THE JOURNAL OP BmmeAL CHZSIPTRY Q 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 12, Issue of March 25, pp. 9184-9189. 1994 Printed in U S A .

Transmembrane Topology and Sites of N-Glyeosylation of Inositol 1,4,5-Trisphosphate Receptor"

(Received for publication, September 17, 1993, and in revised form, January 3, 1994)

Takayuki MichikawaS, Hiroki Hamanakal1, Hiroko Otsun, Akitsugu Yamamotoi\, Atsushi Miyawakil, Teiichi FuruichiS**, Yutaka Tashiroli, and Katsuhiko MikoshibaS $$ From the $Department of Molecular Neurobiology, Iszstitute of Medical Science, University of Tokyo, 4-6-I Shirokanedai, Minato-ku, Tokyo 108, the %Third Department of Medicine and IDepartment of Physiology, Kansai Medical University, 1 Furnizonocho, Moriguchi-shi, Osaka 570, and the @Molecular Neurobiology Laboratory, The Institute of Physical and Chemical Research OUKENI, Tsukuba Life Science Center, 3-1-1 Koyadai, Tkukuba-shi, Ibaragi 305, &pan

To define the transmembrane topology of the inositol 1,4,5-trisphosphate receptor (InsP3R), we determined the subcellular location of the hydrophilic segment (residues 2463-2529 of mouse type 1 InsF3R) believed to be located at the luminal side of the endoplasmic reticu- lum (ER) in the six-transmembrane model but at the cytoplasmic side in the eight-transmembrane model. This hydrophilic segment includes two consensus sites for N-glycosylation (Asn-2475 and Asn-2503). We pre- pared an anti-peptide antibody against residues 2504- 2523, Electron microscope immunocytochemical studies of mouse cerebellar Purkinje cells showed that binding of this antibody frequently occurs in the intracisternal space of the ER. We constructed three mutant receptors by site-directed mutagenesis of Asn to Gln (N2475Q, N2503Q, and N2475~2503Q~. By concanavalin A col- umn chromatography of these receptors, we found that both Asn-2475 and Asn-2503 are glycosylated. These re- sults indicate that residues 2504-2523, Asn-2475, and Asn-2503 are exposed to the ER lumen. We therefore pro- pose that InsP3R has six membrane-spanning segments. Based on the transmembrane topology and subunit or- ganization, we suggest that InsP,R is a member of the superfamily that includes the voltage- and second mes- senger-gated ion channels on the plasma membrane.

Many cellular responses to hormones, neurotransmitte~, and growth factors are mediated by the intracellular second messenger inositol 1 ,~ ,5- t~sphosphate (InsP3)* (1). InsP3 re- leases Ca2+ from intracellular stores by binding to an InsP, receptor (InsP3R) (2) which is an InsP,-gated Ca2+ channel (3-5) consisting of a homotetramer 15) of a glycoprotein subunit (2, 6, 7). Molecular cloning studies showed that there are at

-* This work was supported by grants from the Ministry of Education,

the Torey Scientific Research Foundation, the Senri Life Science Foun- Science and Culture of Japan, the Human Frontier Science Program,

dation, the Yamanouehi Foundation for Research on Metabolic Disor- ders, and the Nissan Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

stitute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444, 5 Present address: Division of Molecular Neurobiology, National In-

Japan. ** To whom correspondence should be addressed: Dept. of Molecular

Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Tel.: 81-3-3443-8111 (ext. 320); Fax: 81-3-3445-5168.

* The abbreviations used are: InsP,, inositol 1,4,5-trisphosphate; InsP,R, inositol 1,4,5-trisphosphate receptor; InsP,Rl, InsP,R2, InsP,R3, types 1,2, and 3 inositol 1,4,5-trisphosphate receptors; C o d , concanavalin A; ER, endoplasmic reticulum; IgG, immunoglobulin G ; mAb, monoclonal antibody.

