Tm Jolnuw OF B I O ~ I C A L CHEMBIRY B 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 5, Issue of February 4, pp. 3304-3310,1994 Printed in V.S.A.

Molecular Cloning, Sequencing, and Functional Expression of a cDNA Encoding the Human VIa Vasopressin Receptor"

(Received for publication, J d y 12, 1993, and in revised form, September 22, 1993)

Marc Thibonnier8, Colette AuzanS, Zuhayr Madhun, Pamela Wilkins, Liliana Berti-Mattera, and Eric ClauserS From the ~ e p a r t ~ n t of Medicine, University Hospitals of Cle~elaRd and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4982 and the Slnstitut National de la Sant6 et de Ea Recherche Mkdicale, Unitti 36, College de France, 3, rue DUlm, 75005 Paris, France

Vasopressin (AVP), the antidiuretic hormone, is a cy- clic nonapeptide that acta through binding to G protein- coupled specific membrane receptors pharmacologi- cally divided into three subtypes (VI,, Vlb, and V2) linked to distinct second messengers.

Within the family of human AVP receptors, the V, AVP receptor has been cloned, but the structure of the hu- man V1, and Vlb AVP receptors remains unknown. We report here the structure and functional expression of a human V1, AVP receptor complementary DNA isolated from human liver cDNA libraries. Cloning and sequenc- ing of a full-length clone isolated a 1472-nucleotide se- quence encoding a 418-amino acid polypeptide with seven putative transmembrane domains typical of G protein-coupled receptors. Amino acid sequence iden- tity with the rat liver Vl, A W receptor, the human and rat V2 AVP receptors, and the human oxytocin receptor was 72,36,37, and 4S%, respectively.

Functional characterization of the cloned receptor was done by transient expression in COS-7 cells and stable expression in Chinese hamster ovary cells. Local- ization of the expressed receptor at the cellular surface was illustrated by using the fluorescent linear analog phenylacetyl-~-Tyr(lt)-Phe-Gln-Asn-Lys-Pro-Arg-NH~ coupled to fluorescein-avidin by dodecabiotin. Competi- tion binding experiments with phenylacetyl-D-Tyr(Et)- Phe-Val-Asn-Liys-Pr~-[~~"I]~-~~ and AVP analogs re- vealed high affinity specific binding sites of the V1, subtype. Saturation binding experiments with [9HlAVP confirmed the presence of a single class of high affinity binding sites. Measurement of AVP-induced inositol phosphate production and calcium mobilization con- firmed that the expressed V1, AVP receptor is coupled to phospholipase C via a pertussis toxin-insensitive path- way.

Thus, the human Vl, AVP receptor belongs to the su- perfamily of seven-transmembrane segment receptors with a significant sequence identity with the other mem- bers of the AVP-oxytocin family of receptors.

*This work was supported by Grants RO1 HL39757 and PO1 HL41618 from the National Institutes of Health. The costs of publica- tion of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ~ a d u e ~ ~ s e ~ e n ~ ~ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The n u c l e a ~ ~ ~ sequencef's) reported in this paper has been submitted to the ~ n B a n k ~ / E ~ B L Data Bank with accession numberls) L25615.

$ lb whom correspondence should be addressed: Rm. W147, Division of Endocrinology and Hypertension, Dept. of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4982. Tel.: 216-368-4748; Fax: 216-368-4752.

Vasopressin (AVP),l the antidiuretic hormone, is a cyclic nonapeptide involved in the homeostasis of body fluid osmolal- ity, blood volume, vascular tone, and blood pressure. AW also belongs to the family of vasoactive and mitogenic peptides in- volved in physiological and pathological cell growth and differ- entiation. AVP exerts its actions through binding to specific Vla, Vfb, and V, membrane receptors coupled to distinct second messengers (If. While V2 receptors stimulate adenylate cyclase and protein kinase A, VI receptors activate phospholipases .Az, C , and D, resulting in the production of inositol 1,4,5-trisphos- phate (IPS) and 1,Z-diacylglycerol (DAG), the mobilization of intracellular Ca2+, the influx of extracellular Ca2+, the activa- tion of protein kinase C , and protein phosphorylation (2,3). VI, AVP receptors have been shown by radioligand binding tech- niques to be present in vascular smooth muscle cells, hepato- cytes, blood platelets, l ~ p h ~ y t e s and monocytes, type If pneumocytes, adrenal cortex, brain (hip~campus? septum et amygdalae), reproductive organs, retinal epithelium, renal me- sangial cells, and the A10, A7r5,3T3, and WRK-1 cell lines (4). VI, AVP receptors mediate cell contraction and proliferation? platelet aggregation, coagulation factor release, and glycogen- olysis. Vlb AVP receptors are located in the anterior pituitary where they stimulate ACTH release. Vz-renal receptors are present in the Madin-Darby canine kidney and LLC-PKI cell lines, as well as in the medullary portion of the kidney, where they control free water and urea reabsorption via stimulation of adenylate cyclase.

Recently, Morel et al. reported the expression cloning and sequence analysis of a rat hepatocyte VlaAW receptor (5). Moreover, Lolait et al. (6) and Birnbaumer et al. (7) very re- cently described the molecular cloning of human and rat V2 AVP receptors. Simultaneously, Kimura et al. (8) presented the structure of the human oxytocin receptor. Comparison of the amino acid sequence identity of the human Vz A W and oxyto- cin receptors, as well as the rat Vlaand Vz A W receptors, re- veals significant differences, especially at the level of the extra- and intracytoplasmic loops. Currently, the structure of the hu- man V1, and Vlb AVP receptors remains unknown. Obtaining this information is important as it is very likely that the family of AW receptors, like other families of seven-transmembrane segments (7-TMS) receptors, includes several members for which structural differences determine their subtype specific- ity. In this paper, we have described for the first time the molecular cloning, s~uencing, and functionaf. expression of a cDNA encoding the human VI, A W receptor. Pharmacological

1 The abbreviations used are: AW, [Arg%asopressin; DMEM, Dub becco's modified Eagle's medium; PBS, phosphate-buffered saline; TMS,

pair(s); CHO, Chinese hamster ovary; IP, inositol phosphate; HPLC, seven-transmembrane segment(s); Phaa, phenylacetyl; kb, kilobase

high performance liquid chromatography.

3304

Human VI , AVP Receptor Clone 3305

studies show that the isolated receptor displays the typical features of the VI, subtype with coupling to phospholipase C and calcium mobilization.

