, E. J., 255 Lowndes, J. M., 63, 83 Lowry, O. H., 338 Lozeman, F. J., 259 Lubbert, H., 141, 144, 145(14) Lupica, C. R., 376 Lupu, M., 141, 145(4) Lussier, B., 72, 80(7) Lynch, J. W., 383

Lynch, K. R., 259 Lynch, T. J., 72 Lyons, J., 294

M

Ma, H.-W., 95, 96(12), 109, 113(2), 115(2), 117, 125(8), 126(8), 244, 246

MacDermott, A. B., 146 MacDonald, M. J., 281,285, 290(17) MacKay, V. L., 395 Mackin, W. M., 228 Maclouf, J., 414 Mac Neil, S., 72 MacPherson, I., 290 MacVicar, B. A., 355 Madamba, S., 409 Maeda, A., 147 Maenhauat, C., 118 Magnusson, K.-E., 303 Maki, Y., 416(7), 417 Malenka, R. C., 353(14), 355 Malinow, R., 376 Malinski, J. A., 18, 19(20) Mailer, J. L., 258 Manclark, C. R., 124, 391,393(16) Maniatis, T., 110, 121,123(22), 124(22), 186,

249,250(23), 251(23), 266,273,274(13), 280, 289(15), 292(15), 293(15)

Marcus, S., 256 Margolis, B., 255 Mark, G. E., 268 Marnett, L. J., 416(4), 417 Marsh, K., 288 Marshall, M. S., 256 Martell, A. E., 73, 74(13) Martin, G. R., 126 Martin, J. M., 349, 365,385, 395 Martin, K. A., 86 Martinez, S., 321 Martino, P. A., 261-262 Martinson, E. A., 34 Marty, A., 308, 360, 367, 384-385, 387(5),

397,415(25), 418 Maruyarna, K., 286 Mason, J. I., 416(4), 417 Masters, S. B., 14, 83,276 Masu, M., 141, 147 Masu, Y., 147

434 AUTHOR INDEX

Mathews, H. R., 228 Mathias, R., 368 Matsuda, S., 260 Matsuo, H., 147 Mattei, M.-G., 117, 125(13) Mattera, R., 385, 391,393, 395 Matuoka, K., 255 Matus-Leibovitch, N., 151 Maune, J. F., 102 Mauilce, D. H., 197 Mayr, G., 317 Mazzoni, M. R., 18, 19(20) McAllister, G., 86 McConnell, D. G., 228 McCormick, F., 294 McEachern, A. E., 147 McFadzean, I., 357-358, 360(4) McFarland, K. C., 86 McGregor, H., 417 McGuire, J. C., 416(5), 417 McNeil, P. L., 363 Meldrum, E., 182, 186(4), 208,219, 220(10),

230(21), 232, 246 Mellon, P., 147, 151(33) Mengod, G., 151 Menon, T., 32 Menon-Johansson, A. S., 363 Merphy, R. F., 363 Merilfield, R. B., 16 Methfessel, C., 142 Metzger, H., 95, 103, 105(22) Michel, H., 259 Michel, T., 83 Michell, R. M., 195 Middlemiss, D. N., 86 Mikami, A., 147, 336 Miki, I., 147, 409, 411(49), 421(49) Miki, N., 4 Mikoshiba, K., 336 Miledi, R., 140-143, 143(10), 153, 326,

327(10) Millar, F. A., 228 Miller, A. D., 85 Miller, C., 343 Miller, D. L., 15 Miller, R. J., 336 Miller, R. P., 147, 151(33) Miller, R. T., 14, 83 Miller, Z., 45 Milligan, G., 94, 357-358, 360(4)

Mills, J. S., 13-14, 24(7), 25(7), 26(7), 27(7) Milman, G., 89 Milona, N., 117, 118(17), 120(17) Min, D. S., 248 Minami, M., 147 Misconi, L., 261 Mishina, M., 142, 147 Mitra, A., 63 Miyamoto, T., 147, 416(6), 417 Mocz, G., 126 Moffat, B. A., 266 Mollner, S., 126 Mongongu, S., 44, 50(37) Monneron, A., 95 Montagnier, L., 290 Montal, M., 343 Montell, C., 228, 246 Moodie, S. A., 256 Moomaw, C., 243 Moon, K. H., 182, 246 Moor, B., 72, 80(7) Moos, M. C., Jr., 40 Moreau, C., 215 Moreton, R. B., 303 Morgenbesser, S. D., 258 Moil, Y., 336 Moilarty, T. M., 141, 145, 145(5), 147, 151,

151(5) Moilson, A. R., 409 Morita, E. A., 183 Moriyama, K., 260 Morris, A. J., 136, 182, 195, 197, 199(12),

206(12, 15, 18, 19), 207(12, 15, 18, 19) Morton, D. R., 413 Mueller, P., 343 Muir, C. A., 228 Mullaney, I., 358, 360(4) Mulligan, R. C., 283 MulvihiU, E. R., 146 Murad, F., 90, 91(31) Murphy, L. W., 228 Murphy, P. M., 147 Musser, J. H., 417 Myers, S. P., 414

N

Nadler, E., 141, 145, 145(4) Naeve, J., 328

AUTHOR INDEX 435

Nagarkatti, S., 15 Nakafuku, M., 256 Nakai, C., 44, 50(36) Nakai, J., 336 Nakajima, T., 354, 385, 395, 403, 403(15),

405,409, 411(53), 422(53) Nakamura, M., 147 Nakanishi, S., 141, 146-147 Nakatani, K., 228 Nakayama, K., 147 Narayanan, N., 72, 80(7) Narumiya, S., 147 Nathanial, D., 321,322(3), 324(3), 335(3) Nathanson, N. M., 349, 365,385,395 Navon, S. E., 3 Needlernan, P., 409 Neer, E. J., 32, 37, 83, 116, 330, 393, 395,

403(4), 408-409,421(50), 422(50) Neher, E., 143, 146(11), 154(11), 308, 360,

367-368, 376, 384-385, 387(5), 397, 415(25)

Neidel, J. E., 131 Nesbitt, J. A. III, 45 Neumann, E., 90 Newell, P. C., 207 Nicholas, R. A., 197 Nicholson, C. D., 34 Nicholson, R., 271 Nicoll, R. A., 353(14), 355, 376, 379 Nikolics, K,, 86 Nilakantan, R., 117, 125(9) Nishida, E., 260 Nishima, H., 399 Nishizuka, Y., 181 Noble, A. J., 86 Noma, A., 368, 386, 418,421 North, A. R., 396 North, R. A., 355 Northrop, J. P., 90 Northrup, J. K., 197 Northup, J. K., 183, 330, 408 Numa, S., 142, 147,336 Nfisse, O., 320 Nye, S. H., 258

O

Obernotte, R., 147 Ochi, K., 416(7), 417

Ochi, R., 386 Offermanns, S., 396 Ogawa, Y., 140 Ohara, M., 386 O'Hara, P., 395 O'Hara, P. J., 146 Ohkubo, H., 146-147 Ohoka, Y., 399 Oibo, J. A., 72 Oinuma, M., 395 Okabe, K., 183,388, 391, 391(11, 12), 393,

393(12), 395,406 Okado, H., 147 Okayama, H., 89, 280 Okuda, H., 416(8), 417 Okuyama, S., 416(6), 417 Olate, J., 321,391 Oliva, C., 368 Omri, G., 141, 145, 145(5), 147(16), 151,

151(5, 16) Ordway, R. W., 409, 419(45) Orellana, M., 416(4), 417 Oron, Y., 141, 145, 145(4), 151 Orosylan, S., 268 Orth, K., 57, 117, 118(2), 119(2), 125(2) Osawa, S., 14 Ostrowski, M. C., 271,273(6) O'Sullivan, A. J., 303 O'Sullivan, W. J., 190 Otero, A. S., 421 Owen, R. D., 271

P

Pace, A. M., 82, 83(1, 4), 86(1), 87(1), 91(1), 94(1), 276

Padrel, E., 141, 145(5), 151(5) Padrell, E., 151,393,395 Pak, W. L., 228, 246, 248 Pal, P. K., 58, 60(13) Pallast, M., 184 Pallest, M., 19 Palm, D., 126 Palmer, S., 211 Panayotatos, N., 258 Papermaster, D. S., 18, 183 Parada, L. F., 277, 285(5)