least three types of InsP3R derived from distinct genes (8-10). We cloned the cDNA encoding the InsP,R, termed type 1 InsP3R (InsP,Rl), from mouse cerebellar cDNA libraries and determined its primary structure (2749 amino acids, 313 kDa) (8). Structural and functional analyses revealed that InsPBRl possesses the InsP, binding site within the N-terminal fourth and the several putative membrane-spanning segments near the C terminus (8, 11, 12). Immunocytochemical analysis with site-specific antibodies (13,141 indicated that both the N and C termini are cytoplasmic. Hence InsPBR has been thought to traverse the membranes an even number of times. Together with the sequence analyses of fly (15) and frog (16) InsPsRs, we have proposed that the InsP3R traverses the membrane six times. On the other hand, an eight-transmembrane model has been proposed by other groups who cloned the cDNAs encoding rat InsP3Rl (17) and two other types of rat InsP3R, designated type 2 InsP,R (InsP3R2) (9) and type 3 InsPsR (InsP,R3) (lo>, and the partial cDNAs encoding mouse InsP,Rs (18).

There are two classical plasma membrane channel super- families. One is composed of ligand-gated ion channels, and its unit is a pentamer consisting of subunits having four putative membrane-spanning segments. The other is composed of volt- age-gated ion channels, and its unit is a tetramer consisting of subunits (or four repeats in a molecule) having six putative membrane-spanning segments. The InsP ,~channe~ has no sig- nificant sequence homology with these ion channels on the plasma membrane. The InsP,R has some sequence homology with only the ryanodine receptor, which is another type of in- tracellular Ca"+ channel t8,13,19). This homology occurs in the C-terminal portion of these proteins. Based on the hydropathy profiles, the ryanodine receptor has been proposed to traverse the membrane 4 (19) or 12 times (20). For both the InsP,R and ryanodine receptor, no direct evidence for defining the trans- membrane topology has been found, and thus the structures of the channel-forming domains of these two intracellular Ca2+ channels are stili controversial.

The InsP,R has been known to interact with various lectins, such as concanavalin A (ConA) (2,6,7), lentil lectin (211, wheat germ agglutinin (22, 231, and Limulus polyphernus agglutinin (23). Because digestion of the cerebellar InsP,R with endo-@- N-acetylglucosaminidase F caused a slight change in its elec- trophoretic mobility (71, the cerebellar InsP,R has been esti- mated to have a small amount of oligosaccharide chains. Although 20 consensus sites for N-glycosylation have been ob- served in the entire sequence of mouse InsPC3R1, the actual sites have not been determined. Since the residues having oli- gosaccharide chains are not located in the cytoplasm, determi- nation of the glycosylation sites should be useful for defining the transmembrane topology of the InsP3R.

In this study, we took two approaches to determine the trans- membrane topology of mouse InsP,Rl. Immunocytochemical

9184

lFansmembrane Topology of InsP, Receptor 9185

2100 2200 2300 2400 2500 2600 27002749

2100-2749 and amino acid sequence FIG. 1. Hydropathy plot for residues

of residues 2463-2569 of mouse InsP,Bl. Hydropathy plot was computed according to Kyte and Doolittle (42); the window size is 17 residues. Putative membrane-spannjng segments (MI-M6)

Mb) proposed by Yoshikawa et at. (15) are and two hydrophobic segments (Ma and

indicated. The amino acids corresponding to the antigenic peptide are underlined by a thick horizontal line. The consensus sites for N-glycosylation (43) are enclosed. Mutation sites (Asn to Gln) are indicated by arrows. The variable region includes residues 2463-2523 and the conserved re- gion includes residues 2524-2569.

HTCETL:MCIVfYLS~GLRSGGC~GDVLRXPSKEEPLFIilRVIYD

Mb Q (N2475Ql

}- Variable region Conserved region .-I

analysis with an anti-peptide antibody showed that residues 2504-2523 are located in the lumen of the endoplasmic reticu- lum (ER). ConA column chromato~aphy of site-directed mu- tants revealed that both Asn-2475 and Asn-2503 are glycosy- lated, indicating that these Asn residues are present in the ER lumen. These results allow us to refine the transmembrane topology and classification of the InsP,R/channel.

EXPERIMENTAL PROCEDURES

dues 250.12523 (CTSPAPKEELLPmETEQDK) of mouse InsP,Rl (8) Peptide and Antibody Preparation-A peptide corresponding to resi-

was synthesized with a peptide synthesizer, model 430A (Applied Bio- systems), and purified by reverse phase high pressure liquid chroma- tography. Poiyclonal rabbit antiserum was raised against the peptide- keyhole limpet hemocyanin conjugate. The antibody was afinity- purified as described previously (24).

