Deficient Guanine Nucleotide Regulatory Unit Activityin Cultured Fibroblast Membranes fromPatients with Pseudohypoparathyroidism Type I

A CAUSE OF IMPAIRED SYNTHESIS OF 3',5'-CYCLIC AMP

BY INTACT AND BROKENCELLS

MICHAEL A. LEVINE, CHARLESEIL, ROBERTW. DOWNS,JR.,and ALLEN M. SPIEGEL, Metabolic Diseases Branch, National Institute ofArthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, Maryland 20205; Endocrinology Branch, Department ofMedicine, National Naval Medical Center and Uniformed Services Universityof the Health Sciences, Bethesda, Maryland 20814

A B S T R A C T Deficient activity of the guanine nu-cleotide regulatory protein (G unit), an integral com-ponent of the membrane-bound adenylate cyclasecomplex, has been implicated as the biochemical lesionin many patients with pseudohypoparathyroidism(PHP) type I. In addition to renal resistance to para-thyroid hormone in this disorder, there is decreasedresponsiveness of diverse tissues to hormones that actvia 3',5'-cyclic AMP(cAMP). To assess whether a de-ficiency of G units could account for impaired ade-nylate cyclase activity, we studied cAMP productionin intact cultured fibroblasts and fibroblast plasmamembranes from five patients with PHP in responseto several activators of adenylate cyclase.

The number of G units in PHP fibroblast mem-branes, measured by cholera toxin-dependent [32P]ADPribosylation of G-unit peptides, as well as the G-unitactivity, determined by the ability of detergent ex-tracts to reconstitute adenylate cyclase activity in G-unit-deficient S49 CYC- membranes, were found tobe markedly reduced compared with control mem-branes (43 and 40%, respectively), The activation of

Presented in part at the 64th annual meeting of The En-docrine Society, 1982, San Francisco, California.

The opinions expressed herein are those of the authors andare not to be construed as reflecting the views of the NavyDepartment, of the Naval Service at large, or of the De-partment of Defense.

Received for publication 19 August 1982 and in revisedform 17 March 1983.

fibroblast membrane adenylate cyclase by effectorsthat act directly through the G unit (guanosine tri-phosphate, guanosine 5'-O-[3-thiotriphosphate] [GTP-y-S], NaF) was significantly greater in control mem-branes than in membranes from patients with PHP.Moreover, we found that hormone (prostaglandin El)-stimulated adenylate cyclase activity was also greaterin control membranes than in PHP membranes. Nei-ther the apparent affinity of membrane adenylate cy-clase for GTP--y-S (apparent Km = 5 X 10' M) northe rate of enzyme activation by GTP-y-S was signif-icantly different in fibroblast membranes from controlsubjects and patients with PHP. In contrast to the no-table differences in hormone and G-unit-activated ad-enylate cyclase shown in fibroblast membranes fromPHP patients and control subjects, the intrinsic cata-lytic activity of membranes, as determined by forsko-lin-stimulated adenylate cyclase, was not significantlydifferent in the two groups.

Intact fibroblasts derived from patients with PHPaccumulated significantly (P < 0.001) less cAMP(46±21 pmol cAMP/mcg DNA, n = 5) than cells fromnormal individuals (170±51 pmol cAMP/mcg DNA,n = 11) when stimulated with PGEI. PGE1-stimulatedaccumulation of cAMPby intact fibroblast monolayerscorrelated closely with PGEi plus GTP-activated mem-brane adenylate cyclase activity in both patients andcontrols (r = 0.97, P < 0.001).

Our data show that, in patients with PHP, (a) fi-broblast membranes show a decreased complement of

316 The Journal of Clinical Investigation Volume 72 July 1983* 316-324

G units, (b) membrane catalytic activity is normal, butadenylate cyclase activity is reduced when stimulatedby hormone or by effectors which activate the G unit,(c) the ability of cells to accumulate cAMP in responseto hormone stimulation is reduced, and (d) reducedmembrane adenylate cyclase activity correlates wellwith impaired cellular cAMPsynthesis. These results,taken together, indicate that a deficiency of G-unitactivity can impair synthesis of cAMP by both intactand broken cells, and may explain the resistance ofmultiple tissues to hormones that act via cAMP ob-served in PHP.