436 AUTHOR INDEX

Park, D., 182, 183(24), 186(6), 219, 220(3, 4, 13), 221(13), 229, 237, 243-244, 244(18), 245-246, 246(4)

Park, D. J., 182, 248 Parker, I., 141 Parker, P., 219, 220(10), 232 Parker, P. J., 132, 182, 183(20), 186(4),

188(21), 208,230(21), 232, 237, 245-246 Parkes, J. H., 13 Parma, J., 117, 125(12, 13) Parmentier, M., 118 Parnham, M. J., 416(9), 417 Pasik, P., 147 Pastan, I., 45 Pate, T. M., 67 Patel, G., 285 Paupardin-Tritsch, D., 357, 358(3), 360(3) Pawley, J., 333 Pawson, T., 219, 255 Payne, D. M., 261-262 Payne, R., 227 Pearson, R. B., 261 Pelech, S. L., 258, 262(1) Pellicer, A., 89, 280 Pelzer, D., 386 Peralta, E., 147, 148(40), 154(40, 41), 307,

321-322, 322(3), 324(3-5), 335(3-5) Peralta, E. G., 86(k), 87 Perdew, M., 228 Perdew, M. H., 246 Perdrew, M., 246 Perez-Reyes, E., 37, 72, 73(3) Perkins, A. S., 83 Perrin, O. D., 238 Peterson, G. L., 338 Peterson, K. E., 147 Pettis, P., 414 Pfaffinger, P. J., 349, 365, 385, 395 Pfeuffer, E., 95 Pfeuffer, T., 95, 103, 105(22), 126 Pfister, C., 13 Phillips, H. S., 86 Phillips, W. J., 13, 21(5) Phillipson, C. A., 126 Philp, R., 182, 186(4), 246 Pilkis, S. J., 67 Piomelli, D., 409 Pitcher, J. A., 395 Pitier, T. A., 376 Pitt, G. S., 117, 118(17), 120(17)

Pittman, Q. J., 355 Pleiman, C. M., 83, 260, 270(13) Plummer, M. R., 336 Poenie, M., 297-298, 298(2), 308, 325,

334(7) Pohl, S. L., 37 Pollack, R., 290 Polverino, A., 256 Ponnapalli, M., 95, 96(12), 109, 113(2),

I15(2), 117, 125(8), 126(8) Possee, R. D., 98 Potter, B.V.L., 147 Pouyss6gur, J., 82, 83(1), 86(1), 87(1), 91(1),

94(1), 134, 258, 259(3) Powell, J. A., 336 Poyard, M., 117, 125(12) Pozzan, T., 297 Pragnell, M., 341,342(20), 343(20), 344(20) Preiss, J., 131 Premont, R. T., 95, 96(12), 109, 113(2),

115(2), 116-117, 118(15), 124(10), 125(8, 10), 126(8, 10), 145, 151,244, 246, 275

Preston, M. S., 32 Proctor, W. R., 376 Pronin, A. N., 94 Pryer, N. K., 356 Purcell, P., 321 Purdon, A. D., 413,414(57) Pusch, M., 368, 376 Putney, J. W., Jr., 319

Q

Qian, N.-X., 271,272(5), 273(5), 275(5) Qin, D., 421 Qiu, H., 61, 62(17), 63(17) Quarmby, L. M., 96, 105(15), 106(15),

107(15) Qui, H., 68, 69(25), 70(25), 71(25) Quick, M., 328

R

Radziejewska, E., 258 Rae, J., 359 Rae, J. L., 312 Rail, T. W., 32 Ramachandron, J., 14

AUTHOR INDEX 437

Randall, R. J., 338 Rao, A. K., 413,414(57) Rapp, U. R., 256, 260, 268, 273,274(12) Rarick, H. M., 13-14, 19(6), 20(6), 21(6),

23(6), 24(7), 25(7), 26(7), 27(7), 402 Rawlings, S. R., 297, 308-309 Ray, J., 280, 288(12) Ray, L. B., 260 Reddy, M. A., 271,273(6) Reed, P. W., 417 Reed, R. R., 57, 86(16), 87, 95, 96(7), 117,

118(2, 17), 119(2), 120(17), 125(2-4), 126, 126(3), 395

Reeves, J. P., 373,386 Reichlin, M., 187 Reid, M. S., 227 Reinisch, L., 298, 308 Reisler, E., 67 Ren, H., 126 Revankar, G. R., 57, 58(10) Rhee, S. G., 90, 131-132, 145, 154-155,

155(1), 168, 181-182, 183(24), 186(6), 195, 196(4), 207, 219, 220(2-7, 13), 221(13), 227, 229,237,243,243(3), 244, 244(18), 245,245(1), 246, 246(4), 248

Rice, G. C., 86(1), 87 Richardson, G. B., 376 Ricketts, M. H., 281 Rifo, M., 394 Ringold, G. M., 90 Rink, T. J., 297 Risser, R., 290 Rittenhouse, A., 336 Robbins, D. J., 258 Robbins, D. T., 260 Robbins, L. S., 86 Robert, M., 13 Roberts, B., 283 Roberts, J. L., 145, 147, 151(33) Robichon, A., 147 Robins, R. K., 57, 58(10) Robishaw, J. D., 102 Robitaille, R., 336 Rodbell, M., 37, 43, 45(35), 46(35), 74, 90,

92(30) Roeckel, N., 117, 125(13) Roman, R., 90 Rosebrough, N. J., 338 Rosemblit, N., 86 Rosenberg, A. H., 266

Rosenberg, G. B., 95 Rosenthal, W., 19, 184, 365, 368-369.

369(15), 370(15), 373,374(27), 394 Rosner, M. R., 261 Ross, A. H., 238, 241(13) Ross, E. M., 37, 124, 155, 182 Rossomando, A. J., 261-262 Rouot, B., 357, 358(3), 360(3) Rozakis-Adcock, M., 255 Rubin, G., 228, 246 Rubin, G. M., 255 Rubin, H., 280 Rubin, L. J., 227 Rubin, R., 217 Rudin, D., 343 Ruley, H. E., 285-286 Ruoho, A., 63 Russell, M., 83 Russell, T. R., 45 Ruth, P., 336 Ryu, S. H., 182, 195, 207, 219, 220(5-7L

229, 244, 246, 248

S

Saari, G. C., 395 Sable, C., 83 Sah, P., 376 Sakai, H., 260 Sakamoto, J., 339, 340(18), 341, 342(20),

343(20), 344(20) Sakmann, B., 142-143, 146(11), 154(11),

308, 360, 367, 376, 385, 387(5), 397, 415(25), 418

Sala, F., 307 Salad, H., 416(3), 417 Salomon, Y., 31, 43, 43(1, 2), 45(1, 2, 35),

46(35), 48(1), 74, 90-92, 92(30), 111 Salon, J., 86 Salter, R. S., 37 Sambrook, J., 110, 121, 123(22), 124(22),

t86, 249, 250(23), 251(23), 266, 273, 274(13), 280, 289(15), 292(15), 293(15)

Samples, D. R., 413,415(56) Samuelsson, B., 414 Sanchez, S., 394 Sandmann, J., 86(k), 87 Sanghera, J. S., 258, 262(1) Sasai, Y., 147

438 AUTHOR INDEX

Sasaki, K., 355 Sassone-Corsi, P., 272, 273(10) Sato, M., 355 Satoh, T., 256 Saur, W., 32, 37, 41, 45(34), 56, 57(3) Sayce, I. G., 238 Scarborough, G. A., 203 Schacht, J., 197, 238 Scheer, A., 132, 182, 183(20), 237, 245 Schekman, R., 356 Scherer, N., 385 Scherer, R. W., 422 Scheriibl, H., 330, 373 Schlegel, W., 37, 297, 303, 308-309, 313,

317, 319 Schlessinger, J., 131,255 Schnabel, P., 132, 182, 183(20), 237, 245 Schneuwly, S., 228, 246 Schneyer, A. L., 86 Schrader, K. A., 86(16), 87, 95, 96(6), 117,