Imrnunoglobu~in G ~ I g ~ ~ . ~ ~ ~ d Technique with Frozen ~ ~ t r ~ - ~ h i n Sections-Frozen ultramicrotomy was performed as described else- where (14). The gold particles conjuga~d to goat IgG against rabbit or rat IgG (5 nm in diameter) (Ultra Biosols) were used as second anti- bodies.

P r e - e r n ~ e d d ~ ~ Imrnunogold Labeling of ~ a r o s e . e m ~ d d e d Fmg- rnents uf Mouse Cerehellum-Immunogold labeling of agarase-embed- ded fragments of mouse cerebellum was performed as described previ- ously (251. For double staining, the gold particles conjugated with goat IgG against rabbit IgG (5 nm in diameter) (Ultra Biosolsf and the Clusterprobe-GTM Fab conjugate against rat I@ (6 nm in diameter) (Nanoprobes) were used as second antibodies.

Site-directed Mutagenesis-To substitute Gln for Asn-2475 and Asn- 2503 of mouse InsP3R1, two oligonucleotide primers including appro- priate substitutjons ( 5 ' - A G C T G ~ C ~ G G G G C ~ C C T - 3 ' for the nucleotides 7742-7762 and 5 ~ - A G A G G ~ G ~ A ~ C T C C C C C G T - 3 ' for the nucleotides 7826-7846~ (26) were s ~ t h e s i ~ e ~ w i t h a DNA synthe- sizer, model 392 (Applied Biosystems). The EcoRI fragment from the internal EcoRI site (6646-nucleotide position) to the 3' end of mouse InsP3R1 was isolated from pBactS-Cl (4), subcloned into pBfuescript SK(+), and used as template DNA. Mutagenesis was performed with a MutanK kit (Takara). After confirmation of the mutations by DNA sequencing (27), the mutated EcoRI fragments were put back into pBactS-C1.

Expression ofthe Mutant Receptors-Transfection of NG108-15 cells with the mutated InsP,Rl cDNA was performed as described by Chen and Okayama 1281. The microsomal fraction from the transfected cells was prepared and analyzed by Western blot as described previously (4, 8).

~ e g l y c o s ~ l a ~ ~ o n ofthe cDNA-derived Receptors-The microsomal pro- teins from the transfected cells were treated with N-glycosidase F, 0- glycosidase, or neuraminidase (Boehringer ~ a ~ h e i m ) as described previously (29).

~ o ~ . ~ e p h a r o s e CoEurnn Chrorna~og~phy-The microsomal frac- tion (3 mg of proteirdml) was solubilized with buffer A (20 m Tris.HC1 (pH 7.5), 1 nm ~-mereaptoe~hanol, 0.1 mM phenylmethyIsulfony1 fluo- ride, 10 PM pepstatin A, 10 leupeptin) containing 1 mM CaCl,, I mM MnCI,, and 1% (wiv) Triton X-I00 by stirring for 30 min at 4 "C. In- soluble substances were removed by centrifugation at 22,000 x g for 60 min at 4 "C. f i r addition of 5 M NaCl to give a final concentration of 0.5 M, the solubilized fraction (ZOO PI) was applied to a ConA-Sepharose

(Pharmacia) column (0.7 x 2.0 em) equilibrated with binding buffer (buffer A containing 0.5 M NaCl, 1% Triton X-100, 1 mi CaCI,, 1 mM MnCl,). The column was washed with 0.2 ml of binding buffer four times. Adsorbed proteins were eluted with 1 mlf5 fractions, 0-2 ml each) of buffer A containing 1 M methyl-~-o-mannoside, 0.1 M NaCl, 15% Triton X-100, 1 mM EDTA, and 1 mM EGTA. Each fraction was analyzed by Western blot using the ECL detection system (Amersharn Corp.).