E ~ E ~ M E ~ ~ P R O C E D ~ S ~ a ~ ~ r i a Z ~ V a s o p r e s s i n , Tris-HCl, and other reagents, unless stated

otherwise, were from Sigma. Ionomycin was from Calbiochem Corp., San Diego, CA. COS-? and CHO cells were obtained from the American Type Culture Collection ( ~ ~ v i l l e , MD). Cell culture media and Gene- ticin were from Life ~ c h n o l o ~ e s , Inc. Fetal bovine serum was from HyCIone, Logan, UT. Restriction and modifi~tion enzymes were from Bcehringer M ~ e i m or New England Biolabs, Iodogen (1,3,4,6-tetra- c~o~3a-6f f -d iphenylg lyco~~l ) was fmrn Pierce Chemical Co. Fura-2 AM and Neutralite fluorescein-avidin were fmrn Molecular Probes, Portland, OR. NAP-10 and Nick columns were from Pharmacia LKB Biotechnology Inc. Nitrocellulosa membrane (EA-85 N i ~ l l u l o s e ~ was from Schleicher & Schuell. Nensorb 20 c&ridges, N a W (activity = 131 mCilml), fya2PLATP (specific activity = 6,000 Ci/mmoD, IsHLAVP (spe- cific activity = 62 Cilmmol), and ~y0-~2-~HIinositol (specific activity = 20 Cifmmol) were obtained fmrn Du Pont NEN. [36SldATP (specifc activity =A 1,000 Cihnmol), ICU-~~PI~CTP (specific activity = 3,000 Gi m o l ) , and the Megaprime labeling kit were from ~ e r s h a m Corp. Ml3mpl8 phage, p B l u e s ~ p t II p h a g e ~ d , and XLl-Blue E s c ~ r i c ~ ~ coli strain were fmm Stratagene, La Jolla, CA. The DNAsequencing kit was from U. S. Biochemical Corp. The V2=AVP antagon~st phenylacetyl- ~ ~ E t ) - P h e C l n - A s n - L y s - ~ a - ~ * ~ ~ and the VZ-renal AVP antaga- nist d(CH2)b[~neZ,Ile4~~a-NH21AVP were gifts from Dr Maurice Man- ning (Medical College of Ohio, Toledo, OH). The new VI, AVP non peptide a n ~ o n i s t SR 49059 waa kindly provided by Dr. Serradeil-Le Gal ( S ~ o f i ~ c h e r c h e , ~ ~ o ~ e , France). The V1, AW antagon~st p h e n y l a ~ e t y l - ~ ~ ~ t ) - P h ~ V a l - ~ n - L y s - ~ ~ ~ - ~ ~ , custom-s~the- sized by Bachem (Torrance, CAI, was rad io i~nated ~ ~ l ~ ~ 1 ~ P h a a ~ using the Iodogen technique and purified by HPLC as previously de- S C r i M (9).

DNA probe Synt~s~-OligonucIeotide probes co~esponding to sev- eral regions of the p u b ~ i ~ e d rat liver AVP receptor nucleotide sequence were pmduced (5). The probes were 45 nucleotides long, recogxizing the N terminus ~ 5 ' - A T G A G ~ C C C ~ G A ~ T C C C A ~ A T C ~ ~ C G - ~ ~ C ~ C - 3 ' ~ and the C terminus ( 5 ' - G ~ T ~ G ~ G T C ~ T " C ~ C ~ T C A T ~ T A T C ~ A G ~ A T C C ~ - 3 ' ) , as we11 as the first ( 5 " ~ ~ G ~ T ~ C T G T G C T G ~ C ~ T A G C A G T G T G C T ~ T ~ - GCC-3'), second ( 5 " A ~ C ~ A G A C C ~ ~ T C ~ C ~ C ~ C C A ~ A G T A ~ A C C ~ A G - ~ ~ , six& ( 5 ~ - C C A C A ~ ~ A C ~ ~ ~ G ~ G - G C ~ C C A ~ ~ ~ A ~ T A ~ . ~ ) , and seventh ~ 5 ' - G ~ C A T - G ~ A T A T C C A C ~ ~ A ~ A ~ ~ T C ~ ~ ~ ~ 3 ' ) transmem- brane segments. They were synthesized with a Applied Biosystems o~igonucl~tide synthesizer and purified on a 15% ~lyacrylamide gel. FoIlowing ~ ~ r o p h o r e s i s , o l i ~ n u c l ~ t i d e s were visualized by short wave W illumination, excised from the gel, and l~philized. For hy- b r i ~ ~ a t i o n purposes, the ol i~onucl~t ides were labeled with "ZP at the 5 ' - h y ~ x y l p u p using [-psZPIATP and T4 pol~uc~eot ide kinase, and then purified over Nensorb 20 cartridges to screen the libraries.

A gtl0 Library ~ r e e n i ~ - ~ m b i n a n t phages (lo6) fmm an ampii- fied A gtl0 human liver cDNA library purchased from Clonetech (HL 1115a) were incubated with E. coli C600 cells for 20 min at 37 "C in SM medium, mixed with 0.7% LB agarose, and plated on 1.5% LB agar in 150-mm Petri dishes at a density of 50,000 plaque-fo~ing uni~dish. Plates were incubated at 37 "C until lytic plaques began to appear (8-9 h). GRer coaling at 4 "C for 1 h, phages were li&d onto duplicate BA 85 n i t ~ l ~ o s e filters, which were denatured, neutralized, and heated at 80 "C for 2 h. Replicate fiiters were prehybridized for 12 h at 42 "C in a solution containing 6 x SSC, 0.1% SDS, 5 x Denhardt's, and 100 &ml sheared salrnon sperm DNA. The p~hybrid~zation solution was dis- carded and replaced by the same hybndizat~on solution, which con- tained labeled first and seventh TMS nucleotides (5 x loe cpdfilter) for further incubation at 42 "C for 24 h. Filters were washed with 6 x SSC + 0.1% SDS at roam temperature for 1 h, then with 2 x SSC + 0.1% SDS at 42 OC and 1 x SSC + 0.1% SDS at 55 "C until b a ~ ~ ~ d radioac- tivity monitored with a hand-held Geiger counter had decreased to acceptable levels. Subsequentl~ the filters were dried and subjected to au toradi~aphy on Kodak XAR-5 films with one Du Pont Quanta I11 screen for 12-24 h at -70 "C. The positive clones were isolated and

the labeled second and sixth TMS oligonucleotides. plaque-purified by secondary and tertiary screening procedures using

In order to isolate a ~ - 1 e n ~ ~ cDNA clone for the human VI, AVP receptor, a second A gtl0 human liver cDNA library (a generous giR from Dr. Savio Woo, BayIor College of Medicine, Houston, TX) was

screened with both radiolabeled oligonucleotides (derived from the N terminus and the first TMS of the rat V1, AVP receptor sequence) and the 1.7-kb partial cDNA fragment isolated during the first screening. This cDNA fragment was random labeled with using CCT-~~PJ~CTP and the Megapnme DNA labeling system from Amersham Corp. Pre- hybridization, hybridization and washing procedures were identical to those described above for the filters hybridized with the labeled oligo- nucleotides. ~ehybridization and hybridization with the cDNA frag- ment were performed in 50% formamide, 6 x SSC, 0.1% SDS, 5 x Denhardt's, 100 pglml sheared salmon sperm DNA at 42 *C. Washing conditions were 2 x SSC + 0.1% SDS for 15 min at 50 "C and 0.1 x SSC + 0,1% SDS for 15 min at 50 "C twice.