125(3), 126, 126(3), 395 Schubert, B., 373 Schultz, A. M., 268 Schultz, G., 37, 41, 45(34), 118, 124, 184,

330, 365, 368-369, 369(15), 370(15), 373-374, 374(27), 375(29), 394, 396

Schultz, W., 19 Schulzki, H.-D., 126 Schwartz, J. H., 409 Schweitzer, P., 409 Scolnick, E. M., 14 Scott, M.R.D., 83, 85(11) Scott, R. H., 352, 356, 371 Sealfon, S., 147 Sealfon, S. C., 145, 147, 151(33) Seeburg, P, H., 86, 285 Seed, B., 88 Sega, M. W., 153 Segaloff, D. L., 86 Seger, D., 259 Seger, R., 259 Seiman, J. G., 83 Seki, T., 72, 80(9), 147, 154(43) Sekiya, K., 416(8), 417 Sekura, R. D., 124, 391,393(16) Selinger, Z., 352 Sendtner, M., 364 Settleman, J., 256 Seuwen, K., 134

Seyama, Y., 147 Shabanowitz, J., 261-262 Shahid, M., 34 Shapira, H., 147 Sharp, A. H., 336 Shaw, K., 238, 241(13) Shelton, E. R., 147 Shibasaki, F., 219, 220(11), 255 Shibasaki, H., 399 Shibata, M., 255 Shichi, H,, 184 Shigemoto, R., 141, 147 Shimizu, T., 147,409, 411(49), 421(49) Shimoji, K., 416(6), 417 Shin, S., 277, 287(1), 290(1), 294(1) Shiraishi, M., 416(7), 417 Shortridge, R. D., 228, 246, 248 Shoshani, I., 56-57, 57(4), 61, 62(17),

63(17), 68, 69(25), 70(25), 71(25) Shuttleworth, S., 330 Sibley, D. R., 126 Sidiropoulos, D., 182, 183(19), 193(19),

195(19), 196, 237, 331 Siegelbaum, S. A., 409 Sigal, I., 14 Siggins, G. R., 409 Sigworth, F. J., 308, 360, 367, 382, 385,

387(5), 397-398, 415(25) Silverstein, S., 89, 280 Silvia, C., 86 Simchowitz, L., 417 Simmoteit, R., 126 Simon, M., 366 Simon, M. A., 255 Simon, M. I., 90, 118, 144-145, 145(14),

182-183, 183(23), 196, 237, 244-246 Simond, W. F., 394 Simons, C., 183, 187(27) Simons, M.-J., 118 Sims, S. M., 415 Singer, D., 365 Singer, J. J., 409, 415, 419(45) Sitaramayya, A., 13 Sizeland, A. M., 255 Skelflo, P., 376 Skiba, N. P., 13-14, 24(7), 25(7), 26(7), 27(7) Skolnick, E. M., 257 Skolnik, E., 255 Skolnik, E. Y., 255

AUTHOR INDEX 439

Slattery, I., 147 Slaughter, C., 57, 117, 118(2), 119(2), 125(2) Slaughter, C. A., 243 Sluss, P. M., 86 Smeal, T., 271,273(9) Smigel, M. D., 95, 103(2) Smith, A. J., 261 Smith, G. E., 98 Smith, H. W., 416(5), 417 Smith, R. M., 73, 74(13) Smith, S. J., 336 Smrcka, A. V., 145, 155, 182, 183(13), 237,

245 Snaar-Jagalska, B. E., 208 Sninsky, J. J., 122, 249 Snodin, C., 86 Snutch, T. P., 141, 144, 145(14), 336 Snyder, D. W., 414 Snyderman, R., 86(m), 87 Soejima, M., 368,418 Soltoff, S., 294 Somers, R., 184 Somkuti, S. G., 32 Southern, P. J., 288 Spada, A., 72 Spada, S. B., 276 Spat, A., 217 Spatt, A., 217 Spector, A. A., 413 Speicher, R., 368 Spicher, K., 365, 369, 396 Spiegel, A. M., 183, 187(27), 394 Sprague, E. A., 413,415(56) Sprengel, R., 86 Sreedharan, S. P., 147 Stanbridge, E. J., 277, 287(2), 290(2), 294(2) Standring, D. N., 68 Stanley, E. F., 336 Staros, J. V., 68 Steer, M. L., 73 Stehno-Bittel, L., 321,326 Stein, R., 131 Steiner, A. L., 90 Steller, H., 228, 246 Stengel, D., 117, 125(12, 13), 183 Stephens, L., 196 Sternweis, P. C., 102, 145,155, 182,183(13),

237,245 Stevens, C. F., 376

Stewart, W. W., 24 Still, W. C., 63 Stinnakre, J., 140, 142, 143(10), 153, 326.

327(10) Stock, J. B., 182, 183(19), 193(19), 195(19),

196, 237,331 Stork, P. J., 86 Storm, D. R., 95 Strathmann, M., 118, 366 Strathmann, M. P., 145, 182, 196, 237 Straub, R. E., 147 Stroobant, P., 203 Stryer, L., 3-4, 23,394 Stryer, L. C., 13,228 Stiibner, D., 56, 57(4) Studier, F. W., 266 Sturek, M., 326 Sturgill, T. W., 258-262 Stutchfield, J., 155, 169 Sudlow, G., 414 Sugden, B., 288 Sugimoto, T., 354, 385, 395, 402-403,

403(15, 33), 404(33), 405, 405(33), 406(33), 407(19, 33), 408(33), 409, 409(33), 411(33, 49, 53), 421(36, 49), 422(33, 53)

Suh, H. W., 182, 246 Suh, P.-G., 182, 195, 207, 219, 220(5-7),

229, 246, 248 Sullivan, K. A., 14, 83 Sultzman, L., 219 Summers, M. D., 98 Sun, F. F., 416(5), 417 Suprenant, A., 355,396 Sutherland, E. W., 32 Suzuki, K., 219, 220(11) Swanson, R. J., 183 Sweat, F. W., 45 Szabo, G., 349, 351,351(1), 356(1), 365,385,

395,421 Szuts, E. T., 227

T

Taari, Y., 376 Tabakoff, B., 117, 124(14), 125(14), 127(14) Taber, D. F., 63 Takahashi, H., 147

440 AUTHOR I N D E X

Takahashi, K., 399, 402, 403(33), 404(33), 405, 405(33), 4O6(33), 407(33), 408(33), 409(33), 411(33), 421(36), 422(33)

Takahashi, T., 142-143, 146(11), 154(11), 376

Takano, M., 421 Takemoto, D. J., 15 Takenaka, K., 260 Takenawa, T., 219, 220(11, 12), 255 Takeuchi-Suzuki, E., 256 Takishima, K., 261 Tallant, E. A., 72 Tamaki, H., 147 Tamir, H., 183 Tanabae, Y., 141 Tanabe, T., 336 Tanaka, K., 147 Tang, M., 368, 369(15), 370(15) Tang, W.-J., 57, 79, 87, 95-96, 97(13),

100(13), 117, 118(2), 119(2), 125(2, 3), 126(3), 395

Tang, W.-J.Y., 126 Taniguchi, J., 386 Tank, D. W., 343 Tanka, K., 147 Tar, A., 3, 399 Tarver, A. P., 227-228 Tatham, P.E.R., 157 Taussig, R., 14, 95-96, 105(15), 106(15),

107(15), 394 Taylor, D. L., 363 Taylor, S. J., 132, 145, 182, 238 Taylor, W. C., 147 Terao, S., 416(7), 417 Terzic, A., 394 Thambi, L., 86 Thastrup, O., 80 Theler, J.-M., 297, 303,309 Thomas, D. Y., 395 Thomas, G., 262 Thomas, G.M.H., 137, 154-155, 155(2),

159(3), 167, 167(3), 168-169, 172(5), 173(6), 174(6), 179(7), 180(7)

Tiberi, M., 86 Tien, H. T., 343 Tiffany, A. L., 147 Ting, D., 399 Ting, T. D., 3 Tinker, D. O., 414 Toda, M., 416(6), 417