RESULTS Strategies to Determine the Tf.ansmernbrune lbpology of

InsP&--Fig. 1 shows a hydropathy plot of residues 2100- 2749 of mouse InsP3R1 (8). We have proposed six putative membrane-spanning segments (Ml-Ms) in this region (15). We have also pointed out that there are two additional hydrophobic segments (Ma and Mb) which are a little inferior in their mem- brane-spanning potential (15). In this study, we focused on residues 2463-2569 between the M5 and M6 segments (Fig. 1). By comparing the sequence of the three InsP3R types, these residues are divided into two regions, residues 2463-2523 as a variable region (V region) and residues 2524-2569 as a con- served region (C region). The G region includes the Mb seg- ment. The V region includes two consensus sites for N-glyco- sylation (Asn-2475 and Asn-2503). Among all potential N-glycosylation sites, they are the only sites that exist in the putative channel-forming domain (between the M1 and M6 seg- ments). The V region is located in the ER lumen following the six-transmembran~ model in which neither the Ma nor Mb seg- ment traverses the membrane. In contrast, the V region is cy- toplasmic in the eight-transmembrane model 19,10,17,18), and hence no consensus site forN"g~ycosy1ation is exposed to the ER lumen. To determine the precise location of the V region, we used two strategies as shown in Fig. 1. One i s immunocytochemical study using an anti-peptide antibody which specifically recog- nizes a part of the V region. We chose residues 2504-2523 as an epitope (Fig. 1) based on the antigenic index (30). The other is G o d column chromatography of the site-directed mutant re- ceptors to determine whether Am-2475 and Asn-2503 are gly- cosylated. For this purpose, we constructed three mutant re- ceptors which have Gln substitutionh) at Asn-2475 (N2475Q), Asn-2503 (N2503Q1, or both ~ N 2 4 7 5 ~ ~ 5 0 3 Q ) .

P r ~ u c ~ ~ o n of an A n t i b ~ y ~ p e c ~ ~ c u ~ ~ y Recognizing Residues 2504-2523 of Mouse InsP&l-A polyclonal antibody (referred to as 1ML1) against the synthetic peptide co~esponding to residues 2504-2523 of InsP3R1 was prepared and a ~ n i t y - p u ~ rified. Western blot analysis showed that antibody lMLl spe- cifically reacted with an InsP,R protein in a microsomal frac- tion of mouse cerebellum (Fig. 28) . rmmunohistochemica1 analysis of mouse cerebellum revealed that antibody lMLl densely stained soma and dendritic arborization of Purkinje cells (data not shown). This staining pattern was not distin- guished from that with a monoclonal antibody (mAb) against

9186

A CBB 1 2

200 *

116-

C

200 - 116-

1 ML1 1 2 3

Danamem hrane Topology of InsP:, Receptor

B 1ML1 *.- -.- -

1 2

200

116-

200-

116-

FIG. 2. Specificity of anti-peptide polyclonal antibody 1MLl. A and R . Innv I . 30 ng IA 1 and 6 ng 1 I{ I ofpurifird mouse crrrhrllar InsP:,R protrins, and Inrw 2. 6 pg IA ) and 1 pg t I 1 I of mousr crrrhrllar micro- somal protrins. A , Coomassir Iirilliant lilur 11-250-stainrd grl; R . Wrst- ern hlot with antihody 1ML1. Whrn prcimmunr s w u m was usrtl. no signal was drtrctrd. (I and I ) . Inrlrs I and 2. 1 pg of t h r microsomal protrin of NGIOX-16 crlls transfected with t h r wild type and intrrnal dclrtion construct 1)2112-2605 ( 1 2 1 of m o w r InsI'.,Rl cDNA, rrsprc- tivrly; lnrw 3 . 1 pg of thr microsomal protrins of NG10X-15 cclls. C'. Wrstrrn hlot with antihodv I M L l ; I ) . Wrstcrn hlot with mAh IC11. NGIOX-16 crlls contain :I small amount ofrndogrnous Insl'.,R 1x1. hut a t th i s condition. only thr cDNA-drrivrd rrcrptors wrrr drtrctrd. Molrcu- Iar sizr markrrs arr shown on thc /cf/ ( x I O 'I.

mouse InsP,,Rl, such as 18A10 (7) or 4Cll (31). To confirm the epitope for antihody l M L l on the InsP,,Rl

protein, we determined the immunoreactivity of antibody IMLl with the internal deletion mutant D2112-2605 that lacks residues 2112-2605 (12). As shown in Fig. 2C. antihody l M L l reacted with the intact receptor derived from the InsP,,RI cDNA hut did not react with the mutant receptor D2112-2605. In contrast, mAh 4 C l l . whose epitope is located within residues 679-727 (8), reacted with both the intact and mutant receptors (Fig. 2D ). Furthermore, hinding of antibody I M L l to the intact rrceptor was completely hlockcd by preah- sorption of antihody lMLl with the antigenic peptide (data not shown). These results indicate that antihody lMLl specifically recognizes residues 2.504-2523 of InsP,,Rl.