DNA S u ~ c ~ o n ~ n g a n d Sequencing-A phage lysates generated from tertiary screening dishes were used to obtain midi-preparations of re- combinant A phage DNA. The EcoRI fragments of the cDNA inserts were identified by agarose-gel electrophoresis and Southern hybridiza- tion analysis with the sZP-labeled oligonucleotides. These fragments were subcloned by standard recombinant techniques into M13mp18 phage and into pB~uescnpt 11 phagemid vectors prior to tr~nsformation of XLl-Blue E. coli strain. Nucleotide sequencing was done using the dideoxy chain t e ~ a t i o n method of Sanger and the DNA sequencing kit from U. S. Biochemical Corp. E l e ~ p h o r e s i s of reaction products was carried out in 5% ~lyacrylamide gels containing 50 g of urea, 45 m 'k is borate, 1.25 m~ EDTA followed by autoradio~aphy on Kodak XAR film. S e q u e n ~ g of both strands was initiated with universal primers, and the oligonucl~tides were used for library screening. The deduced amino acid sequence b e ~ n ~ n g with an ATG and extending to the t e ~ n a t ~ o n codon was ana~yzed by hydrophobicity piot and com- pared to the known sequences of the rat liver VI, AVP receptor, the rat and human V2-renal AW receptors, the human oxytocin receptor, and other 7"TMS receptors.

~ a n ~ ~ n ~ ~ p ~ s s ~ ~ ~ in COS-7 CelbDNA was prepared and used for t ~ n s f e ~ i o n in COS-? cells, an i m m o ~ ~ ~ e d cell line derived from Gfrican green monkey kidney that we have shown to be devoid of en- dogenous V,-vascular AVP receptors. COS-7 cells were obtained from ATCC and grown in DMEM with 10% fetal calf serum, penicillin, and s~eptomycin in 5% C 0 2 , 95% O2 at 37 "C. Transfection of DNA into COS-7 cells was performed using the DEAE-dextran method. The EcoRI fragment coding for the entire AVP receptor was inserted in the pECE expression vector (10, ll), and this construct was mixed with serum- free DMEM and D33AE-dextran, then applied to lo6 COS-7 cells in a T 75-cmz flask for 90 min. h r exposure to chloroquine for 4 h, Me2S0 treatment was performed at room temperature for 5 min. Expression of ~ n ~ o n ~ AVP receptors in the transferrted cells was assessed 48 h later by radioligand binding experiments and inositol phosphate production as described below.

Stable Expression in CHO Cells-Stable transfection of DNA into CHO cells was perfomed using the CaP04 prec~pitation method (12). The express~on plasmid pECE conta~ing the AVP receptor clone was mixed with the resistance plasmid pSV2Neo and 250 m~ CaCIZ in HBS buffer tin m: Hepes 25, NaCll40, N a m 4 1.5, NaHZP04 1.5, pH 7.4) for 30 min at room ~ m p e r a ~ ~ ~ then applied to lo6 CHC, cells in a T 75-cm2 flask. f i r i n ~ b a t i o n for 4 h at 37 OC, 12.5% glycerol treatment was ~ r f o ~ e d at Mom tempera~re for 2 min. Pure clones were se- lected for their resistance to the antibiotic Geneticin and purified by the l ~ i t i n g dilution techn~que. Clones expressing the greatest receptor density in binding experiments were further explored by fluorescent labeling and radioligand binding experiments, as well as measurement of tCa2+li mobilization and inositol phosphate production, as described below.

~ a ~ ~ o ~ ~ g a n ~ Bid ing Assays-We prepared the radioiodinated compound p h e n y l a c e t y ~ - ~ - ~ ~ t ~ P h e - V ~ - ~ n - L y s - ~ [ ~ ~ ~ I l ~ - N H ~ (E12sI~Phaa) by the Iodogen technique and HPLC purification as described previously (9). Control and transfected COS-7 and CHO cells were grown to confluence in 24-well dishes and washed twice with DMEM .t 25 RIM Hepes + 0.25% bovine semm albumin, pH 7.4. Com- petition binding experiments were performed in duplicate by incubating the cells (final volume 250 $) in the same medium with one fixed concentration of [12bI]TyrPhaa (0.30 nrd and increasing concentrations of unlabeled AVP or the linear V1, AVP antagonists phenylacetyl-w Tyr(Et)-Phe-Val-Asn-Lys-~o-Tyr-~~ or phenylacetyi-~-~Et)-Phe- G I n - ~ n - ~ y ~ - P r o - ~ ~ - ~ ~ , the n o n ~ p t ~ d e V,, AW antagonist SR 49059, the V2 AW antagonist d f C H 2 ) 6 [ ~ - n e 2 , n e q ~ a - ~ 2 ~ V P , or oxy- tocin for 30 min at 30 "C. The cells were washed thme times with ice-cold PBS and lysed with 0.5 ml of 0.2 N NaOH, 1% sodium dcdecyi sulfate. C e l l - ~ ~ d [lZsIl'QrPhaa was counted in a y counter. IC, val- ues were derived fmm nonlinear least square analysis, and K, values

3306 Human V I , AVP Receptor Clone

LJKd). were calculated by the equation of Cheng and plusoff: Ki = ICsd(l +

Additionally, saturation binding experiments ofAVP receptors of con- trol and transfected CHO cells were done in 24-well dishes in duplicate as described abbve but in 250 pl of PBS + 10 m~ MgCl, + 0.20% bovine serum albumin, pH 7.4, with increasing concentrations of [3H]AVP f 1 p~ unlabeled AW. The radioactivity was measured by liquid scintilla- tion spectrometry (Beckman counter LS 5801, yield = 64%). Affinity (Kd) and capacity (BmJ of the AVP receptors were calculated by a nonlinear least square analysis program (13). Protein concentration was measured with Pierce's BCA reagent using ovalbumin as an inter- nal standard.

