Molecular Cloning, Sequencing, and Functional Expression of a cDNA ...

4 downloads 0 Views 1MB Size Report
Marc Thibonnier8, Colette AuzanS, Z u h a y r Madhun, Pamela Wilkins, Liliana Berti-Mattera, and. Eric ClauserS. From the ~ e p a r t ~ n t of Medicine, University ...
Tm Jolnuw OF B I O ~ I CCHEMBIRY AL

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

B 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Molecular Cloning, Sequencing, and Functional Expressionof a cDNA Encoding the HumanVIa Vasopressin Receptor" (Received for publication,J d y 12, 1993, and in revised form, September22, 1993) Marc Thibonnier8, Colette AuzanS, Zuhayr Madhun, Pamela Wilkins, Liliana Berti-Mattera, and Eric ClauserS From the ~ e p a r t ~ofn Medicine, t University Hospitals of Cle~elaRd and Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4982and the Slnstitut National dela Sant6 et de Ea Recherche Mkdicale, Unitti 36, College de France, 3, rue DUlm, 75005 Paris, France

Vasopressin (AVP),the antidiuretic hormone, is a cyVasopressin (AVP),l the antidiuretic hormone, is a cyclic clic nonapeptide that acta through binding Gto protein- nonapeptide involved in the homeostasis of body fluid osmolalcoupledspecificmembranereceptorspharmacologiity, blood volume, vascular tone, and blood pressure. AW also callydivided into three subtypes (VI,, Vlb, and V2) belongs to the family of vasoactive and mitogenic peptides inlinked to distinct second messengers. volved in physiological and pathological cell growthand differWithin the family of human AVP receptors, the V, AVP entiation. AVP exerts its actions through binding to specific receptor has been cloned, but the structure of the hu- Vla, Vfb, and V, membrane receptors coupled to distinct second man V1, and Vlb AVP receptors remains unknown. We messengers (If.While V2 receptors stimulate adenylatecyclase report here the structureand functional expression of a and protein kinase A, VI receptors activate phospholipases .Az, human V1, AVP receptor complementary DNA isolated C , and D, resulting inthe production of inositol 1,4,5-trisphosfrom human liver cDNA libraries. Cloningand sequencing of a full-length clone isolated a 1472-nucleotide se-phate (IPS) and 1,Z-diacylglycerol (DAG), the mobilization of intracellular Ca2+,the influx of extracellular Ca2+,the activaquenceencodinga418-aminoacidpolypeptidewith seven putative transmembrane domains typical of G tion of protein kinase C , and protein phosphorylation (2,3). VI, protein-coupled receptors. Amino acid sequence iden- AVP receptors have been shown by radioligand binding techtity with the rat liver Vl, A W receptor, the human and niques to be present in vascular smooth muscle cells,hepatoe s monocytes, type If rat V2AVP receptors, and the human oxytocinreceptor cytes, blood platelets, l ~ p h ~ y t and pneumocytes, adrenal cortex, brain (hip~campus? septum et was 72,36,37,and 4S%, respectively. amygdalae), reproductive organs, retinal epithelium, renal meFunctional characterization of the cloned receptor was done by transient expression in COS-7 cells and sangial cells, and the A10, A7r5,3T3, and WRK-1 cell lines (4). stable expression in Chinese hamsterovary cells. Local- VI, AVP receptors mediate cell contraction and proliferation? ization of the expressed receptorat the cellular surface platelet aggregation, coagulation factor release, and glycogenwas illustrated by using the fluorescent linear analog olysis. Vlb AVP receptors are located in the anterior pituitary phenylacetyl-~-Tyr(lt)-Phe-Gln-Asn-Lys-Pro-Arg-NH~ where they stimulate ACTH release. Vz-renal receptors are coupled to fluorescein-avidinby dodecabiotin. Competi- present in the Madin-Darby canine kidney and LLC-PKI cell tion binding experiments with phenylacetyl-D-Tyr(Et)- lines, as well as in the medullary portion of the kidney, where Phe-Val-Asn-Liys-Pr~-[~~" and I ] ~AVP - ~ ~analogs re- they control free water and ureareabsorption via stimulation of vealed high affinity specific binding sites of the V1, adenylate cyclase. subtype. Saturation binding experiments with[9HlAVP Recently, Morel et al. reported the expression cloning and of high affinity sequence analysis of a rat hepatocyte VlaAW receptor (5). confirmed the presence of a single class bindingsites.Measurement ofAVP-induced inositol Moreover, Lolait et al. (6) and Birnbaumer et al. (7) very rephosphate production and calcium mobilization con- cently described the molecular cloning of human and rat V2 firmed that the expressed V1,AVP receptor is coupled to AVP receptors. Simultaneously, Kimura et al. (8)presented the phospholipase C via a pertussis toxin-insensitive pathstructure of the human oxytocin receptor. Comparison of the way. Thus, the human Vl, AVP receptor belongsto the su- amino acid sequenceidentity of the human Vz A W and oxytoperfamily of seven-transmembrane segment receptors cin receptors, as well as the rat Vlaand Vz A W receptors, rewith a significant sequence identitythewith other mem- veals significant differences, especiallyat the level of the extraand intracytoplasmic loops. Currently, the structure of the hubers of the AVP-oxytocin family ofreceptors. man V1, and Vlb AVP receptors remains unknown. Obtaining this information is important as it is very likelythat thefamily of AW receptors, like other families of seven-transmembrane segments (7-TMS) receptors, includes several members for which structural differences determine their subtype specific*This work was supported by Grants RO1 HL39757 and PO1 ity. In this paper, we have described for the first time the HL41618 from the National Institutes of Health. The costs of publica- molecular cloning, s ~ u e n c i n gand , functionaf.expression of a tion of this article were defrayed in part by the payment of page cDNA encoding the human VI, A W receptor. Pharmacological charges. This articlemust 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 paperhas been submitted to the ~ n B a n k ~ / E Data ~ B Bank L with accession numberls) L25615. $ lb whom correspondenceshould 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.