INTRODUCTION

Pseudohypoparathyroidism (PHP)' is an uncommonmetabolic disorder characterized by the biochemicalfeatures of hypoparathyroidism (hypocalcemia andhyperphosphatemia), despite apparently normal para-thyroid hormone (PTH) secretion, and resistance ofthe target organs, bone and kidney, to PTH (1-3). Thebiologic action of PTH is mediated by 3',5'-cyclic AMP(cAMP), and patients with PHPType I exhibit a mark-edly blunted nephrogenous cAMP response to intra-venous infusion of PTH in comparison with normalindividuals and patients with other forms of hypo-parathyroidism (4). This observation suggested thatPHP Type I (as distinguished from PHP Type II, inwhich patients are characterized by an absent phos-phaturic response to PTH despite a normal urinarycAMP response) is caused by a biochemical defectproximal to cAMPgeneration, i.e., in the plasma-mem-brane bound hormone-receptor adenylate cyclasecomplex. An integral component of the adenylate cy-clase complex is the guanine nucleotide regulatory unit(G unit), a protein that couples hormone receptors tothe catalytic unit of the enzyme. Recently, it has beenfound in this and other laboratories that many patientswith PHP Type I show a reduced complement of Gunits in membranes prepared from erythrocytes (5, 6).Deficient erythrocyte membrane G-unit activity inerythrocyte membranes is associated with decreasedresponsiveness of diverse tissues (e.g., kidney, thyroid,gonad, and liver) to hormones (e.g., PTH, TSH, go-nadotropin, and glucagon) acting via cAMP; patientswith normal erythrocyte membrane G-unit activitygenerally manifest hormone resistance limited to PTHtarget tissues (7).

Because human erythrocyte membranes do not con-tain a hormone-sensitive adenylate cyclase, one cannot

I Abbreviations used in this paper: G unit, guanine nu-cleotide regulatory unit; GTP-'y-S, guanosine 5'-O-(3-thio-triphosphate); IMEM, improved modified Eagle's medium;PGE,, prostaglandin El; PHP, pseudohypoparathyroidism;PTH, parathyroid hormone.

test for abnormalities in hormone response in this cell.Hormone receptors and adenylate cyclase have beenfound, however, in human diploid fibroblasts. Wethuschose to study cultured fibroblasts from five patientswith PHPType I and from 11 normal subjects in orderto examine the functional consequences of G-unit de-ficiency. We report here detailed biochemical studiesof cultured fibroblasts, which include measurement ofG-unit activity, membrane adenylate cyclase activity,and cellular cAMPaccumulation. Wefound decreasedG-unit activity in fibroblast membranes from patientsmanifesting PHPand reduced erythrocyte membraneG-unit activity. We further found reduced synthesisof cAMP with intact fibroblasts, as well as fibroblastplasma membranes. These observations provide strongsupport for the hypothesis that a deficiency of G unitsis the cause of impaired hormone-responsive adenylatecyclase activity in vivo.

METHODS

Patients. Five female patients were studied; four weresiblings from a large kindred and the fifth was unrelated.The diagnosis of PHPType I was confirmed by documentinga markedly blunted urinary cAMP response to a standardintravenous infusion of PTH (4). Characteristic features ofAlbright's hereditary osteodystrophy were present in all pa-tients, including obesity, brachydactyly, subcutaneous ossi-fications, and short stature in each. These five patients hadbeen previously shown to have deficient (-50% of control)G-unit activity in their erythrocytes and resistance to mul-tiple hormones (i.e., PTH, TSH, and glucagon) with actionsmediated by cAMP(5, 8). The control group comprised fivemales and six females. All fibroblast strains were not usedin all experiments.

Cell culture. Skin specimens from each subject were ob-tained by 4-mm punch biopsy in the deltoid region, andfibroblast cultures were established by explanation of mincedskin fragments. Informed consent was obtained from pa-tients and volunteers for this procedure. At the time of skinbiopsy, all PHP Type I patients were receiving vitamin Dand thyroid hormone replacement. The ages of the patients(17-28 yr) were comparable to the control group (20-25 yr).Cells were grown in Improved Modified Eagles Medium(IMEM) supplemented with 10% fetal calf serum, 10- Mcrystalline zinc insulin, penicillin (100 U/ml), and strepto-mycin (100 ug/ml) at 37°C in a humidified atmosphere of5% CO2/95% air. Confluent monolayers of fibroblasts wereharvested for passage by trypsinization and subcultured 1:3.Cells were periodically examined for the presence of my-coplasma contamination (9), and the cell lines used in allexperiments were maintained for less than 20 passages inculture.

Intact cell studies. Cell cultures from PHP patients andnormal subjects were used at passages 6 through 10. Con-fluent cell monolayers were dispersed with trypsin andseeded onto 24-well plates (16-mm diam, Costar, Cam-bridge, MA) at a density of 50,000 cells/well. Fibroblastsgrown in these wells in complete growth medium under theusual incubation conditions reached confluency within 24 h.