Neutralite Fluorescein-Avidin Labeling-We coupled phenylacetyl-o- Tyr(Et)-Phe-Gln-Asn-Lys-h-Arg-NH, (PhaaGln) via its lysine e-amino group to dodecabiotin (PhaaGln-dodecabiotin) and purified this complex over a C8 HPLC column as previously described (9). VI, AVP receptors of CHO cells were visualized by PhaaGln-dodecabiotin made fluorescent by labeling with fluorescein-avidin. CHO cells used for fluorescence measurements were grown on glass coverslips to 50% confluence. The coverslips were washed with ice-cold PBS buffer, then incubated in six-well plastic trays with 1 ml of PBS buffer and 1 1.1~ PhaaGln-do- decabiotin alone or in the presence of 5 p~ PhaaGln for 30 min at 37 "C. Thereafter, the coverslips were washed five times with 1 ml of buffer and incubated with 25 pg of Neutralite fluorescein-avidin for 2 h in the dark. The coverslips were washed again five times, and furation was performed for 15 min at 4 "C in 3% paraformaldehyde. The preparations were examined with the microscope/computer system described previ- ously (9) with excitation and emission wavelengths at 490 and 515 nm, respectively.

Znositol Phosphate Production-Subconfluent monolayer cultures of control and transfected COS-7 and CHO cells were grown for 48 h in 12-well dishes, washed with inositol-free DMEM, and labeled with myo- [2-3Hlinositol ( 2 3 pCi/ml) in inositol-free DMEM for 20 to 24 h (14). Thereafter, the cells were preincubated for 1 h in DMEM, followed by a 15-min preincubation in Hanks' balanced salt solution containing 10 m~ glucose, 1.2 m~ CaCl,, and 10 m~ LiC1,. Increasing concentrations of AVP were added for a 30-min incubation at 37 "C. The reaction was stopped by addition of 1 ml of ice-cold methanolHC1 (lOO:l), followed by 400 pl of H,O and chloroform, to obtain a ratio for ch1oroform:methanol: HCl of 200:lOO:l. After phase separation, the upper phases were re- moved and loaded onto AG1-X8 Dowex columns (chloride form, 200-400 mesh). Inositol monophosphates (IPl), inositol bisphosphates UP,), and inositol trisphosphate (IP3) were eluted with 30 m ~ , 90 m ~ , and 0.5 M HCl, respectively, for scintillation spectroscopy counting. The lower phases containing labeled phosphoinositides were washed with a theo- ric upper phase solution containing 1 m~ inositol and counted. The amount of inositol phosphates released was expressed as dpdwell or as a fraction of total labeled phosphoinositides. Typical incorporation into phosphoinositides amounted to approximately 3.5-5 x lo' 3H-labeled dpdwell. In addition, experiments were performed in transfected CHO cells pretreated with 0.1 pg/ml pertussis toxin for 18 h prior to AVP stimulation to determine the sensitivity of AVP-induced inositol phos- phate production to pertussis toxin (15).

CAMP Production-Subconfluent monolayer cultures of control and transfected CHO cells were grown for 48 h in 12-well dishes to measure adenylyl cyclase activity in the presence and absence of AVP as de- scribed before (16).

Measurements of Intracellular Calcium-Free intracellular calcium concentration was measured as described previously in serum-starved monolayer cultures of control and transfected CHO cells loaded with 1 p~ fura-2 AM (3,9). Autofluorescence by the cells or agonists, as well as fura-2 leak outside the cells, was negligible.

Data Analysis-Nucleotide and amino acid sequences were analyzed and compared with the computer package Geneworks on a MacIntosh computer (IntelliGenetics, Inc. Mountain View, CA).

RESULTS AND DISCUSSION Structure of the Human VI , AVP Receptor-Screening of the

Clonetech A gtl0 human liver cDNA library yielded several positive clones for which further subcloning and restriction endonuclease analysis isolated a 1.7-kb EcoRI fragment. DNA sequencing of this 1.7-kb EcoRI fragment indicated a 79% iden- tity of the coding sequence with the nucleotide sequence of the rat liver V1, AVP receptor (data not shown). Sequencing re- vealed that this 1.7-kb EcoRI fragment encompassed two thirds of the coding region of the human liver V1, AVP receptor and

the 3'-untranslated region. To achieve the complete cloning and sequencing of the hu-

man liver V1, AVP receptor, we screened another human liver cDNA library (developed and kindly provided by Dr. Savio Woo). This library was screened with the 32P-labeled 1.7-kb fragment isolated above as well as the radiolabeled oligonucleo- tides derived from the N terminus and the first transmembrane segment of the rat VI, AVP receptor sequence.

Subsequent subcloning and sequencing identified a 1.5-kb insert, the composition of which is shown in Fig. 1. The 1472- nucleotide sequence of the human V1, AVP receptor cDNA en- codes a 418-amino acid protein (M, = 46,745) deduced from the open reading frame of 1254 nucleotides spanning from nucleo- tide 62-1315 of the cloned cDNA. The ATG of GACAGCATGC is assigned as codon 1 on the basis of its close match to the GCC(A/G)CCATGG Kozak consensus sequence for initiation of translation in vertebrates (17) and by homology with the se- quences of the other members of the AVPloxytocin family of receptors. However, another in-frame ATG is present a t posi- tion 44 of the cloned sequence, and the definite answer regard- ing the true translation initiation site will be obtained by amino acid sequencing of the purified receptor protein.

The nucleotide coding sequence identity of the human V1, AVP receptor with the rat liver V1, AVP receptor, the human oxytocin receptor, the human V, AVP receptor, and the rat V2 AVP receptor nucleotide sequences is 83,68,62, and 65, respec- tively. Homology with other families of seven-transmembrane domain receptors is less than 50%.

Hydropathicity analysis done according to Eisenberg using a window of 20 residues (18) shows that the translated protein has the typical features of a G protein-coupled transmembrane receptor with seven putative hydrophobic domains, connected by three extracellular and three intracellular loops. There are several possible sites of post-translational modifications of the human V1, AVP receptor. The N-terminal region preceding the first putative transmembrane domain contains two potential N-linked glycosylation sites (N-X-(S/T)) at Asn-14 and Asn-27. An additional N-linked glycosylation site is present at the level of the second extracellular loop (Asn-196). There are 8 threo- nine and 17 serine residues in the third cytoplasmic loop and the C-terminal region of the human V1, AVP receptor. These residues could be sites for regulatory phosphorylation, there- fore suggesting that the functions of the human VI, AVP re- ceptor are regulated by protein kinases. Indeed, there are con- sensus sequences specific for protein kinase C-dependent phosphorylation ((S/T)-X-(R/K)) in positions 382,404,407, and 410. Cysteine residues present in the second (Cys-124) and third (Cys-203) extracellular loops are possibly involved in the tertiary structure of the receptor and ligand binding. As a mat- ter of fact, we reported that [3HIAVP binding to V1, AVP recep- tors of human platelets was altered by the presence of free sulhydryl group alkylating agents like N-ethylmaleimide (19). Potential sites for palmitoylation (Cys-359, Cys-365, and Cys- 366) are present in the C-terminal region of the receptor se- quence and may anchor this part of the receptor protein to the inner face of the plasma membrane (20). An aspartate residue located in the second transmembrane domain and playing a key role in binding affinity and functional coupling of other 7-TMS receptors is present in position 97 of the human V1,AVP receptor sequence (12,21). By the same token, an alanine resi- due at the carboxyl end of the third intracytoplasmic loop in- strumental in G protein coupling is present in position 285 of the human VI, AVP receptor sequence (22). Moreover, the ca- nonical sequence Asp-Arg-Tyr present at the end of the third transmembrane domain of the substance P, substance K, dopa- mine D2, dopamine DS, and endothelin B receptor genes is also present at the end of the third transmembrane domain of the