1 The abbreviations used are: AW, [Arg%asopressin; DMEM, Dub becco's modified Eagle's medium; PBS, phosphate-bufferedsaline; TMS, seven-transmembranesegment(s);Phaa,phenylacetyl;kb, kilobase pair(s); CHO, Chinese hamster ovary; IP,inositol phosphate; HPLC, high performance liquid chromatography.

3304

Human V I ,AVP Receptor Clone

3305

screened with both radiolabeled oligonucleotides (derived from the N terminus and the first TMS of the ratV1, 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. PreE ~ E ~ PM R O CE E D~~ S ~ hybridization, hybridization and washing procedures were identical to ~ a ~ ~ r i a Z ~ V a s o p r e Tris-HCl, ssin, and other reagents, unless stated those described above for the filters hybridized with the labeled oligonucleotides. ~ehybridizationand hybridization with the cDNA fragotherwise, were from Sigma. Ionomycin was from Calbiochem Corp., San Diego, CA. COS-? and CHO cells were obtained from the American ment were performed in 50% formamide, 6 x SSC, 0.1% SDS, 5 x Type Culture Collection ( ~ ~ v i l lMD). e , Cell culture media and Gene- Denhardt's, 100 pglml sheared salmon sperm DNA at 42 *C. Washing ticin were from Life ~ c h n o l o ~ eInc. s , Fetal bovine serum was from conditions were 2 x SSC + 0.1% SDS for 15 min a t 50 "C and 0.1 x SSC HyCIone, Logan, UT. Restriction and modifi~tionenzymes were from + 0,1% SDS for 15 min at 50 "C twice. Bcehringer M ~ e i or m New England Biolabs, Iodogen(1,3,4,6-tetraDNA S u ~ c ~ o n ~ n g Sequencing-A and phage lysates generated from c ~ o ~ 3 a - 6 f f - d i p h e n y l g l y cwas o ~ ~fmrn l ) Pierce Chemical Co. Fura-2 tertiary screening dishes were used to obtain midi-preparations of reAM and Neutralite fluorescein-avidin were fmrnMolecularProbes, combinant A phage DNA. The EcoRI fragments of the cDNA inserts Portland, OR. NAP-10 and Nick columns were from Pharmacia LKB were identified by agarose-gel electrophoresis and Southernhybridizae~ Biotechnology Inc. Nitrocellulosa membrane (EA-85 N i ~ l l u l o s was tion analysis with the sZP-labeled oligonucleotides. These fragments from Schleicher & Schuell. Nensorb 20 c&ridges, N a W (activity = 131 were subcloned by standard recombinant techniques into M13mp18 mCilml), fya2PLATP(specific activity = 6,000 Ci/mmoD, IsHLAVP (spe- phage and into pB~uescnpt11phagemid vectors prior to tr~nsformation cific activity = 62 Cilmmol), and ~y0-~2-~HIinositol (specific activity = of XLl-Blue