Todaro, G. J., 280 Togashi, C. T., 67 Toh, H., 147 Tohyama, K., 399 Tokota, Y., 147 Tomhave, E., 86(m, n), 87 Tomlinson, J., 336 Tomlinson, S., 72 Tonks, N. K., 258 Toro, M. J., 385 Totty, N., 182, 186(4), 230(21), 232, 246 Totty, N. F., 167-169, 179(7), 180(7) Trautwein, W., 365, 368, 369(15), 370,

370(15), 373,386, 418 Trentham, D. R., 352, 356, 371 Troung, O., 167-169, 179(7), 180(7) Trussel, L. O., 383 Trussell, L. O., 350, 352(3) Tsien, R. W., 336, 376 Tsien, R. Y., 297-298,298(2), 303,308, 325,

334(7) Tsuchida, K., 141, 147 Tsutsumi, M., 147, 151(33) Tubb, D. J., 45 Tung, R. T., 402, 403(33), 404(33), 405(33),

406(33), 407(33), 408, 408(33), 409(33), 411(33), 422(33)

Turk, J., 409

U

Uhing, R° J., 86(m, n), 87 Ui, M., 199, 371, 395, 399, 402, 403(33),

404(33), 405, 405(33), 406(33), 407(19, 33), 408(33), 409, 409(33), 411(33, 49), 421(36, 49), 422(33)

Ullrich, A., 131,255 Unson, C. G., 394 Ushikubi, F., 147

V

Vaillancourt, R. R., 255, 258, 260, 262(11), 265(11)

Vaitukaitis, J. L., 187 Vajda, S., 147 Vallar, L., 72, 276, 294 Vallis, Y., 357, 363

AUTHOR INDEX 441

Van Aelst, L., 256 van Deenen, L.L.M., 171 van der Eb, A. J., 280, 282(14) Van Dijken, P., 215 VanDongen, A.M.J., 373 van Dyke, T., 141 Van Eijk, R., 215 Van Haastert, P.J.M., 207-208, 211,215 Van Lookeren Campagne, M. M., 215 van Paridon, P. A., 179, 180(10) Van Patten, S. M., 261 Van Sande, J., 118 Van Tol, H. H., 86 Varnai, P., 317 Vassart, G., 118 Verjans, B., 215 Verma, I. M., 272, 273(10) Viciana, P. R., 256 Vincent, J. D., 358, 359(5) Vincent, L. A., 259 Visser, A.J.W.G., 179, 180(10) Voigt, M. M., 86 Volterra, A., 409 von Zastrow, M., 86 Vratsanos, S. M., 417

W

Wade, J., 255 Wahlestedt, C., 147, 154(43) Wakelam, M.J.O., 211 Walaas, S. I., 376 Waldo, G. L., 136, 182, 183(18), 195-197,

197(9), 199(12), 202(14), 206(9, 12, 15, 18, 19), 207(9, 12, 15, 18, 19), 237

Wallace, M. A., 137 Wallace, R. A., 153 Wallert, M. A., 409 Walseth, T. F., 40 Walsh, D. A., 261 Walsh, J. V., 409,415,419(45) Wang, Y., 57, 58(10) Wank, S. A., 147 Warne, P. H., 256 Wassermann, N., 417 Wasylyk, B., 271,273(3, 4) Wasylyk, C., 271,273(3, 4) Watanabe, T., 147 Watsky, M., 359

Watson, A. J., 246 Watson, P. A., 117, 120(11), 124(11),

125(11), 126(11) Webb, W. W., 343 Weber, M. J., 256, 258, 259(3), 261-262 Webster, C., 182, 246 Wechter, W. J., 58, 60(13) Weinberg, R. A., 255,277,285(5) Weiner, J. L., 376 Weinstein, D., 262 Weinstein, I. B., 83 Weiss, B., 41 Weissman, B., 281,290(17) Welden, J., 35 Welsh, M. J., 147 Wenz, M., 90 Wescott, W. C., 343 Whitcomb, R. W., 86 White, A. A., 41, 45(33), 49, 54(43) White, T. J., 122,249 Whiteway, M., 395 Whiting, P., 86 Wigler, M., 89, 256, 280 Wilcox, M. D., 183 Wilkie, T. M., 118 Wilkie, T. W., 246 Wilkinson, J., 277, 287(2), 290(2), 294(2) Williams, J. T., 355, 396 Williams, P. J., 355 Willumsen, B. M., 256 Wilson, E., 86(o), 87 Wincek, T. J., 45 Winitz, S., 83,255 Wirtz, K.W.A., 169, 171, 179, 180(10), 200 Witcher, D. R., 335, 341, 342(20), 343(20),

344, 344(20) Wittig, B., 330, 373,374(27), 394 Wittinghofer, A., 364, 374, 375(29) Witzemann, V., 142 Wolff, D. J., 37, 72 Wolff, J., 56, 76 Wolfman, A., 256 Wollheim, C. B., 303 Wong, T. K., 90 Wong, Y. H., 81-82, 83(1, 2, 4), 86(1-3, p),

87, 87(1-3), 91(1, 2), 94(1), 111 Woo, S.L.C., 183 Wood, J. N., 134 Wood, S. F., 227 Wood, W. I., 86(1), 87

442 AUTHOR INDEX

Woon, C. W., 14 Wootton, J. F., 352, 356, 371 Wray, V. P., 106 Wray, W., 106 Wu, D., 90, 182, 183(23), 237, 244-245 Wu, J., 259 Wuestehube, L. J., 356 Wulfern, M., 365, 368, 369(15), 370(15) Wurtman, R. J., 86(k), 87 Wynalda, M. A., 413-414

X

Xu, K., 280

Yeager, R. E., 95 Yee, D., 90 Yee, R., 9 Yeung, S.-M.H., 56-57, 57(4), 58(6), 60(6),

65(6), 66(6), 67(6) Yokota, Y., 147 Yokoyama, C., 416(7), 417 Yoo, O. J., 182, 219, 220(3), 229, 243,

244(18), 245, 246(4) Yoon, J., 246 Yoshimasa, T., 126 Yoshimoto, T., 416(7), 417 Yoshimura, M., 95, 96(11), 117, 120(6),

124(14), 125(6, 14), 126(6), 127(14) Yoshioka, T., 227 Yuen, P.S.T., 40

Y

Yadagiri, P., 416(4), 417 Yajima, M., 368, 369(15), 370(15) Yajnik, V., 255 Yamada, M., 394, 406 Yamamoto, K., 184 Yamamoto, S., 416(7), 417 Yamamoto, T., 256 Yamazaki, A., 4 Yancopoulos, G. D., 258 Yang-Feng, T. L., 86 Yatani, A., 183,337, 357, 365, 373,385-386,

388, 391,391(6, 11, 12), 392-393,393(6, 12, 19), 395,406, 421

Yates, J., 288 Yau, K. W., 228 Yazawa, K., 386

Z

Zachary, I., 82-83, 83(1), 86(1), 87(1), 91(1), 94(1)

Zenser, T. V., 41, 45(33) Zhang, L., 376 Zhang, X.-F., 256, 260 Zhang, Y., 79, 80(20), 86 Zho, W., 147, 151(33) Zhou, Q. Y., 86 Zhou, W., 352 Zijlstra, F. J., 416(9), 417 Zubiaur, M., 83 Zwaagstra, J. C., 117, 120(11), 124(11),

125(11), 126(11) Zwiers, H., 200

SUBJECT INt~EX 443

Subject Index

A

Acetylcholine receptors, muscarinic, s e e

Muscarinic acetylcholine receptors Adenine, radioassay for cAMP, 91-92 Adenosine deaminase, ATP-regeneration

system, 35 Adenylyl cyclase

assay ATP-regeneration systems, 34-35 chromatography

on alumina column, 45-47, 49-52, 93

on Dowex 50, 45, 48-49, 92-93 contaminating enzymes, inhibition,

33-34 data analysis

blank, 53-54, 93-94 calculations, 54-55 sample recovery, 54 value reporting, 55-56, 93

enzyme concentration, 36 incubation temperature, 37 inhibition assays, 90-94 radioimmunoassay, 31 reaction stopping