Intmcrllulnr Location of thc IMLI Epitopc in Mnusc Ccwhcl- lar Purizin.jc Crlls-To determine the location of residues 2504- 2523 of InsP,lR1, we performed imrnunogold Iaheling of ultra- thin frozen sections of mouse cerehellum. The gold particles that reacted with antibody lMLl were ohserved predominantly in the intracisternal space of the stacked ER in Purkinje cells (Fig. 3A). In contrast, the gold particles reacting with mAh 4Cll were ohserved mainly in the cytoplasm (Fig. 38). For quantitative analysis, we selected cross-sectional profiles of

flattened smooth ER and mrasurrd thc, distnnw hc.t\vrrn thc, ER memhranr and thr gold particlrs. As shown in Fig. 4. hind- ing of antihody 1MI,1 frcquently occurrrd in thcb luminal si(h nf the ER memhrane. whilr hinding of mAh 4C.l I orcurrrd mainly in the cytoplasmic sidr.

To demonstratcl the differrncc. in thc. location of thc rpitopcs more directly, wr douhle-lahrlrd ngarnsc-rmhrddrd microso- mal fractions of mousr crrrhrllum hy antihody l.CII,l (rahhit I g G ) and mAh 4 C l l ( r a t IgG), Gold particlrs conjugatrtl with goat 1 8 , against rahhit Id; and mrtal clustrrs conjugatrd with the Fah frapnrnt against rat IgG wrrr usrd as srcond antihnd- ies. As shown in Fig. 5 , thrsc. two s i p a l s clearly srparntrd across the mrmhrane. Thr gold particlrs as signals of antihody l M L l were located insidr microsomal vrsiclrs. whik thr mrtal clusters as signals of mAh 4 C l l wrrr locatcd outsidc~ the. vesicles. Thew rrsults showed that rcsiducs 2.504-2523 of InsP,,Rl are in the ER lumen.

S i t c s of N-Glyrosylntinn nf Monsc 1n.~1':~RI--Thr luminal lo- cation of rrsidues 2504-252.3 suggrstrtl that Asn-2475 and/or Asn-2503 are glycosylnted. To vrrifv this possihility. wr intro- duced site-dirrctcd mutations at thrsr potrntial N-glvcosyla- tion sites and expressed the mutant rrceptors in N(;IOH-I5 cells. NG108-15 cells contain a small amount of rndogrnous InsP,,R whose molecular s iz r is smallrr than that of c D N A - derived InsP,,RI (Fig. 6A. Ioncs .5 and 6 ) ( X I. Whcm I pg of microsomal proteins prepared from the cDNA-transfctctrd crlls were analyzed by Western hlotting, only t h r cDNA-dcrivrd rc- ccptors were detected (Fig. 6A, Inncs 1 3 I . Wr nntrd that thc

lFansmemhrane Topology of InsP:{ Receptor 9187 100 , I

t A

D l c t a n r c trim)

FIG. 4. Frequency distribution of the gold particles over cross- sectional profiles of the smooth ER memhrane of mouse cerehel- lar Purkinje crlls. IXstnncrs hr twrrn thr crntrrs of thc, El< mrrnhrnnc and of t h r gy)ld particlrs rracting with antihody IML1 ( A I or mAh 4 C l l I n ) w r r r mrnsurrd. T h r c.ytoplasrnic sidr is indicntrd as positivc, and thc luminal sidr as nrgntivr. The total numhrr of gold pnrticlrs countcd was 189 for antihody I M L I and 236 for mAh 4C11.