Human V,, AVP Receptor Clone 3307

-52 ggcgcgaqggctqgagctCcgaagagggccgagtaggagctgcatggacagc

ATG CGT CTC TCC GCC GGT CCC GAC GCG GGG CCC TCG GGC ARC TCC AGC CCA TGG TGG CCT Met arg l e u ser ala gly pro asp ala gly pro ser gfy asn ser se r p ro t rp t rp p ro

CTG GCC ACC GGC GCT GGC AAC ACA AGC CGG GAG GCC GAA GCC CTC GGG GAG GGC AAC GGC l e u a l a t h r gly ala gly asn t h r ser arg glu a l a glu a la l e u 9ly glu gly asn gly

CCA CCG AGG GAC GTG CGC AAC GAG GAG CTG GCC AAA CTG GAG ATC GCC GTG CTG GCG GTG pro pro arg asp val arg asn glu glu l e u ala lys l e u glu i le a la val l e u a l a Val

I""""""""

"""1""""""""""""""- I ACT TTC GCG GTG GCC GTG CTG GGC ARC AGC AGC GTA CTG CTG GCT CTG CAC CGG ACG CCG t h r phe ala Val a la va l l e u gly asn ser ser val l e u leu a la l e u h i s arg t h r pro

CGC AAG ACG TCC CGC ATG CAC CTC TTC ATC CGA CAC CTC AGC CTG GCC GAC CTG GCC GTG arg lys thr ser arg m e t h i s l e u phe i le arg h i s l e u ser l e u ala asp l e u a la Val

GCA TTC TTC CAG GTG CTG CCG CAA ATG TGC TGG GAC ATC ACC TAC CGC TTC CGC GGC CCC a la phe phe gln va l l e u pro gln met cys t r p asp i le t h r tyr arg phe arg gly pro

I"""""""""""

"11""""""""" I

I""""""""""""""""" GAC TGG CTG TGC CGC GTG GTG AAG CAC CTG CAG GTG TTC GGC ATG TTT GCG TCG GCC TAC asp t r p l e u cys arg val val l y s his leu gln val phe gly met phe ala ser a l a t y r "... 111""""""""""" i ATG CTG GTA GTC ATG ACA GCC GAC CGC TAC ATC GCG GTG TGC CAC CCG CTC AAG ACT CTG met leu val val met t h r ala asp arg tyr i le ala val cys h i s pro leu lys thr leu

1""""""" CAA CAG CCC GCG CGC CGC TCG CGC CTC ATG ATC GCG GCC GCC TGG GTG CTG AGC TTC GTG gln gln pro a l a arg arg ser arg l e u met i l e ala ala a l a t r p v a l l e u scar phe Val """IV"""""""""" I CTG AGC ACG CCG CAG TAC TTC GTC TTC TCC ATG ATC GAG GTG AAC AAT GTC ACC RAG GCC

FIG. 1. Primary nucleotide stmc- l e u set t h T pro gln t y r phe v a l phe ser met i ie glu v a l asn asn val t h r lys a la

ture of the hamum V E - V ~ C ~ ~ AVP CGC GAC TGC TGG GCC ACC TTC ATC CAG ccc TGG GGT TCT CGT GCC TAC GTG ACC TGG ATG

1 """"

receptor cDNA. Positions of the pu&- arg asp cys trp ala t h r phe i le gln pro t r p g l y ser arg a la t y r val t h r t r p met tive T&fS 1-mI are indicated by iiws ."---."----- -----."____ v __---_-_-".-I -- ---_ ------- I above the nucleotide sequence. ACG GGC GGC ATC ?TT GTG GCG CCC GTG GTC ATC TTG GGT ACC TGC TAC GGC TTC ATC TGC

t h r g ly g ly i l e phe val ala pro val Val i i e l e u gly t h r cys t y r gfy phe i l e cys

TAC AAC ATC TGG TGC AAC GTC CGC GGG AAG ACG GCG TCG CGC CAG AGC AAG GGT GCA GAG t y r asn i le t r p cys asn Val arg gly lys thr ala ser arg gln ser lys gly a la glu

CAA GCG GGT GTG GCC TTC CAR AAG GGG TTC CTG CTC GCA CCC TGT GTC AGC AGC GTG AAG gln ala gly V a l ala phe gln lys gly phe leu leu ala pro cys val ser ser val lys

TCC ATT TCC CGG GCC AAG ATC CGC ACG GTG AAG ATG ACT TTT GTG ATC GTG ACG GCT TAC ser ile ser arg a l a l y s ile arg t h r Val l y s met t h r phe Val i l e Val t h r ala t y r "-""""""VI-"""""""""""""""" I ATC GTC TGC TGG GCG CCT TTC TTC ATC ATC CAG ATG TGG TCT GTC TGG GAT CCC ATG TCC i le va? cys t r p ala pro phe phe i le i l e g ln met t r p s e r val t r p asp pro met ser

GTC TGG ACC GAA TCG GAR AAC CCT ACC ATC ACC ATC ACT GCA TTA CTG GGT TCC TTG ART Val t r p t h r q l u ser glu asn pro t h r i l e t h r i le thr a la leu l e u gly ser l e u asn

1 """"""*

""""~"""""-vII"-"""""""""~""" 1 AGC TGC TGT AAT CCC TGG ATA TAC ATG TTT TTT AGT GGC CAT CTC CTT CAA GAC TGT GTT ser cys cys asn pro t r p i l e t y r met phe phe ser g l y h i s leu leu g l n asp cys va l

CAA AGC TTC CCA TGC TGC CAA AAC ATG AAG GAA AAA TTC AAC AAA GAA GAT ACT GAC AG? gln ser phe pro cys cys gln asn met l y s 9lu l y s phe asn l y s glu asp thr asp ser

ATG AGC AGA AGA CAG ACT ?TT TAT TCT AAC AAT CGA AGC CCA ACA AAC AGT ACG GGT ATG met ser arg arg gln t h r phe t y r ser asn asn arg ser pro t h r asn ser th r g ly met