ATP/sodium dodecyl sulfate/cAMP, 43

hydrochloric acid, 43-45, 50-51 zinc acetate/sodium carbonate/

cAMP, 41-42, 45, 52-53 reaction time, 36-37 substrate radiolabel, see ATP transfected cells, 1 t 1-112

ATP requirements, 32 calcium-sensitive, s e e Calcium-sensitive

adenylyl cyclase cloning, 108, 116-117, 124-126 degenerative primers, 118-121 divalent metal cation requirements,

32 expression systems, s e e COS cells;

HEK-293 cells; S p o d o p t e r a

f r u g i p e r d a

G protein regulation, 37-38, 81, 116 isoforms

cloning, 124-126 distribution, 96 forskolin response, 114 functions, 126-127 Gs response, 127 immunoblot analysis, 99-101 regulation, 96 sizes, 96, 106-107 types, 95-96, 116-117, 124-126

membrane topology, 118-119 oxidation, 68 phosphoenolpyruvate effects, 34-35 polymerase chain reaction, 121-124 "P"-site, see "P"-site purification, recombinant histidine-

tagged enzyme, 102-108 sequence homology between species,

117-121, 124-126 substrate affinity, 32 ultraviolet irradiation

inactivating effects, 68 protection from, 69

fl-Adrenergic receptor, adenylyl cyclase stimulation, 115

Affinity chromatography adenylyl cyclase, 102-105 cGMP phosphodiesterase, 7 inositol phosphates, 197-200 N-type calcium channel, 338-340 phosphatidylinositol transfer protein,

172-173, 175-176 phospholipase C-/3, 204,223,225,233,

235, 239-241 Albumin, arachidonic acid solubilization,

413-414 Alumina

column care, 49, 52 preparation, 46-47, 93

nucleotide binding, 45-46

444 SUBJECT INDEX

Aluminum fluoride, G protein activation, 31

Angiotensin II receptor, superfrog expression, 150

Angrelide, cAMP phosphodiesterase inhibition, 34

Antibodies cell loading

cell permeation, 358 controls, 358-359 microelectrode injection, 360-363 patch pipette, 359, 369-370 scrape loading, 363-364 visualization, 357-358

phospholipase C-fl isoform generation, 221-222

Antisense oligonucleotides, G protein, 145, 329-331,373-374

Arachidonic acid albumin binding, 413-414 compatible perfusion systems, 414-415 inhibitors, 414, 416 metabolism, 409 patch clamp configurations, 415, 418-419 pK, 413 potassium channel regulation, 409, 411 solubilization, 41 I-413 stability, 411

Armyworm, see Spodoptera frugiperda 3'-Arylazidoiodo-2',5'-dideoxyadenosine

photocoupling, 61 structure, 62 synthesis, 61-62

ATP 32p-labeled

detection, 40, 48, 50-51 disposal, 53 half-life, 40 a-phosphate labeling specificity, 39-40 quality of preparations, 40

regeneration systems, 34-35 stability, 352-354 tritiated

assay advantages, 38 detection, 39, 48, 50-51 disposal, 52-53 half-life, 38 stability of label, 39

3 '-(p-Azido-m-iodophenylacetyl)-2' ,5'- dideoxyadenosine

adenylyl cyclase inactivation, 68-71 photocoupling, 67-68 "P"-site modification, 71 radioactive labeling, 68 solubility, 68 synthesis, 65

3'-(p-Azido-m-iodophenylbutyryl)-2',5'- dideoxyadenosine

adenylyl cyclase inactivation, 68-71 photocoupling, 67-68 "P"-site modification, 71 radioactive labeling, 68 solubility, 68 synthesis, 65

B

Baculovirus, Spodoptera frugiperda expression system using, see Spodoptera frugiperda

Barium, nucleotide precipitation, 41 Brain, whole cell recording, see Whole cell

recording

C

Caged compounds confocal microscopy, 333 GTP analogs, 371

Calcium chelation, 74, 76, 297 chloride current dependent on, 140, 143,

146, 321 fluorescence imaging, 303-304, 322-325,

334-335 homeostasis mechanisms, 80 hormonal elevation, 79 inositol phosphate effect on release, 207 levels

cytosolic, 73 determination

computer methods, 73-75 with fura-2, 73, 146 with indo-1,298, 316-317 with quin-2, 297

hormone effects, 79 ionomycin effects, 79 membrane depolarization effects, 80 thapsigargin effects, 79-80

light adaptation mediation, 228

SUBJECT INDEX 445

in phospholipase C assay calcium-45, 153-154 free ion determination, 139-140

Calcium channels dihydropyridine-sensitive

function, 335-336 G protein stimulation, 337

hormonal effects in PC-12 cells, 371-372 N-type

blockers, 336-337 function, 336 immunoblotting, 341-342 labeling, 337 purification from rabbit brain

heparin-agarose chromatography, 339-340

labeling, 340 membrane isolation, 337-338 solubilization, 338, 340 sucrose density gradient centrifuga-

tion, 341 wheat germ agglutinin chromatogra-

phy, 338-339 subunits, 341

reconstitution bilayer formation, 345-346 channel insertion, 346-348 monolayer formation, 344 technique selection, 343-344 tip-dip method, 344-345

subunit composition, 336, 341,343 types, 335-336

Calcium Green cell loading, 324-325 photobleaching, 324

Calcium phosphate, coprecipitation transfection technique, 89-90, 274,282

Calcium-sensitive adenylyl cyclase assay, 74, 101-102 calcium concentration effects, 72-77, 80

inhibition, 72, 76-77 stimulation, 72

calmodulin effects, 77-78, 95 expression system, see Spodoptera

frugiperda hormone effects, 79 immunoblot analysis, 99-101 ionomycin effects, 79 magnesium effects, 76 manganese effects, 76

Calmodulin contamination in creatine phospho-

kinase, 76 endogenous, removal, 77-78 regulation of adenylyl cyclase, detection,

77-78 Camera, fluorescence detection, 302-303 cAMP

assays protein binding, 90 radioimmunoassay, 90 with tritiated adenine, 91-92

chromatography, 45-47, 49-52, 92-94 phospholipase C stimulation, 212-213

cAMP phosphodiesterases adenylyl cyclase preparation contamina-

tion, 33 inhibitors, 33-34

Cell lines, see COS cells; HEK-293 ceils cGMP phosphodiesterase

activation by rhodopsin, 3, 11-13 by transducin, 3, 11-13, 21-23 by trypsin, 9, 11, 13, 19-21

bovine retina assay, 9, 11-12, 19 chromatography, 6-9 extraction from rod outer segments,

4-6, 19 structure, 3, 13

inhibitory subunit, 3, 13, 19, 23-27 factor Xa fusion protein, 23 fluorescence labeling, 24 transducin c~ subunit binding

affinity, 24-26 assay, 24 site, 23, 25-28

Chloramphenicol acetyltransferase, assay, 274-275

Chloride current calcium dependence, 140, 143, 146, 321 inositol 1,4,5-trisphosphate effect, 145 Xenopus oocyte, 143, 145, 325-331

Chloroform, lipid extraction, 167 Cholera toxin, G protein sensitivity, 373 Collagenase, defolliculation of Xenopus

oocytes, 150 Confocal microscopy

caged compound activation, 333 cost, 308

446 SUBJECT I N D E X

detectors, 333 light source, 307-308, 331 muscarinic receptors, 322-325, 331,

333-334 optics, 331-332 resolution, 307, 333 scanning, 307 scan speed, 334 voxel, 307

co-Conotoxin GVIA blocking of N-type calcium channel, 336 labeling of N-type calcium channel, 340 radiolabel, 337, 339

COS cells, expression systems adenylyl cyclase

assay, 111 cDNA preparation, 109-110 cell culture, 109, 124 transfection, 110-111, 116

phospholipase C-~ cell culture, 186 expression vector construction, 186 immunochemical analysis, 187 transfection, 186-187

Creatine kinase ATP-regeneration system, 34-35 calmodulin contamination, 76

D

DEAE-dextran transfection technique, 88- 89

Degenerative primers adenylyl cyclase

design, 118-121 sequences, 119-121

phospholipase C-/3 design, 246-248 sequences, 248, 250, 252

Dictyostelium discoideum, phospholipase C

assay, 208-212 calcium effects, 213 cAMP stimulation, 212-213

2' ,5'-Dideoxyadenosine adenylyl cyclase affinity, 57 synthesis, 57-58

2',5'-Dideoxy-3'-p-fluorosulfonylbenzoyladenosine

adenylyl cyclase inactivation, 66-67 "P"-site modification, 65-67 purification, 59-60 reactivity, 65 structure, 60 synthesis, 58-59 tritiation, 59-60, 67