mutations of the consensus sites for N-glycosylation did not affect the amount expressed hut increased the electrophoretic mobility of the receptors. Both single amino acid suhstitutions, N2475Q and N2503Q, produced a slight reduction in apparent molecular mass of the receptors (Fig. 6A, Inncs 2 and .? ) com- pared with that of the wild type receptor f Fig. 6A, lnnr I ). The double mutation N2475Q/N2503Q caused a further reduction in apparent molecular mass compared with the single muta- tions N2475Q and N2503Q (Fig. 6A, lnnc 4 ) . To confirm the effects of these mutations, we performed enzymatic deglycosy- lation of the cDNA-derived receptors. When the wild type re- ceptor was treated with N-glycosidase F. its apparent molecu- lar mass was slightly reduced (Fig. 6R. lanes 1 and 2 ) . N-Glycosidase F cleaves all high mannose. hyhrid, hi-, tri-, and tetra-antennary complex oligosaccharides between the inner- most NJ'-diacetylchitohiose and the Asn residue. Conse- quently, it liberates nearly a11 N-linked oligosaccharides from glycoproteins. Neither 0-glycosidase nor neuraminidase treat- ment affected the electrophoretic mobility of the wild type re- ceptor (data not shown). We found that the mutant receptor N2475QRV2503Q migrates with similar mobility to that of the wild type receptor digested with N-glycosidase F f Fig. 6R. Innc 3 ). No detectahlc change in the migration of the mutant recep- tor N2475QRV2503Q was ohserved by the N-glycosidase F treatment (data not shown).

To test the attachment of N-linked oligosaccharide chains more directly, we suhjccted the cDNA-derived receptors to ConA column chromatography. The wild type InsP:,R1 ex- pressed in NG108-15 cells was adsorhed on the ConA column and eluted with 1 M methyl-tr-1)-mannoside (Fig. 7 R ) in the same manner as the cerebellar InsP:,R (Fig. 7A ). Both mutant receptors N2475Q and N2503Q appeared in either column washes (lanes 1 and 2 ) and elution (Fig. 7, C and D). In con- trast, the mutant receptor N2475QN2503Q was recovered only in the column washes (Fig. 7E). These results indicate that mouse InsP,,Rl has only two ConA-adsorptive oligosaccharide chains at Asn-2475 and Asn-2503 among the 20 consensus sites for N-glycosylation in the entire sequence.

l~ l s~*~ ' ss lo~

In thr presrnt study, wr drtrrminrd thr transmrmhranr topology of InsP:,R by two approachrs using immunocytochrmi- cal and molecular biological trchniqurs hnsrd on thr primary structurr of mouse InsP,,RI drcided by molrcular cloning. Thr immunocytochemical study using the sitr-sprcific antihody showed that residues 2504-2523 arr locatrd in thr intracistrr- nal space of the ER. R v ConA column chromatography of thc site-directed mutant rrcrptors. we found that both Asn-2475 and Asn-2503 are glycosylated, indicating that thew two Asn residues arc located in t h r ER lumrn. Thr rrsults of thtbsr two approaches are consistent with thr suggrstion that thr V r r ~ o n between the M5 and M6 segments is rxposrd to thr luminal side of the ER memhranr. Together with thc widrncr that hoth the N and C termini arc cytoplasmic f 13. 14 1, we again proposr a transmembrane topology model in which t h r InsP,,R traverses the memhrane s i x timrs (Fig. HA 1.

To which ion channel suprrfamily dors the InsP,,K hrlong'! Since the InsP:,R is considerrd an ion channrl that is composcd of four suhunits 1 . 5 ) with six memhranr-spanning scpnrnts. we suggest that the InsP:,R is a mcmhrr of thr suprrfamily that includes the voltage-gated ion channrls. Hcwnt findings sug- gest that this superfamily also includes the second mrssrngrr- gated ion channels on the plasma mrmhranr. such as cyclic nucleotide-gated cation channels, putativr Ca"'-activatrd K'

9188

A

Dansmembrane Topoloxy of InsP:, Receptor

1 2 3 4 5 6

200 *

1 2 3 . . . - ." .