Hormone response experiments were performed in the 16-mmwells with intact adherent cells. The growth medium

Deficient Fibroblast Membrane C-Unit Activity in Pseudohypoparathyroidism 317

was first removed and the cells were washed twice with 1.0ml of cold Hepes (25 mM) buffered IMEM (without bicar-bonate; pH 7.4). This solution was replaced by 0.5 ml ofHepes-buffered IMEM containing 0.2% bovine serum al-bumin, 5 X 10-4 M3-isobutyl-l-methylxanthine and the de-sired concentration of freshly dissolved agonist (standardmedium) and the plates were placed in a 37°C water bath.At the end of incubation, 0.5 ml of 10% perchloric acid (finalconcentration of 5%) was added to each well. To determinecAMP in intracellular and extracellular pools, the standardmedium was removed before adding 0.5 ml of 5% perchloricacid to each well.

The contents (cells and/or medium) of each well weretransferred to a 12 X 75-mm glass test tube and precipitatedprotein was removed by centrifugation at 10,000 rpm for15 min. A 500-Ml aliquot of each supernate was neutralizedwith 250 id of 2 N potassium bicarbonate, and after a secondcentrifugation this supernate was diluted with 50 mMso-dium acetate buffer (pH 4.6), and acetylated according tothe method of Harper and Brooker (10). Aliquots of eachsample were then assayed for cAMPby automated radioim-munoassay using the Squibb gamma-flo (E. R. Squibb andSons, Princeton, NJ) (11). Recovery of exogenous [3H]cAMPadded to the perchloric acid averaged 90%.

The contents of three 16-mm wells/cell line per experi-ment were reserved for determination of DNA. Cells wereharvested with trypsin and disrupted by sonication. CellularDNA content was measured by a highly sensitive fluoro-metric technique (12).

Preparation of fibroblast membranes. Plasma mem-brane-enriched fractions were prepared by a modificationof the hypotonic lysis method of Kartner et al. (13). Flasks(Costar T150) containing confluent fibroblasts were washedthree times with 20 ml of phosphate-buffered saline (withoutcalcium or magnesium) at room temperature. The washedcells were then swollen by incubation in 15 ml of 1 mMsodium bicarbonate (pH 7.4) for 5 min with frequent me-chanical agitation at 4°C. Cells that were still adherent tothe plastic flask after 5 min were detached by manual scrap-ing with a rubber policeman. Complete cell harvesting andlysis were confirmed by light microscopy and trypan blueexclusion. Sodium EDTA was added to the disrupted sus-pension to a final concentration of 0.5 mM, and the suspen-sion was centrifuged at 27,000 g for 20 min at 4°C. Thepellet (crude membrane preparation) was resuspended in aminimal volume of 20 mMTris-HCI (pH 7.4), 0.5 mMEDTA, 10% (wt/vol) sucrose at 4°C, layered onto a cushionof 20 mMTris-HCI (pH 7.4), 0.5 mMEDTA, 44.5% (wt/vol) sucrose and centrifuged at 70,000 g for 30 min in a SW40 rotor. Material banding at the 10%:44.5% interface(plasma membrane-enriched fraction) was collected by Pas-teur pipette, diluted 1:5 with 20 mMTris-HCI (pH 7.4) with0.25 Msucrose, and washed three times in this buffer. Mem-branes were collected by centrifugation and were resus-pended in this buffer and stored in liquid nitrogen until used.

Adenylate cyclase assay. Adenylate cyclase activity offibroblast membranes was assayed in a 100-Mul final volumecontaining 30 mMTris-HCI (pH 7.5), 10 mMMgCI2, 1 mMdithiothreitol, 20 uM cAMP, 5 mMcreatine phosphate, 3.3U of creatine phosphokinase, 100 MM[a-32P]ATP (0.1 Ci/mmol), and 6-7 Mg of membrane protein. Other modulatorswere added as shown in the table legends. Samples wereincubated for 20 min at 30°C unless otherwise indicated.cAMPwas isolated and measured by the method of Salomonet al. (14).