TGG AAG GAC TCG CCT AAA TCT TCC AAG TCC ATC AAA TTC ATT CCT GTT TCA ACT TGA gcC t r p l y s asp ser pro l y s ser ser lys ser i le l y s phe i l e pro Val ser t h r OPA

t tg ca t t ca tgc aac t t g a t t c t t g t g a t t gac t t t t tq gc t ca t t ag c tg aa t tga gc t aga aat cac dag dac aaa t a c a c t t t a t t a ata tad cca t aa a t c aa t t ca t t g t g t a tg aga c tg t q t t t c t aq t t q ca t t t c a t a t t q e t a cca aac c

human Vla AW receptor sequence (23). Alignment of the amino acid sequences of the human VI,

AVP receptor, the rat VI, A W receptor, the human oxytocin receptor, the human V2 AVP, and the rat V, AVP is presented in Fig. 2. The amino acid sequence identity of the human Vla A W receptor with the rat liver VI, A W receptor, the human oxyto- cin receptor, the human V, A W receptor, and the rat V2 AW receptor sequences is 72, 45, 36, and 37%, resp~ively. This degree of identity is comparable to those observed between the members of other 7-Th([S receptor groups like the adrenergic and muscarinic receptor families. Amino acids enclosed in boxes

-1

20 60

I20 40

190 60

240 80

300 100

360 120

620 140

480 160

540 180

2 00 600

660 220

720 240

2 60 780

840 280

900 300

960 320

1020 340

1080 360

1140 380

1200 400

1260 418

1320 1360 1420

are identical for the human VI, AVP receptor and the other members of the AW-oxytocin receptors' famils

Among the five members of the AW/oxytocin family of re- ceptors isolated so far, several amino acid sequences are re- markably conserved (Fig. 2). AH receptors share the sequence FQVLPQ present at the 3' end of the second transmemb~~e domain. This sequence is thought to play a major role in ligand binding and signal transduct~on. The sequence FXGPRXL- CRXW present at the 3' end of the first extracellular loop and the sequence R C W ~ ~ W ~ located in the second extraeel- Iular loop are present in all AVP/ox+in receptor sequences.

3308

FIG. 2. Alignment of the amino acid sequences of 7-TMS receptor pro- b i n s of the AW-oxytoein family. De-

V1.AVP receptor (HVla), the rat liver Vi. duced amino acid sequences of the human

A W receptor (Rat Vla), the human ( W 2 ) and rat V, receptors 1RatV2), and the human oxytocin receptor (HOxy) were aligned. Consellred amino acids through- out the family of A V P - 0 ~ ~ receptors family are bored, and positions of the pu- tative TMS I-VI1 are indicated by lines above the amino acid sequences.

HvIa

Hv2 RatVfs

mv2 Ho*Y

HVla RatVlo Hv2 Ratvz HOxy

Hvla

Hv2 RatVia

Ralvz HOxy

Hvh RatVfa H v Z Ratv2 H*Y

HVlo

HV2 RatVla

Ram HOxy

Hvto RatVla Hv2 Ratv2 HOxy

HVI a

Hv2 Ratvia

Ralw H*Y

H v l a RatVla Hv2 Ratv2 €foxy

XKFXPVST

These two extracellular sequences are not found in other mem- bers of the superfamily of 7-TMS receptors and are plausible elements of the ligand r ~ ~ i ~ o n site. Additiona~ly, all five sequences have in common the motif NPWN in their seventh transmembrane domain, which is thought to be instrumental in receptor-mediated endocytosis. Finally, two adjacent cys- teine residues are present in all C termini and, through palmi- toylation, may anchor the receptor tail to the inner face of the plasma membrane (20).

At variance, other regions within the five sequences bear very little similarity. For instance, the third intracytoplasmic loop, which plays a key role in G protein coupling, differs dras- tically between the Vx and Vz sequences (Fig. 2). It also comes as no surprise that the N- and C-terminal regions differ widely between the five sequences.

~ a ~ i # l i g a n d Binding Characteristics of the Expressed Ha- man VI, AVP Receptor in COS7 and CHO Cells-The radioli- gand binding c h a r a c ~ ~ s t i c s of the cloned human VI, AVP re- ceptor were first explored in the transiently transfected COS-7

cells. Competition binding experiments confirmed that the cloned receptor has the typical pharmacologic profile of the VI, subtype (Fig. 3a). The most potent competi~r of [12511QrPhaa- specific binding was the linear V1, antagonist phenylacetyl-D- ~ E t ~ - P h e - ~ l n - A ~ n - L y s - ~ o - ~ - ~ ~ (Xi = 1.24 * 0.16 ma), followed by the linear VI, non-peptide an~gonis t SR 49059 (ICi

= 1.29 c 0.17 nM), then AVP itself (Ki = 1.79 f 0.36 ma), then the linear VI, antagonist phenylacetyl-D-Tyr(Et)-Phe-Val-Asn-Lys- Pro-Tyr-NHi% (Ki = 2.96 = 0.52 nM), and the Vz antagonist d ( C H z ) 6 [ ~ I l e 2 , n e 4 , ~ a - ~ ~ l A W (Ki = 67.61 2 16.91 m). Fi- nally, oxytocin was the weakest competitor (Ki = 128.93 r+ 22 m). No specific binding was observed in nontransfected cells and in cells transfected with the pECE vector alone (data not shown).

To characterize further the ph~acologica l characteristics of the cloned human V1, AVP receptor, we used CHO cells stably transfected with the full-length 1.5-kb clone we isolated. We verified in competition experiments with [12611TyrPhaa that control CHO cells did not have endogenous VI, AW receptors

Human VI , AVP Receptor Clone a

3309

Competitor Concentration (M)

b

I . - . ,o.12 IO"^ 1g* 10.* 1 0 ” lo5

Competitor Concent~ation fM)

FIG. 3. Specific binding of A W to COS? and CHO cells trans- fected with the human V,, receptor clone. Transfected COS-7 and CHO cells prepared as described under ~ ~ ~ r i m e n ~ l Procedures” sec- tion were grown to confluence in 24weE dishes. The figures show the mean of compet~tion binding experiments performed with one concen- tration of [laaIj”haa and increasing Con~ntTations of A W OT its analogs (n s 3-5 for each anafog) in COS-7 cells (panel a ) or CHU cells (panel b).