B

200 *

FIG. 6. Electrophoretic mobility of the mutant receptors. The exprrssrd rccrptors wcrc annlyzrd hy Wrstrrn hlot using mAh 18A10. A, Lanes 1 4 . 1 pg of microsomal protrins from NClOX-15 crlls trans- frctrd with the wild typr. N2475Q. N2503Q. and N2475Q/N2503Q o f mouse Insl':,Rl cDNA. rcspcctivcly; Innr.5.20 pg o f microsomal protcins from NG108-15 cells transfrctrd with pRactS lvrctor control I; Innr 6.20 pg of memhrane protrins from NGlO8-15 crlls. R . /ones I and -7, 1 pg of microsomal protrin from NGl08-15 crlls transfrctcd with the wild typr and N2475QN2503Q o f mouse InsP:,RlcI)NA, rcspcctivrly, Lnnr 2, ahout 1 pg of N-glycosidasc F-trratrd microsomal proteins from NG108-15 cells transfrctcd with wild tvpr cDNA. When N-glycosidase F was omitted from the reaction. the mobility o f the rrceptor was not changrd. Molrcular sizc markers arr shown on the /rft ( x 1OFt~.

channels, a putative Ca2' channel for phosphoinositide-medi- ated Ca2+ entry, and plant K' ChanneMtransporters (32, 33). The voltage- and second messenger-gated ion channels share a basic design that consists of a set of six putative membrane- spanning segments (Sl-S6) and a pore-forming region between the S5 and S6 segments. The pore-forming region (known as H5, P, or SS1-SSZ) is thought to form an ion-conduction path- way across the membrane (34). The structural similarity pre- dicts the presence of the pore-forming region in the InsP:,R. A hydrophobic region (residues 2530-2Fifi2J exists between the M5 and M6 segments of mouse InsP:,Rl (Fig. 1). This region, however, has no significant sequence homology with the pore- forming regions of the voltage- and other second messenger- gated ion channels. Heginbotham et al . (35) showed that the acidic residue of the Gly-Glu pair in the HF, region is critical for formation of a functional channel. In mouse InsP,Rl, a Gly-Asp pair (at amino acid positions 2549 and 2550) exists in the hy- drophobic region between the M5 and M6 segments (Fig. 1). This Gly-Asp pair is highly conserved among all types of InsP,,R (9, 10, 15-17). We therefore propose that the hydropho- hic region around the Gly-Asp pair acts as a pore-forming re- gion of the InsP:,R (Fig. 8R). According to this proposal, the InsP,,R shares the hasic design of the channel-forming domain

1 M methyl I.-D-mannoside

I 1

1 2 3 4 5 6 7 a 9 1 0

A

200 - I

B

200 - 200 -

m

D

200 - 200 -

FIG. 7. Cod-Sepharose column chromatography of the mutant

rhrllum ( A I or NG108-15 crlls transfrctcd wlth thr wlld tvpv r H I. InsP,R proteins. .\.licrosomal fractions w r r r prvparrd from mousc crr-

N2475Q 1 C I , N2503Q I D I, a n d N 2 4 7 5 c p v m " N ~ I E I of mnuw lnsP , R I cDNA. The microsomal protrins wrrr soluhilizrd in I 5 Tntnn X-lOOand applird to a ConA-Srpharosr column. After thr column was washrd ( Innrs 1-5 1. thr adsorhrd protr ins wrrr r lutrd wlth 1 \I mrthyl-wl)-mnn- noside Ilnnrs 6-10 I . Each fractlon W:IR nnalyzrrl hy Wrstc.rn hlot uslng mAh 18A10. Molrcular sizc markrrs arc, shown on th(* It,/! ( x 1 0 ' I .

,,,,,,,,,,, ~

4Cll (r

rll

1 ML1 V r.Iyrr+.ltnn

FIG. 8. Transmembrane topology model for mouw InnP,Rl within the ER membrane. A. thr putat ivr mrmhranc~-spnnn~ng WR- mcnts arr intftcatrd as o ~ ~ r n hoxrs 1 1 4 1 . Thr rpi toprs fnr antthodv lMLl r res idurs 2504-252:1,, mAh 4Cll lwithln r rs idurs 679-7271 ( H I . and mAh 18A10 lwlthin rrsidurs 273G27.171 14211 a r r ~nd lca t rd . Thr

l inr. R . thc putativc porr-forming rrrion lthrri: ItnrI is vmhrddrd in the. InsP,, binding sitc 1 within rrsidurs 1-650r 1121 is lndtcatrd ;IS a hnff.hvr/

ER membrane from thr luminal sidr.

with the voltage- and second messenger-gated ion channels on the plasma membrane.