Measurement of G-unit activity. G units extracted fromfibroblast membranes in detergent were assayed by addition

to CYC- membranes and determining catecholamine- andsodium fluoride-sensitive adenylate cyclase activity. Mem-branes from the CYC- clone (94.15.1) of S49 mouse lym-phoma cells, genetically deficient in G-unit activity (15),were prepared as previously described (16). Fibroblast mem-brane pellets were prepared by centrifugation and solubi-lized in buffer A (1.5 mg protein/ml) (10 mMTris-HCl [pH7.5], 10 mMMgCI2, 0.1 mMEDTA, 1 mMdithiothreitol,0.2% [wt/vol] Lubrol PX) and incubated overnight at 40C.The membrane suspensions were then centrifuged for 30min at 50,000 g and the supernatant solutions (membraneextract) collected and retained at 0-4°C for immediate assayof G-unit activity. CYC- membranes (200 ug), and the in-dicated amount of fibroblast membrane extract were incu-bated for 20 min at 30°C in a volume of 80 MAI of buffer Bcontaining 30 mMTris-HCI (pH 7.5), 10 mMMgC92, 1 mMdithiothreitol, 20 MMcAMP, 5 mMcreatine phosphate, 3.3U of creatine phosphokinase, 100 MMATP, and the appro-priate agonists. After the 20-min incubation, 20 Ml of bufferB containing [a-32P]ATP (0.1 Ci/mmol) was added and in-cubation continued for a further 20 min at 300C. cAMPwasisolated and determined by the method of Salomon et al.(14). Fibroblast extracts alone did not show measurable ad-enylate cyclase activity in response to stimulators, and theslight intrinsic activity of CYC- membranes in response toisoproterenol/guanosine 5'-O-(3-thiotriphosphate (GTP--y-S)or NaF ('1.0-1.5 pmol cAMP/tube) was subtracted fromthe activity of combined fibroblast extract and CYC- mem-branes to obtain the increment due to addition of fibroblastG units (G-unit activity).

Cholera toxin-dependent [32P]ADP-ribosylation. Fibro-blast membranes (32 Mg protein) were incubated for 20 minat 30°C in 100 mMpotassium phosphate (pH 7.5), containing0.1 mMguanosine triphosphate (GTP), 25 MM[32PJNAD (5-10 Ci/mmol), 20 mMthymidine, 100 U/ml Trasylol, andeither cholera toxin (100 Mg/ml; preactivated during a 10-min 30°C incubation in 20 mMdithiothreitol) or an equiv-alent volume of 20 mMdithiothreitol without cholera toxin(final dithiothreitol concentration in cholera toxin labelingmix was 2 mM). The reaction was terminated by dilutingthe membrane suspension with 10 vol of ice-cold 100 mMpotassium phosphate (pH 7.5), centrifuging (30,000 g, 15min), and resuspending the treated membranes in 100 mMpotassium phosphate buffer, pH 7.5. Samples were preparedfor electrophoresis by the addition of gel sample buffer (finalconcentration 1% sodium dodecyl sulfate [SDS] [wt/vol], 10%sucrose [wt/vol] in 62.5 mMTris-HCl [pH 6.81, 2.5% [vol/vol] j3-mercaptoethanol, and 0.005% Bromphenol-Blue) andincubation for 5 min at 95°C.

SDS-polyacrylamide gel electrophoresis was performed in10% acrylamide gels using the system of Laemmli (17). Stan-dards for molecular-weight determination (phosphorylase B,bovine serum albumin, ovalbumin, and carbonic anhydride)were obtained from Bio-Rad Laboratories, Richmond, CA.The areas corresponding to the 52,000- and 42,000-Mr[32P]ADP-ribosylated subunits of the G unit were determinedby weighing tracings cut from densitometric scans of the gelautoradiogram. The membrane G-unit value for each patientand control was the average value obtained from duplicatelanes. Protein was fixed and stained in 25% isopropyl alcohol,10% acetic acid containing 0.05% (wt/vol) Coomassie Bril-liant Blue, then destained in 10% acetic acid before dryingand autoradiography.

Protein was measured by the method of Lowry et al. (18)with bovine serum albumin as standard. The t test was usedto compare the means of PHP patients vs. control activi-ties (19).

318 M. A. Levine, C. Eil, R. W. Downs, Jr., and A. M. Spiegel

Materials. Prostaglandin El (PGE1) was purchased fromthe Upjohn Company, Kalamazoo, MI. PGE1 was dissolvedin absolute ethanol before dilution in standard medium oradenylate cyclase assay media. Ethanol content in the in-cubation did not exceed 0.5% (vol/vol) and this concentra-tion did not significantly affect cAMP synthesis. [32P]NADwas obtained from NewEngland Nuclear, Boston, MA. GTP-,y-S was obtained from Boehringer Mannheim Biochemicals,Indianapolis, IN. Forskolin was obtained from Calbiochem-Behring Corp., San Diego, CA. Cholera toxin was purchasedfrom Schwarz/Mann Div., Becton, Dickinson, and Co., Or-angeburg, NY. Growth media (IMEM) and trypsin-EDTAwere obtained from the NIH Media Unit, and fetal calfserum was purchased from Biofluids (Rockville, MD). Otherchemicals were the best commercial grade available.