(data not shown). Competition binding experiments confirmed that the cloned receptor belongs to the VI, subtype (Fig. 3b). The most potent competitor of [12511~Phaa-specific binding was the linear V1, a n ~ g o n i s t phenylacetyl-D-~Et)-Phe-Gin~ Asn-Lys-~o-~g-NH2 (Ki = 0.79 rt 0.23 m), followed by the linear Vla non-peptide antagonist SR 49059 (Xi = 1.12 rt 0.08 m), then AW itself (Ki = 1.77 rt 0.12 m), then the linear VI, antagonist phe~ylace ty l”D-~Et) -Phe-Val -Asn-Lys-~~- NH, (Ki = 2.37 * 0.07 m), and the V, antagonist d~CH2)5[~ n e z , n e ~ * ~ a - ~ 2 1 A W (Ki = 52.94 t 7.60 DM). Finally, o ~ i n was the weakest c o m ~ t i t o r CKi = 87.14 * 22 mal. This order of aanities is therefore identical to that observed in COS-7 cells and confirms the VI, subtype of the cloned receptor.

In addition, saturat~on binding experiments with f3H.@SW were performed in CHO cells transfected with the cloned hu- man V1, AW receptor cDNA (Fig. 4). A single class of b ~ n d ~ g sites was identified with a ~ssociation constant, K d = 1.21 rt 0.06 m and a total capacity B,, = 780 t 39 fmol/mg of protein f n = 24). N o n s ~ i f i c binding was negli~ble in transfected CHO cells. No specific binding was observed in nontransfected cells and in cells transfected with the pECE vector alone (data not shown). Ki and & values derived from com~ti t ion and satu- ration binding experiments are well in agreement with those for the human platelet VLa AW receptor we extensively char- acterized (2,9,13,24). The ra~oligand binding characteristics of the cloned human VI, AW receptor in CHO cells we present here are similar to those reported by Morel et al. (5 ) for the cloned rat V1, AW receptor in transfected CHO cells (& = 0.67 ma, = 584 fmo2lmg of protein).

Fluorescent Labeling ofthe Human VI, A ~ P ~ c e p t o F in CHO Cells-As we reported recently, linear V1-vascular AW antago- nists can be coupled to dodecabiotin to visualize AW receptors by addition of a ~ u o r e s c ~ n - a ~ d i n tag (9). Fluorescent labeling of CHO cells with PhaaGln-dodecabiotin-~uorescein-avidin re-

0 0 5 10 15 20 25

Free [3MAVP (nM)

FIG. 4. Characteristics ofAVP binding sites in CHO cells trans- fected with the human VI, receptor clone, Transfected CHU cetls prepared as described under “Experimental Procedures” were grown to confluence in 24-well dishes. Saturation binding experiments were per- formed with increasing concentrations of [SHIAVf? Specific binding is shown as open circles, and nonspecific binding was deterrnined in the presence of 1 AW (open squures). The inset is a Scatchard linear transformation of the data (n = 24).

vealed a striking increment of fluorescence at the surface of CHO cells transfected with the cloned V1, AVP receptor cDNA when compared to the control CHO cells. The fluorescence was speci~cal~y displaced by an excess of unlabeled PhaaGln (data not shown).

AVP-~n~uced ~nosj to~ ~ ~ o s p ~ a t e P r ~ u ~ ~ w ~ in COS7 and CHO Ce€ls--The signal transduction of the cloned human VI, AW receptor was explored in the transiently transfected COS-7 cells by measuring AW-jnduced inositol phospha~ pro- duction (Fig. 56s). A W s t ~ ~ a t i o n for 30 min induced a dose- dependent increase in the f o ~ a t i o n of IPI (2-fold, 3607 dpnt, well), fP2 (7-fold, 4375 dpmlwell~, and IPS (Bfold, 802 d p ~ w e l l ) fmm respective base lines of 1805,645, and 309 d p ~ w e l l ( n = 6). These data suggest that the cloned receptor is indeed coupled to phospholipase C, No st im~at ion of IP production was observed in control COS-7 cells (1545 r 68 dpmlwell in the presence of 1 ).m AVP versus 1583 rt 180 dpmlwell in control conditions).

A ~ ~ ~ n d ~ c e ~ inositol phosphate p ~ u c t i o n was also studied in control and transfected CHO cells. No stimulation of IP pmduction was observed in control CHO cells (data not shown). As shown in Fig. 5b, AW induced a dose-de~ndent increase in the f o ~ a t i o n of IP1 (20-fold, 16,787 dp~wel l ) , IP, (50-fold, 2,053 d p ~ w e l l ) , and IPS (3~fold, 1,026 dpdwel l~ from respec- tive base lines of 834, 393, and 422 dpdwell f n = 6). The s t ~ u l a t i ~ action of A W was blocked by the non-~ptide VI, an~gonis t SR 49059. AW stimulation of inositol phosphate p ~ u c t i o n was greater in stably transfected CHO cells than in transiently t r ~ s f e c t e d COS-7 cells, because of the selection of the best CEO clones in terms of AW receptor expression. In addition, the profile of st~mulation was different between the two cell lines (CNO cells: IP1 > IP2 > IPS uersus COS7 cells: IP2 > IPS > IF’& p r e s ~ a b l y because of different phospha~se con- tents in the two cell lines. These data suggest that the cloned receptor is indeed coupled to phospholipase C. Ib test if AVP act~vation of inositol phosphate product~on in t r a n s f e ~ d cells was pertussis toxi~-sensitive or not, t r~sfec ted CHO cells were pretreated with 0.1 d m 1 pertussis toxin prior to AVP stimulation. The dramatic increase of inositol phosphate pro- duction induced by AW was not altered after pretreatment with pertussis toxin (three i n d e ~ n d e n t sets of triplicate ex- p e ~ e n t s ~ . This pertussis toxin ~nsens i t i~ ty of the cloned re- ceptor is in agreement with our previous obse~ation that the VI, receptor of human platelets is coupled to a pertussis toxin- insensitive G protein belonging to the G,11 family (19).

AVP Eflect on A ~ ~ y l y ~ Cyclase ~ c ~ ~ u i ~ y in CHO CeZls-Ib confirm that the cloned human VI, AVP receptor cRNA is

3310 Human VI , AVP Receptor Clone a

l e - 1 0 l e - 9 5 e - 9 i e . 8 l e - 7 l e - 6

AVP Concentration (M)

l e -10 l e - 9 Se-9 i e - 8 l e - 7 le-? + SR

AVP Concentration (M) FIG. 5. AVP-induced inositol phosphate production in COS7

and CHO cells transfected with the human VI, receptor clone. Transfected COS-7 and CHO cells prepared as described under "Experi- mental Pmdures" were grown to confluence in 12-well dishes and incubated in inosital-free DMEM buffer, pH 7.4, with my0-[2-~H]inosi- tal. Formation of [3H]inosital phosphates was measured after addition of increasing concentrations of AVP for 30 min in COS-7 cells (punel a) or CHO cells ( p a n e 2 b ) fn = 6 for each series).