In spite of the similarity of the proposed hasic d r s i m of the channel-forming domain, the gating machinery of the InsP:[R is different from that of the voltage- and second mrsscnger-gated channels on the plasma memhrane. The InsP,,R has no homolo- gous sequence with the S4 segment which has hcen proposcd to

Transmembrane Topology of I m p 3 Receptor 9189

serve as a voltage sensor in the voltage-gated ion channels. Actually, the cerebellar InsP3RS reconstituted into the planner lipid bilayer were only weakly voltage dependent (36). The InsP3Wchannel is specifically activated by the intracellular second messenger molecule InsP3. The second messenger-gated ion channels on the plasma membrane and InsP,R are similar in that both the N and C termini of the channel protein are cytoplasmic with their ligand binding sites exposed to the cy- toplasm. But the InsP3 binding site is located within the N- terminal fourth of the InsP,R, while those of the second mes- senger-gated channels are located in the C-terminal region. The positional difference in the ligand binding sites within the channel proteins might reflect the diversity of the gating mechanisms.

The electrophoretic mobility of the three mutant receptors (Fig. 6A ), their ability to bind to a Codcolumn (Fig. 7), and the effects of the N-glycosidase F treatment (Fig. 6B revealed that mouse InsP3Rl has two Cod-adsorptive oligosaccharide chains a t Asn-2475 and Asn-2503 (Fig. 1). These two glycosyl- ation sites, which are located in the V region between the M5 segment and the putative pore-forming region, are conserved in rat (17), frog (161, and human2 InsP3R1. The amino acid se- quence of the V region is well conserved beyond the species in each type of InsP3R but is diversified among the three InsP,R types. In spite of the sequence diversity, two consensus sites for N-glycosylation exist in rat (9) and human (37) InsP3R2 at the positions equivalent to those in InsP3R1. Only one consensus site for N-glycosylation is present in rat (10) and human (37) InsP3R3 at the position corresponding to Asn-2475 of mouse InsP3R1. These observations suggest that both InsP3R2 and InsP3R3 are glycosylated. In contrast to the vertebrate InsP,Rs, the Drosophila InsP,R has no consensus site for N- glycosylation in the loops that are exposed to the ER lumen according to the six-transmembrane model (15).

What functions do the oligosaccharide chains of the InsP,R have? Mishina et al. (38) showed the necessity of N-glycosyla- tion for the subunit assembly of the acetylcholine receptor by site-directed mutagenesis. Unlike the InsP3R, the ligand-gated ion channels on the plasma membrane, such as acetylcholine, y-aminobutylic acid, glutamate, glycine and serotonin recep- tors, are glycosylated at the N-terminal domain that is located in the extracellular space. Our preliminary experiments with the cross-linker disuccinimidyl tartarate revealed that the mu- tation of neither Asn-2475 nor Asn-2503 influences the tet- ramer formation of mouse 1 n ~ P , R l . ~ The glycosylation sites of the vertebrate InsP3Rs are between the M5 segment and the putative pore-forming region. In some voltage- and second mes- senger-gated ion channels on the plasma membrane, such as the cyclic nucleotide-gated ion channels and the a subunit of the voltage-gated Na' channels, the glycosylation sites are also observed in the loop between the S5 segment and the pore- forming region. By using the glycosylation inhibitor tunicamy- cin, it was revealed that the oligosaccharide chains of the volt- age-gated Na+ channel are required to maintain the normal steady state of biosynthesis and degradation of the channel proteins (39) and to control the intracellular distribution of the Na+ channel (40). Neuraminidase treatment of purified Na' channels had significant effects on both steady-state activation and single channel conductance (41). Determination of the in- tracellular distribution and ion channel activity of the site- directed mutant receptors used in this study should provide clues to understand the significance of the oligosaccharide chains of the InsP3R.

' N. Yamada, Y. Makino, R. A. Clark, D. W. Pearson, M. G. Mattei, J. L. Guenet, E. Ohama, I. Fujino, A. Miyawaki, T. Furuichi, and K. Mi- koshiba, submitted for publication. ' T. Michikawa, T. Furuichi, and K. Mikoshiba, unpublished data.

Acknowledgments-We thank Dr. T. Endo for critical comments on the manuscript, Dr. Y. Ryo for immunohistochemical analysis, Drs. K. Sudo and T. Iizuka for technical support in preparing the rabbit anti- serum, and S. Suzuki for technical assistance in preparing the mono- clonal antibodies. We gratefully acknowledge the secretarial assistance of K. Kawamoto.

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