RESULTS

G-unit activity in fibroblast membranes. Fibro-blast G-unit activity was measured by two independentmethods: (a) the ability of detergent extracts from fi-broblast membranes to reconstitute adenylate cyclaseactivity in membranes from S49 CYC- cells and (b)cholera toxin-dependent [32P]ADP-ribosylation of theG unit in fibroblast membranes. Fibroblast G-unit ac-tivity was linear and proportional to the amount ofdetergent extract added to CYC- membranes witheither of the activators, NaF (Fig. 1 A) or isoproter-enol-GTP-'y-S (Fig. 1 B). The G-unit activity extractedfrom PHP samples was clearly reduced at any con-centration tested and with either activator system (Fig.1, A and B).

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G-unit activity from control fibroblast membraneswas 53.6±9.5 and 27.0±5.3 pmol cAMP/tube, whereasG-unit activity in the PHP group was 22.3±2.6 and15.2±2.6 pmol cAMP/tube, for NaF- and isoprotere-nol-GTP-'y-S-stimulated activity with 10 ul of extractadded per tube, respectively (mean±SD). These dif-ferences between control and PHP fibroblast G-unitactivity were highly significant (P < 0.001).

The results of fibroblast G-unit measurement as de-termined by membrane [32P]ADP-ribose incorporationare presented in Fig. 2. Cholera toxin catalyzes thetransfer of [32P]ADP-ribose from [32P]NAD to 42,000-and 52,000-M, membrane polypeptides, which arepart of the G unit.

The electrophoretically separated 42,000- and52,000-Mr bands were of greater density in the laneswith normal membranes than in the lanes correspond-ing to membranes from PHPsubjects. The area of thepeaks on densitometric tracings corresponding to the42,000- and 52,000-Mr bands was significantly(P < 0.005) greater for control than for PHP mem-branes (58.2±13.3 vs. 24.7±6.6 arbitrary units,mean±SD). As the amount of [32P]ADP-ribose incor-porated into the two polypeptides is a function of theirrelative concentrations, this experiment confirms thereduced complement of G units in fibroblast mem-branes from PHP subjects.

Adenylate cyclase activity in fibroblast membranes.To test the hypothesis that a deficiency of G units

135

30

25

20

15

10

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5.0 10.0

FIGURE 1 G-unit activity (pmol cAMP/tube) in detergent extracts of fibroblast membranesmeasured by reconstitution of adenylate cyclase in S49 CYC- membranes. Indicated amountsof extract from control (open symbols) or PHP (closed symbols: patient A, *; B, *; C, *; D,V) membranes were assayed in triplicate with either 10 mMsodium fluoride (A) or 100 JMl-isoproterenol plus 100 ,M GTP-y-S (B).

Deficient Fibroblast Membrane G-Unit Activity in Pseudohypoparathyroidism 319

PHP- - B D - - A C -

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CONTROL

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FIGURE 2 (A) Autoradiogram of SDS-polyacrylamide electrophoresis gel. Fibroblast mem-branes were treated with cholera toxin and [32P]NAD as described in Methods. [32P]ADP-ri-bosylation of the 42,000- and 52,000-dalton bands, corresponding to subunits of the G unit,was cholera toxin dependent. Labeling of other bands in each fibroblast membrane preparationwas less uniform, and was not dependent upon the presence of cholera toxin. Coomassie Bluestaining of the gel shown above gave patterns of equal intensity and distribution, consistentwith the application of equivalent amounts of membrane protein to each lane. (B) Represen-tative densitometric tracings from a control subject and a patient with PHP. The arrow indicatesthe peaks corresponding to the 52,000- and 42,000-M, bands. Densitometric scans of the au-toradiogram were done (as described in Methods) and the areas corresponding to the 52,000-and 42,000-Mr peaks were quantitated. The individual values (averaged from duplicate lanes)for patients with PHPwere: patient A = 19.3, B = 29.3, C = 29.4, D = 17.9; for controls: 66.1,38.5, 61.0, 49.0, 76.6.

should result in reduced synthesis of cAMP, we com-pared the effect of multiple stimulators of adenylatecyclase in cultured fibroblast membranes from fourPHP patients and five control subjects (Table I). Wefound highly significant differences in the adenylatecyclase activity of membranes prepared from PHPfibroblasts as compared with normal.

Guanine nucleotides (GTP or its nonhydrolyzableanalog GTP-,y-S) and NaF stimulate catalytic activityby interacting directly with the G unit, independentof any requirement for hormone receptor occupancy.The control membranes showed greater adenylate cy-clase activation than PHP membranes when eitherGTP or GTP-,y-S was present in the assay mix (TableI). Moreover, the response to the nonphysiologic stim-ulator NaF was also significantly greater in controlmembranes than in PHPmembranes, emphasizing fur-ther a G-unit deficiency as the cause of impaired mem-brane adenylate cyclase activity.