400 r

J

- 1 0 1 2 3 4 5 Time (min)

FIG. 6. Am-induced [Cas+], mobilization in CHO cells trans- fected with the human V,, receptor clone. Control and transfected CHO cells prepared as described under "Experimental Procedures" were grown to confluence on A C W coverslips, loaded with fura-2, and incubated in Krebs-Henseleit-Hepes buffer, pH 7.4. Basal fCa2+li was measured fluorometrically after addition of 1 AVP in transfected cells (solid line) and control cells (dofted line). Figure shows represent- ative tracings of four different experiments.

AVP-induced Calcium Mobilization in CHO Cells-% con- firm that the cloned human VI, AVP receptor cDNA is coupled to phospholipase C in transfected CHO cells, AVP-induced mo- bilization of intracellular free calcium was measured in h a - 2 loaded cells. Control CHO cells loaded with h a - 2 displayed no response to 1 l;l~ AVP (Fig. 6). In CHO cells transfected with the VI, AVP receptor cDNA clone, AVP induced a mobilization of intracellular calcium (357 f 153 m, n = 4) with return to the base-line level over 5 min.

In conclusion, this is the first report of a cDNAcoding for the human VI, AVP receptor. This receptor presents the typical features of the 7-TMS G protein-coupled receptors. It possesses a high degree of homology with other receptors of the AVP- oxytocin family, especially at the level of the seven transmem- brane segments. We present several lines of evidence suggest- ing that the isolated clone belongs to the VI, subtype. Radioligand binding competition experiments performed in transiently transfected COS-7 cells and stably transfected CHO cells with several AW analogs and oxytocin reveal the typical pharmacologic profile of a VI, AVP receptor. Moreover, saturation binding experiments in CHO cells characterize a single class of high affinity binding sites, like in human plate- lets we explored before. Results of inositol phosphate produc- tion and calcium mobilization experiments confirm that the cloned receptor is ~nctionally coupled to phospholipase C in a pertussis to~n-independent fashion.

A c ~ n o w ~ ~ ~ ~ s - W e thank Andrea Bayer for skilled technical as- sistance, Dr. Woo for granting us the permission to use his liver cDNA library, and Prof. Corvol for welcoming us in his research unit at the Coll&ge de France, Paris, where this project was initialed.

REFERENCES

1. Michell, R. H., Kirk, C. J. & Billah M. M. (1979) Biochem. Sac. 'Dana. 7,

2. Thibonnier, M. (1992) Regul. Pept. SS, 1-11 3. Thibonnier, M., Bayer, A. L., Simonson, M. S. & Kester, M. (1992) Edocrinof-

agy 129,2845-2856 4. Thibonnier, M. (1993) in N e u r ~ ~ a c r ~ n o ~ ~ of the Concepts in Neurosurgery

Series 5 (Selman, W., ed) pp. 1940, Williams & Wilkins, Baltimore 5. Morel, A., O'Cml1,A. M., Brownstein, M. J. & Lolait, S. J. (1992)Nafure 866,

523-526 6. Lolait, S. d., O'Camll, A. M., McBride, 0. W., Konig, M., Morel, A. & Brown-

7. Bimbaumer, M., Seibold, A., Gilbert, $., Ishido, M., Barberis, C.,Anataramian, stein, M. J. (1992) Nature Sa7,33fG339

A,, Brabet, P. & Rosenthd, W. (1992) Nature 557,333-335 8. Emma, T., Tanizawa, O., Man, K., Brownstein, M. J. & Okayama, H. (1922)

9. Thibonnier, M., Bayer, A. L. & Madhun, Z. (1993)Am. J. Physiol. 266, in press Nature SSS, 526-529

10. Ellis, L., Clauser, E., Morgan, D., Edery, M., Roth, R. A. & Rutter, W. J. (1986)

11. Teutsch, B.. Bihoreau, C., Monnot, C., Bemstein, K E., Murphy, T. J., Alex- Cell 45,721-732

ander, R. W., Corvol, P. & Clauser, E. (1992) Biochem. Bwphys. Res. Com- mun. 187,1381-1388

12. Bihoreau, C., Monnot, C., Davies, E., Teutsch, B., Bemstein, K, Corvol, P. & Clauser, E. (1993) Pmc. Natl. Acad. Sci. U. S. A. SO, 5133-5137

13. Thihmnier, M. & Roberts, J. M. (1985) J. Clin. Invest. 78, 1857-1864 14. G a n , H. D. & Hawthorne J. N. (1978) Biachem. J. 178,541-552 15. Dubvak G. R.. Cowen. D. S. & Meuller. L. (1988)J. Bid. Chem. 263,18108-

861-865

lilli coupled to phospholipase c independently of Gi, AVP effect on 16. mou, J., Emsberger, P. & Douglas, J, G. (1993) Hypertension 21,1035-103s adenylyl cyclase activity was studied in control and transfected 17. Kozak, M. (1987) Nucleic Acids Res. 15,81258148

(-THO In control cells, luvr A~ did not significantly alter 18. Eisenberg, D., Weies, R. M. & Tedlliger, T. C. (1984) Proc. Nafl. Acad. Sci.

CA" oroduction (23.563 uersus 15.261 fm01 of &b'fP/mE Of 19. Thibonnier. M.. Govara T.. L. & Berti-Mattera L. M. (1993)Am. J. Phvsid. 284, U. S. A. 81, 140-144

. . protein, n = 6) whereas forskolin induced a 7-fold rise of CAMP C1336G13k

to 110,746 fmol of cAMP/mg of protein. Similarly, in transfected 2 0 . ODowd, B. F., Hnabwich, M., Caron, M. G., Lefkowitz, R. J. & Bouvier, M.

cells, AVP did not alter d P production (25,350 ue?-sus 20,258 21. Homtman, D. A., Brandon, S., Wilson, A- L., Guyer, C. A, C r e e , E. J., Jr. & (1989) J. Biol. Chem. 264,7564-7569

fmol O f ~ P ' m g of protein* n 6) whereas forskolin induced a 22. melshrg, M. A., CoteccMa, s., OstrowsE, J., caron, M. G. LeRowitz, R. J. Limbird, L. E. (1990) J. Bid. Chem. 8ea, 21590-21595

?-fold rise of CAMP to 121,603 fmol of CAMP/m~ of protein. (1992) J. BioZ. Chem. 287.1430-1433

-

Thus, the lack of &b'fP production decrease in the presence of 23. Mizuno, T., saito, Y., Itakura'M., 1% F., I%, T.. Moriyma, E., H&wma, H. &

in transfected suggests that the 'Ioned Aw receptor 24. Thibonnier, M., Hinko, A & Pearlmutter, A. F. (1987) J. Cardiouasc. Pitarma-

"

Hirose, S. (1992) Biochem. J. 287,306-309

is not coupled to Gi. cd. 10,24-29