E

Eicosanoids metabolism, 409 potassium channel regulation, 409, 411 solubilization, 411-413 stability, 411

Electroporation transfection technique, 90 Elongation factor-TU, effector region, 15 Epidermal growth factor receptor

Ras activation, 255 stimulation of phospholipase C, 196

Exocytosis patch clamp monitoring, 320 regulation by Ge, 168

F

Fast atom bombardment mass spectrometry, peptides, 16

Fast protein liquid chromatography phosphatidylinositol transfer protein, 173 phospholipase C-fl, 206-207

Fibroblasts NIH 3T3

focus formation assay, 277-281 growth in nude mouse, 292-293 maintenance, 281-282 oncogene response element assays,

272, 274-275 transfection

assay, 283-284 DNA carrier, 282

Rat- 1 focus formation assay, 277-281 maintenance, 284 transfection, 285

REF52 maintenance, 286-287 oncogene cooperation assay, 285-286 transfection, 287

SUBJECT INDEX 447

transformation assays anchorage-independent growth, 290-

291 focus-formation method, 277-281 growth rate acceleration, 288, 290 oncogene cooperation method, 285-

287 saturation density, 288, 290 serum growth factor requirements,

289 stable cell line establishment, 289-290

Fluorescence, s e e a l s o Confocal microscopy

assay, 24 cell loading of probes, 299, 315-316 cGMP phosphodiesterase, inhibitory

subunit labeling, 24 data acquisition with patch clamp mea-

surements, 312-313,318-320 detection

camera, 302-303, 304, 306 photomultiplier tube, 302, 304, 306

dual emission advantages, 306-307 dichroic mirror, 304, 310 dye calibration, 316-317 excitation source, 309-310 imaging, 304

dual excitation detector synchronization, 303 disadvantages, 306-307 image processing

ratio calculation, 303 video mixing technique, 303-304

light source, 300 quantitation principles, 297-298 wavelength selection

filters, 302 monochromators, 300, 302

imaging, 302-304, 306 microscope requirements, 299-300, 309 perfusion chamber, 313-314 probes, 72, 146, 297, 325

Focus-formation assays cell lines for, 277

maintenance, 281-282, 284 morphological changes, 277-279 selection, 280 transfection, 282-285

disadvantages, 279

time course, 284 transfection assay, 283-284

Forskolin, in analysis of adenylyl cyclase activation, 95-96 affinity chromatography, 102-105 isoform response, 114 protection against thermal inactivation,

37 Frog

identification, 150 maintenance, 148 oocyte collection, 148-150 superfrog types, 150

Fura-2 cell loading, 299 in intracellular calcium determination.

73, 146, 298 ratio fluorescence application, 298

G

GDP/~S G protein binding, 352-353 patch pipette perfusion, 369

Ge, exocytosis regulation, 168 Gel filtration

cGMP phosphodiesterase, 7 phosphatidylinositol transfer protein.

173-174, 176-177 phospholipase C-fl, 204

Genes, oncogenic, s e e Oncogenes Gi

adenylyl cyclase regulation, 37-38, 81, 116

a-subunit mutation, 81-82 cell expression systems, 82-83

COS cells, 85, 87 HEK-293 cells, 85-87 PAl2 cells, 84-85 retroviral infection of mammalian cells

cell culture, 84-85 DNA preparation, 83-84

cloning, 81 detection, 94 pertussis toxin sensitivity, 145, 155, 196,

237 signal transduction role, 81 subunit sequence homology, 81 transfection, 82, 88-90 types, 81


Gk a subunit, 393 arachidonic acid modulation, 420-421 effect on potassium channels, 392-393,

420-421 Go

antisense oligonucleotide, 145, 374 pertussis toxin sensitivity, 145, 155, 196,

237 G proteins, see a lso Ge; Gi; Gk; Go; Gq; Gs;

Transducin activation in X e n o p u s oocyte, 144 activators, 31 ADP ribosylation, 31, 94, 371-373 c~ subunit

diversity, 365-366 immunoblotting, 399 patch pipette perfusion, 369

antisense oligonucleotides, 145, 329-331, 373-374

BY subunit denaturation, 407-408 detergent solubilization, 405-406 phospholipase C stimulation, 238-239 purification, bovine brain, 238

classes, 196 expression in HEK-293 cells, 85-87 membrane association, 155, 196 oncogene response element, transactiva-

tion assay, 275-276 regulation

adenylyl cyclase, 37-38, 81, 116 ion channels, 308, 349, 357, 365, 371-

372, 385, 394-396 phospholipase A2, 422 phospholipase C, 131-132, 144-146,

155, 182, 208 structure, 196

G~, pertussis toxin insensitivity, 155, 196 Growth factor receptor-bound protein, Ras

protein regulation, 255 Gs

adenylyl cyclase isoform response, 127 ADP ribosylation, 373 regulation of adenylyl cyclase, 37-38,

81 Gt, see Transducin GTP

analogs caged compounds, 371 types, 37-38, 350-353,369, 390

in sharp microelectrode recording, 354- 356

in single-channel recording, 354 stability, 352-354 thin-layer chromatography, 258 in whole-cell recording, 348-349

GTPase-activating protein, Ras protein regulation, 255

GTP-binding proteins, s ee G proteins GTPyS

adenylyl cyclase activation, 38 complex with G protein a subunit,

stability, 407 G protein binding, 356 patch pipette perfusion, 369 phospholipase C-/3 response, 163-164,

169 potassium channel activation, 390 receptor response, 350-352

Guanylyl imidodiphosphate patch pipette perfusion, 369 receptor response, 350-351

H

Heart, perfusion, 396-397 HEK-293 cells

adenylyl cyclase expression activity, 112-113 assay, 111 cDNA preparation for, 109-110 forskolin response, 114-115 hormonal stimulation, 115

cell culture, 88, 109, 124 G protein expression, 85-87 transfection

calcium phosphate coprecipitation, 89-90

DEAE-dextran technique, 88-89, 110- 111, 116

electroporation, 90 lipofection, 90

High-performance liquid chromatography IP3, 216-217 phospholipase C, 225-226, 230-231,

235-236 synthetic peptides, 16

Histidine, tagging of recombinant proteins, 102-105,267

HL-60 cells, s ee Neutrophils

SUBJECT INDEX 449

HPLC, s e e High-performance liquid chromatography

Human chorionic gonadotropin, adenylyl cyclase stimulation, 115

Human chorionic gonadotropin receptor, COS cell expression, 116

Hydrochloric acid, assay stopping, 43-44

I

IBMX, s e e 3-Isobutyl-l-methylxanthine Indo-1

calibration, 316-317 cell loading, 316 confocal microscopy application, 334 detection, 310 excitation, 310 in intracellular calcium determination,

298,316-317 lnositol phosphates

calcium-releasing effects, 207 Dowex separation, 165-166, 190, 215 fluorescent probes, 335 neomycin affinity chromatography, 197-

200 radiolabel, 159-160, 197-198 synthesis by turkey erythrocytes, 196-

197 Inositol 1,4,5-trisphosphate

effect on chloride current amplitude, 145 high-performance liquid chromatogra-

phy, 216-217 identification, 213,215-217 isotope dilution assay, 213,215-216 light-induced regulation in retina, 227 quantitation, 210-213

Inositol 1,4,5-trisphosphate-binding protein, isolation, 217-218

Inside-out patch recording G protein

criteria for defining active subunits, 400-402

diffusion, 399-400 potassium channels

apparatus, 387, 397-398 buffers, 391 cell preparation, 386, 396-397 concentration clamp, 388-389 data

acquisition, 387 analysis, 387-388

GTP analogs, 390-391,398 method suitability, 385-386, 397 patch solution, 387, 398 pertussis toxin uncoupling, 391-393 pipette glass, 387 time course, 389

lodoazidodideoxyadenosine, precursor preparation

p-azido-m-iodophenylacetic acid, 62-64 p-azido-m-iodophenylacetic anhydride.