PGE1 stimulates adenylate cyclase activity in fibro-blast membranes by interacting with a membrane-bound receptor. Occupancy of this receptor by PGE,increases G-unit-mediated catalytic activity (7). PGE1stimulation of adenylate cyclase activity in culturedfibroblast membranes was almost totally dependentupon the addition of exogenous guanine nucleotides

to the reaction media. Wefound that PGEI-stimulatedadenylate cyclase activity was significantly greater incontrol membranes than in PHPmembranes, whetheror not GTP or its nonhydrolyzable analog GTP-'y-Swas added (Table I).

TABLE IAdenylate Cyclase Activity in Cultured Fibroblast Membranes

Controls PHP PatientsEffector' (n = 5) (n = 4)

Basal 196±44 61±84GTP 230±60 62±11 tGTP-y-S 1,700±140 670±45§PGE, 580±140 225±604PGE, + GTP 2,260±380 1,010+1704PGE, + GTP-y-S 3,470±270 1,800±170llNaF 2,270±190 1,670±1601Forskolin 1,160±110 1,080±300

All results are expressed as mean±SE for the group. Units are pi-comoles of cAMPper milligram of membrane protein per 20 min.'Concentrations: GTP, GTP-y-S (100 AM), PGE5 (10 AM), NaF(10 mM), forskolin (10 AM).I P < 0.05, different from control.§ P < 0.001, different from control.

P < 0.01, different from control.

320 M. A. Levine, C. Eil, R. W. Downs, Jr., and A. M. Spiegel

Decreased adenylate cyclase activity in cultured fi-broblast membranes from PHPpatients in response toPGEI, guanine nucleotides, and NaF could be due todecreased membrane catalytic activity. Wethereforeexamined the response of cultured fibroblast mem-branes to forskolin, a diterpene activator of adenylatecyclase whose effects are considered to be due to directstimulation of the catalytic moiety (20). The responseof adenylate cyclase to forskolin did not differ amongcontrol and PHP membranes (Table I).

Wealso examined the apparent affinity of the ad-enylate cyclase complex for GTP-y-S and the activa-tion time of the enzyme with GTP-'y-S in culturedfibroblast membranes from control subjects and pa-tients with PHP. Fig. 3 shows the results of an exper-iment comparing the adenylate cyclase activity ofmembranes from four patients with PHP with mem-branes from five control subjects. Adenylate cyclaseactivity of the membranes from patients with PHPwaslower than that of control membranes for all concen-trations of GTP-y-S tested, but there was no differencein affinity detected (apparent Km for GTP-y-S was 5X 10-8 M for both groups).

Weanalyzed the rate of enzyme activation by GTP--y-S in cultured fibroblast membranes from four pa-tients with PHPand five control subjects (Fig. 4). Ad-enylate cyclase activity in membranes from bothgroups was linear with respect to time, but the activityof PHP membranes was significantly less than that ofnormal membranes at each time point.

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FIGURE 3 Adenylate cyclase activity (nmol cAMP/mg pro-tein per 20 min) at differing concentrations of GTP-y-S.Fibroblast membranes were assayed for adenylate cyclasefor 20 min at 30'C. Open symbols (0) represent membranesprepared from control fibroblast lines, and closed symbols(patient A, 0; B, *; C, *; D, V) represent membranes pre-pared from cell lines derived from patients with PHP. Eachpoint is the mean of triplicate determinations.

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FIGURE 4 Membrane adenylate cyclase activity as a func-tion of time. Fibroblast membrane adenylate cyclase activitywas assayed in the presence of 100 gM GTP-y-S for incu-bation periods indicated. Adenylate cyclase activity of mem-branes prepared from normal cell lines is represented byopen symbols (0), and activity of membranes prepared fromPHP cell lines is represented by closed symbols (patient A,*; B, U; C, *; D, V). Each point is the mean of triplicatedeterminations.