64 p-azido-m-iodophenylbutyric anhydride.

64-65 Ion channels, s e e Calcium channels: Potas-

sium channels Ion-exchange chromatography

adenyly[ cyclase assay, reaction stopping, 45-49

cGMP phosphodiesterase, 6-7 inositol phosphates, 165-166, 190 phosphatidylinositol transfer protein,

175, 179 phospholipase C

assay, 133, 165-166 purification, 203-204, 206-207. 226-

227,236-237 Ionomycin, effect on adenylyl cyclase, 79 3-1sobutyl-l-methylxanthine, cAMP phos-

phodiesterase inhibition, 33-34 Isoelectric point, peptide solubility effect.

17

K

Kidney, s e e HEK-293 cells

L

Lipofection, transfection technique, 90 Liposomes, preparation, 172 Lucifer Yellow vinyl sulfone, cGMP phos-

phodiesterase subunit labeling, 24 Luteinizing hormone receptor, adenylyl

cyclase stimulation, 115

M

Magnesium effect on adenylyl cyclase, 76 role in GTP activation of ion channels,

389, 403

450 SUBJECT INDEX

MAPK, s e e Mitogen-activated protein kinase

Mass spectrometry, fast atom bombardment, peptides, 16

MEK-I assay

coupled, 263 separations, 262-264

mitogen-activated protein kinase specificity, 259-260

phosphorylation, 260 recombinant, preparation

cell growth, 266-267 expression plasmid, 265 histidine tagging, 267 induction, 266 purification, 237

solubility, 265 MEK kinase

assay, 270 MEK-1 activation, 260 purification, 270

Membranes, reconstitution bilayer, 344-346 monolayer, 344

Microfluorimetry, s e e Fluorescence Microsomes, preparation, 171-172 Mitogen-activated protein kinase

activation phosphorylation, 258-260 receptor, 258-259 regulation, 259

assay separations, 260-261 substrates, 260-262

functions, 258-259 recombinant, preparation

cell growth, 266-267 expression plasmid, 265 histidine tagging, 267 induction, 266 MEK-1 assay substrate, 262-264 purification, 237

solubility, 265 Mitogen-activated protein kinase kinase,

s e e MEK-1 Muscarinic acetylcholine receptors

confocal microscopy, 322-325, 331,333- 334

tissue ditribution, 321

types, 321-322 X e n o p u s oocyte

chloride current, 322, 325-330 expression, 322 receptor expression stability, 327-328 signal transduction, 322

Myelin basic protein, mitogen-activated protein kinase substrate, 262

Myocytes; cardiac, preparation, 396-397 Myokinase, s e e Adenylyl cyclase

N

Nerve growth factor receptor, Ras activation, 255

Neutrophils, HL-60 cells calcium influx, 317-319 intracellular pH, measurement, 319-320 membranes, preparation, 186 phospholipase C, preparation, 184-186

Nickel, histidine affinity, 102-105, 267

O

Oligonucleotides, antisense, G protein, 145, 329-331,373-374

Oncogene-responsive elements activation

assay chloramphenicol acetyltransferase,

274-275 cotransfection analysis, 272 G protein, 275-276 reporter gene expression, 273 transactivation, 275-276

mechanism, 271 transcription increase, 271

Oncogenes cooperation assay, 285-287

cell line for, 286-287 transfection, 287

transcription activation of other genes, 271-272

Oocytes, X e n o p u s

calcium oscillations, 147, 323 chloride current

acetylcholine response, 325-330 calcium dependence, 140, 143, 146,

321

SUBJECT INDEX 451

recording amplifier, 153,324 data storage, 153 micropipettes, 153 tissue bath medium, 142, 152-153

response with different receptors, 143-144, 322

values, 143, 326 collection, 148-150 defolliculation, 149-150, 323 development, stages, 141 glutamate receptor expression, 141 G proteins

activation, 144 c~ subunit cloning, 321 antisense oligonucleotides, 329-331

ion permeability, 142-143 layers, 141-142 muscarinic acetylcholine receptors

chloride current, 322, 325-330 expression, 322 receptor expression stability, 327-328 signal transduction, 322

phospholipase C assay

calcium-45, 153-154 chloride current, 152-153

G protein sensitivity, 144-146 receptor expression system, 146-147 resting potential, 142 RNA injection, 150-151,324 size, 141, 147 small molecule injection, 151-152

P

p21 ra~, effector region, 14-15 Papaverine, cAMP phosphodiesterase

inhibition, 33-34 Patch clamp, s e e a l s o Chloride current,

X e n o p u s oocyte amplifiers, 153,310 arachidonic acid techniques, 415,418-

419 cell loading

antisense oligonucleotides, 374 microelectrode injection, 360-363 patch pipette

antibodies, 359, 369-370

dyes, 315-316 G protein, 369 guanine nucleotides, 348-350

scrape loading, 363-364 trituration method, 374

data acquisition with fluorescence measurements, 312-313, 318-320

exocytosis monitoring, 320 Faraday cage, 310-311 GTP supplementation

sharp microelectrode, 354-356 single-channel, 354 whole-cell, 348-349

inside-out, s e e Inside-out patch recording

micromanipulator, 310 oscilloscope, 312 perfusion chamber, 313-314 solutions

pipette, 314-315 recording, 142, 152-153

stimulator, 311 temperature control, 314 tip-dip method, 344-348 whole-cell, s e e Whole-cell recording

Peptides fast atom bombardment mass spectrome-

try, 16 purification, 16 quantitation, 18 solubility

determination, 18 factors affecting, 17 isoelectric point effect, 17 solvent, 17

synthesis, 16-17 Pertussis toxin

ADP ribosylation of G proteins, 237, 371-373

G protein sensitivity, 144-145, 155, 196, 237,357,371-372

potassium channel blocking, 391-393 structure, 371

Phosphatidylinositol, s e e Inositol phosphates; Phosphatidylinositol transfer protein

Phosphatidylinositol 4,5-bisphosphate, s e e

Inositol phosphates Phosphatidylinositol transfer protein

activity, 169

452 SUBJECT INDEX

assay isoelectric focusing, 180 microsome preparation, 171-172 quantitation, 172 reconstitution, 173, 181

bovine brain, purification cytosol preparation, 175 gel filtration, 176-177 heparin-Sepharose chromatography,

175-176 ion exchange chromatography, 175,

179 Phenyl-Superose chromatography,

177-179 phosphatidylcholine and phosphatidyl-

inositol forms, 169 conversion, 179-180 separation, 179

phospholipase C-/3 reconstitution assay, 166-167, 169

rat brain phospholipase C contamination, 172 purification

cytosol preparation, 170-171 gel filtration, 173-174 heparin-Sepharose chromatography,

171-173 size, 173

substrate specificity, 169 Phosphoinositidase, see Phospholipase C Phospholipase A2, G protein regulation,

422 Phospholipase C

activation by hormone receptors, 196, 244-245

cellular distribution, 154 cloning, 131, 195 Dictyostelium discoideum

assay, 208-212, 218 calcium effects, 213 cAMP stimulation, 212-213

G protein regulatory effects, assays applications, 133-134, 137 calcium effects, 139-140 data interpretation, 134-135, 140 detergent interference, 138 Dowex chromatography, 133 enzyme concentration, 138-139 quantitation, 135, 137, 140

reaction stopping, 132-134 substrates

endogenous, 132-134 exogenous, 135-137

temperature, 138 G protein sensitivity, 131-132, 144-146,

155, 182, 208, 237 high-performance liquid chromatogra-

phy, 230-231 isozymes

bovine retina, 229-233 families, 131, 154, 168, 182, 195, 219-

220, 244 sequence homology, 181,220, 245-248 sizes, 220

nomenclature, 131 phosphorylation, 155, 196 phototransduction role, 227-228 reconstitution, 164-167, 169 second messenger generation, 131, 168,

181, 195, 207, 219, 237 substrate specificity, 131, 159 temperature sensitivity, 138 in Xenopus oocytes, chloride current

assay, 140-154 Phospholipase C-/3

assays calculation of data, 166-167 with phosphatidylinositol 4,5-bisphos-

phate substrates, 160-161, 187, 197-200, 228-229

with phosphatidylinositol substrates, 159-160, 197-200, 228-229

degenerative primers, see Degenerative primers

G protein/3y subunit-stimulated assay, 238 /3y response, 243-244 bovine brain

family, 243-244 purification, 238-241 size, 241

G protein stimulation a subunit, 182-183, 245 fly subunit, 182-183, 188-191,220,

245 calcium effects, 190-191 detergent effects, 191 salt effects, 191

SUBJECT INDEX 453

isoforms, 182,220 antibodies, 221-222 sequence homology, 221,246-248 tissue distribution, 221-222

membrane-associated activity, 162 particulate, assay, 192-193 phospholipase C-/33

bovine brain, truncated form, 243- 244

rat brain antibody generation, 221 assay, 222-223 cloning, 220 immunoblot analysis, 227 purification, 223,225-227 sequence, 221

phospholipase C-/34, bovine retina purification

heparin-Sepharose chromatography, 233,235

high-performance liquid chromatography, 231-233,235-236

ion-exchange chromatography, 236- 237

salt extract, 233 tryptic peptides, 232-233

polymerase chain reaction, see Poly- merase chain reaction

recombinant, expression assay, 193-195 cell culture, 186 immunochemical analysis, 187 transfection, 186-187 vector construction, 186

reconstitution system assay, 164-167, 169 cytosol depletion, 155-157 GTP3,S, loss of responsiveness, 163-

164, 169 immunoblot monitoring of enzyme

release, 161-162 protein efflux, time course, 157-158

solubilization bovine tissue, 185-186 HL-60 cells, 184-185

solubilized assay, 187-192 substrate preparation, 187-188

substrate specificity, 159

turkey erythrocyte assay, 200-201 properties, 206-207 purification

ammonium sulfate precipitation, 202-203

cytosolic fraction, 202 erythrocyte preparation, 201 gel filtration, 204 heparin-Sepharose chromatography,

204 hydroxylapatite chromatography,

204 ion-exchange chromatography, 203-

204, 206-207 Phospholipase C-8

bovine retina, 231-232 high-performance liquid chromatogra-

phy, 231-232 Phospholipase C-3,

high-performance liquid chromatography, 230-231

immunoblotting, 230-231 phosphorylation, 220, 245

Phospholipase D assay

reconstitution, 167-168 substrate labeling, 167

GTP-binding protein requirement, 167 Photomultiplier tube, fluorescence detec-

tion, 302, 304, 306 pI, see Isoelectric point Platelet-derived growth factor receptor

Ras activation, 255 stimulation of phospholipase C, 196

Polymerase chain reaction, see also De- generative primers

adenylyl cyclase amplification, 122-123 cDNA synthesis, 122 RNA isolation, 121-122 subcloning, 123-124 type 6 enzyme cloning, 124

nested screening, 126 phospholipase C-/3

amplification, 249, 252 cDNA synthesis, 249 product characterization, 250-251 RNA isolation, 249


Potassium channels ATP-activated, G protein subunit activa-

tion, 408-409 inside-out patch recording, G protein

effects buffers, 391,398 GTP analog studies, 390-391 method suitability, 385-386, 397 pertussis toxin uncoupling, 391-393 time course, 389

muscarinic, cardiac arachidonic acid activation

direct action, 420 Gk effects, 422 indirect action, 420-421

G protein subunit activation, 402-409 patch clamp studies, 397-399 perfusion system, 397

regulation arachidonic acid metabolites, 409, 411,

419-422 G protein subunit activation, 365, 385,

391-395,402-409 c~ subunit, 392-396, 404 fly subunit, 395-396, 402-404 concentration dependence, 404-405 detergent solubilization, 405-406 reproducibility, 404

Protein kinase, mitogen-activated, see Mitogen-activated protein kinase

"P"-site adenylyl cyclase inhibition, 34, 37

structural requirements, 56 homology with catalytic domain, 57

Pyruvate kinase, ATP-regeneration system, 34-35

Q

Quin-2, calcium quantitation, 297

R

R0-20-1724, cAMP phosphodiesterase inhibition, 33-34

Raf-1 assay, 269 immunoprecipitation, 267-268 MEK-I activation, 260

Ras protein assay, in guanine nucleotide analysis,

256-258 GTP binding, 255 mitogen-activated protein kinase activa-

tion, 262 Raf protein binding, 256 receptor activation, 255 size, 255

Retina, phototransduction, 227-228 Rhodopsin, cGMP phosphodiesterase

activation, 3, 11-13 Rod outer segment

cGMP enzyme cascade, 3, 13 cGMP phosphodiesterase extraction

from membranes, 4-6, 19 light adaptation, mediation by calcium,

228 purification, 4, 18 transducin extraction, 183-184

$

Sf9 cells, see Spodoptera frugiperda SNARF- 1

detection, 310 excitation, 310 intracellular pH determination, 298, 319-

320 Sodium dodecyl sulfate, enzyme assay,

reaction stopping, 43 Spodoptera frugiperda

adenylyl cyclase expression, baculovirus system

assay, 101-102 cell culture, 97-98 enzyme purification

affinity chromatography, 102-105 detergent extracts, 103

expression level, 108 G protein activation, 107-108 histidine tagging, 97, 102 immunoblot analysis, 99-10l plasmid construction, 96-97 recombinant baculovirus production,

98 time course of expression, 99-100

membrane isolation, 98-99

SUBJECT INDEX 455

Streptolysin O cytosol depletion, 155-157, 163, 169 membrane lesion size, 158, 167

T

Thapsigargin, effect on adenylyl cyclase, 79

Thin-layer chromatography chloramphenicol acetyltransferase assay,

274-275 2',5'-dideoxy-3'-p-fluorosulfonylbenzoyl-

adenosine, 59 GTP, 258 phosphatidylinositol transfer protein

assay, 172 Tip-dip method, see Patch clamp Transducin

a subunit cGMP phosphodiesterase activation,

13 effector region, 14-15 peptides

activation constants, 23 affinity assay, 24 cGMP phosphodiesterase activation,

21-23 competition assays, 19-21 inhibitory subunit binding site, 25-

28 mechanism of action, 18 purification, 18-19 synthesis, 16-17

phospholipase C activation, 182-183 purification, 184, 399 size, 13 three-dimensional structure, 14

f ly subunit phospholipase C stimulation

assay, 188-192 calcium effects, 190-191 detergent effects, 191 linearity with time, 192 particulate enzyme, 192-193 recombinant enzyme, 193-195 salt effects, 191

purification, 183-184, 399 solubility, 406

cGMP phosphodiesterase activation, 3, 11-13

GTP binding, 13-14 heterotrimeric, purification, 183-184 subunit structure, 3, 12-13

Trituration method, intracellular application of proteins, 374

Trypsin, cGMP phosphodiesterase activation, 9, 11, 13, 19-21

Tumor cells growth potential assay, 291-293 transformation

assays anchorage-independent growth,

290-291 focus-formation method, 277-281 growth rate acceleration, 288, 290 oncogene cooperation method, 285-

287 saturation density, 288 serum growth factor requirement,

289 stable cell line establishment, 289-

290 types, 277,294

growth characteristics, 277,287-290 morphological changes, 277-279

W

Whole-cell recording average current values, 375 brain slices

apparatus, 377-378 blind technique, 376-377 discontinuous single-electrode voltage

clamping, 382 gigaseal, 380-381 junction potentials, 383-384 pipette

diameter, 375 fabrication, 378 solutions, 378-379

protein injection, 384 recording, 379-382 response rundown, 382-383 series resistance, 381-382 sharp electrode technique, 375-376

456 SUBJECT INDEX

slice preparation, 378 thin slice technique, 376-377

GTP depletion of cells, 348-350 patch pipette

intracellular perfusion, 368-370, 384 sealing, 367

voltage pulse, 367-368

X

Xenopus, see Frog

Z

Zinc, nucleotide precipitation, 41-42

Preface - the-eye.euthe-eye.eu/public/Books/BioMed/Heterotrimeric G-Protein Effectors [Methods...

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