Accumulation of cAMP in intact cultured fibro-blasts in response to PGEI. We measured the ac-cumulation and release of cAMPby confluent cells inresponse to a submaximal stimulating concentrationof PGEI. The doubling time of cells used in these ex-periments was 24-36 h for both PHP- and control-derived cell lines. Response to PGE, was tested withnondividing cells, obtained from steady state mono-layers and plated at cell numbers near confluence. Thismethod of cell cycle synchronization was routinelyused; stable content of DNA from 24 to 96 h afterplating confirmed the establishment of the steady state.Hormone responsiveness was quantitatively similarwhen cells were challenged with PGE, at 24, 48, or72 h after plating (not shown).

cAMPaccumulation in response to PGE, was rapidand, in the presence of the phosphodiesterase inhibitorisobutyl methyl xanthine, reached a plateau at 15 minand then remained stable beyond 60 min (data notshown). Wetherefore elected to study confluent mono-layers 48 h after plating and to incubate cells for 10min. Two submaximal concentrations of PGE, (0.5X 10-6 Mand 0.5 X 10-5 M) were selected to enhancepotential differences in cellular cAMP synthesis andto avoid hormone refractoriness or desensitization.

Cells derived from PHP patients accumulated sig-nificantly less cAMPthan cells from normal individualswhen challenged with PGE, (Fig. 5, Table II). Thetotal cAMPaccumulated was determined by measur-

Deficient Fibroblast Membrane G-Unit Activity in Pseudohypoparathyroidism 321

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200 k

150 k

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0

CONTROL

FIGURE 5 PGEI-mediated accumulation (pmol cAMP/megDNA) (cells plus medium) by cultured fibroblasts from pa-tients with PHP (patient A, 0; B, *; C, *; D, V; E, *) vs.control (0). Each point represents the mean of three wellson individual subjects.

ing both the extracellular fraction and the intracellularfraction. In these experiments, cAMP in the extracel-lular compartment represented 8.5±1.5 and 14.4±3.3%of the total cAMPaccumulated by PHPcells and nor-mal cells, respectively. When only the intracellularcAMP was considered, PHP cells were still found toaccumulate significantly less cAMP than normal cells.

Intact cell cAMPaccumulation in response to PGE1correlates (r = 0.97, P < 0.001) with adenylate cyclaseactivity in membranes in individual patients with PHP(Fig. 6).

DISCUSSION

Wefound a reduced complement of functional G unitsin cultured fibroblast membranes from patients withPHP. These findings are in agreement with observa-tions of deficient G-unit activity in membranes pre-

TABLE IIPGEI-stimulated cAMPAccumulation in Intact

Cultured Fibroblasts

Controls (n = 11) PHP Patients (n = 5)PGE,

concentration Total Intracellular Total Intracellular

1sM

0.5 170±51 153±48 46±210 42±1905.0 268±83 228±78 110±49 100±220

Results are expressed as mean±SD for the group. Units are picom-oles cAMP/mcg DNA.e P < 0.001, different from control.

pared from erythrocytes (5, 6) of many patients withPHPType I. Furthermore, similar findings of deficientG-unit activity have been previously reported in mem-branes prepared from platelets of two patients (21)and renal cortex of one patient (22). These results thussupport the hypothesis that an alteration of G-unitactivity is the fundamental molecular lesion in manypatients with PHPType I.

Our studies document the consequences of deficientG-unit activity on adenylate cyclase activity in bothmembranes and intact cells. We found that accumu-lation of cAMP in response to PGE, is reduced in fi-broblasts derived from patients with PHP, and thatmembranes prepared from these cells contain im-paired adenylate cyclase activity. These results supportthe hypothesis that hormone resistance in PHP TypeI is a consequence of impaired adenylate cyclase ac-tivation resulting from deficient G-unit activity.

Bourne et al. (23) have recently published similarfindings of reduced G-unit content in PHP-culturedfibroblast membranes but, in contradistinction to ourresults, found no alteration in either membrane ade-nylate cyclase activity or cAMP response to PGE, orcholera toxin with intact cells. There are a number ofpossible explanations for the differences between ourfindings and those of Bourne et al. We harvested fi-broblast monolayers by manual scraping, disruptedcells by hypotonic lysis, and prepared a purified mem-brane fraction by discontinuous sucrose gradient cen-

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50 100 150 200 250 300

cAMPACCUMULATIONIN INTACT CELLS

FIGURE 6 Relationship between cAMPaccumulation by in-tact cells (pmol cAMP/mcg DNA) in response to PGE, (0.5X 10-' M) and membrane adenylate cyclase activity (nmolcAMP/mg protein per 20 min) stimulated by PGE, (0.5X 10-6 M) plus GTP (10 uM). Membrane adenylate cyclaseactivity and cellular cAMP accumulation of fibroblast celllines from PHP patients are represented by closed symbols(patient A, 0; B, *; C, *; D, V) and from control subjectsby open symbols (0). Each point represents the mean ofthree wells (cAMP accumulation) and the mean of triplicatedeterminations (adenylate cyclase activity). All correlationswere significant (P < 0.001): r(PHP) = 0.93, r(control)= 0.94, r(PHP + control) = 0.97.

322 M. A. Levine, C. Eil, R. W. Downs, Jr., and A. M. Spiegel

trifugation. Bourne et al. freed cultured cells withtrypsin, used a Dounce homogenizer to disrupt cells,and studied particulate extracts. A receptor-enzymesystem located in the plasma membrane might be per-turbed by treatments, chemical or mechanical, thatcould interrupt the integrity of the membrane. Ofthese procedures, the exposure of fibroblasts to trypsinbefore preparing particulate extracts most deservesspecific comment. Treatment of intact mammaliancells with trypsin under conditions similar to those usedby Bourne et al. to harvest monolayers causes increasesin basal and GTP-, Gpp(NH)p-, PGE1-, and NaF-stim-ulated adenylate cyclase (24). These effects could ob-scure potential differences in membrane adenylate cy-clase between PHP and control groups and may ac-count for the large interstrain variability noted byBourne et al. (23).

Since many factors can modify cyclic nucleotidemetabolism in cultured cells, it is difficult to comparedirectly our study with that of Bourne et al. (23). Cul-ture conditions such as passage number (25), cell den-sity (26), time after subculture (27), serum concentra-tion (28), and agonist used have been shown to influ-ence significantly the response of the cAMPsystem.

With human fibroblasts in particular, response to,B-adrenergic agonists is nonuniform, and cell contentof cAMP varies with the cell cycle, the exponentialgrowth phase showing higher cAMPcontent than cellsat confluence (25). Moreover, the hormone respon-siveness of cAMP accumulation by human diploid fi-broblasts has been shown to vary with time after di-vision (25, 27). We therefore carefully standardizedour incubation conditions, synchronized cell growthto minimize artif actual influences of the cell cycle, andused PGE1, which has given more uniform responses(29-31) to stimulate cAMP production in order toavoid variations that might preclude detection of sta-tistically significant differences between cell lines.

The observation that cultured fibroblast membranesand erythrocyte membranes from patients with PHPType I show a comparable reduction in G-unit com-plement further supports the genetic basis of this le-sion. The growth of diploid fibroblasts from these pa-tients in long-term culture did not interfere with theultimate expression of the altered adenylate cyclasegenotype. These patients represent a syndrome of hor-mone resistance generalized to diverse tissues (i.e.,kidney, thyroid, gonad, liver) that can be explainedby a defective adenylate cyclase component commonto all these tissues. This component is the G unit, andit is apparent from our current results that a deficiencyof G units can impair the ability of an intact cell toaccumulate cAMP in response to hormone stimulationand can impair adenylate cyclase activation of mem-branes by agents acting via the G unit. A similar con-

clusion can be drawn from data recently reported byMotulsky et al. (32). Although Bourne et al. (23) wereunable to see a significant difference in cAMPresponseto PGE1 in intact fibroblasts derived from control sub-jects vs. patients with PHP, the same investigators (32)found deficient G-unit activity in platelet membranesas well as reduced cAMP response to prostacyclin inintact platelets from patients with PHP.

Weknow of no evidence to suggest that fibroblastsof PHP type I patients function abnormally in vivo.It is therefore not readily apparent what the functionalconsequence of an impaired cAMP system in thesecells may be. However, the cultured fibroblast canprovide the model system in which the specific mo-lecular defect responsible for deficient G-unit activitymay be elucidated. Furthermore, studies in culturedfibroblasts may lead to further appreciation of the stoi-chiometry and functional interactions of the multipleadenylate cyclase components, and may enable greaterunderstanding of adenylate cyclase per se. It is entirelyconceivable that other PHPgenotypes with normal G-unit activity but resistance to multiple hormones willbe identified with defects in other components of thecAMP system, e.g., the catalytic unit or phosphodi-esterase. As yet, we have not had the opportunity tostudy cultured fibroblasts from patients with normalG-unit activity.

Finally, our studies with cultured fibroblasts suggestthat expression of the genetic defect in PHP Type Imay be anticipated in cells from amniotic fluid. If thiscan be demonstrated, prenatal diagnosis of low G-unitPHPcan be achieved, and genetic counseling will be-come possible.

ACKNOWLEDGMENTSWeare indebted to Ms. S. Fedak, Ms. S. Eastman, and Mr.C. Woodard for excellent technical assistance, and to Ms. L.Perry and Ms. J. Rucker for manuscript preparation.M. A. L. gratefully acknowledges Dr. G. D. Aurbach andDr. S. J. Marx for their helpful guidance and suggestions.

Portions of this work were supported by project CIC-81-06-1516 from the Bureau of Medicine and Surgery, NavyDepartment, Washington, DC.

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