E. coli strain. Nucleotide sequencing was done using the 20 Cifmmol) were obtained fmrn Du Pont NEN. [36SldATP(specifc dideoxy chain t e ~ a t i o method n of Sanger and theDNA sequencing (specific activity = 3,000 Gi kit from U. S. Biochemical Corp. E l e ~ p h o r e s i of activity =A 1,000 Cihnmol), ICU-~~PI~CTP s reaction products m o l ) , and the Megaprime labeling kit were from ~ e r s h a mCorp. was carried out in 5% ~lyacrylamidegels containing 50 g of urea, 45 Ml3mpl8 phage, p B l u e s ~ p II t p h a g e ~ d and , XLl-Blue E s c ~ r i c m ~ ~'kis borate, 1.25 m~ EDTA followed by autoradio~aphyon Kodak coli strain were fmm Stratagene, La Jolla, CA. The DNAsequencingkit XAR film. S e q u e n ~ gof both strands was initiated with universal was from U. S. Biochemical Corp.The V2=AVPantagon~stphenylacetyl- primers, and the oligonucl~tides were used for library screening. The ~ ~ E t ) - P h e C l n - A s n - L y s - ~and a - ~the *VZ-renal ~~ AVP antaga- deduced amino acid sequence b e ~ n ~ with n g an ATG and extending to nist d(CH2)b[~neZ,Ile4~~a-NH21AVP were gifts from Dr Maurice Man- the t e ~ n a t ~ codon o n was ana~yzedby hydrophobicity piot and comning (Medical College ofOhio,Toledo, OH). The new VI,AVP non pared to the known sequences of the rat liver VI,AVP receptor, the rat peptide a n ~ o n i sSR t 49059 waa kindly provided by Dr. Serradeil-Le and human V2-renal AW receptors, the human oxytocin receptor, and Gal ( S ~ o f i ~ c h e r c h~e , ~ o France). ~ e The , V1, AW antagon~st other 7"TMS receptors. p h e n y l a ~ e t y l - ~ ~ ~ t ) - P h ~ V a l - ~ n - custom-s~theL y s - ~ ~ ~ - ~ ~ ,~ a n ~ ~ p ~ ~nin COS-7 s~ s C e~l b D ~ N A was ~ prepared and used sized by Bachem (Torrance, CAI, was r a d i o i ~ n a t e d~ ~ l ~ ~ 1 ~forP t ~ h nas fae ~~ i in o nCOS-? cells, an i m m o ~ ~ cell ~ eline d derived from using the Iodogen technique and purified by HPLC as previously de- Gfrican green monkey kidney that we have shown to be devoid o f enS C r i M (9). DNA probe Synt~s~-OligonucIeotideprobes co~espondingto sev- dogenous V,-vascular AVP receptors. COS-7 cells were obtained from ATCC and grown in DMEM with 10%fetal calf serum, penicillin, and e d liver AVP receptor nucleotide sequence eral regions of the p u b ~ i ~ rat 5% C 0 2 , 95% O2 at 37 "C. Transfection ofDNA into were pmduced (5). The probes were 45 nucleotides long, recogxizingthe s~eptomycinin ~ ~ C cells G - was performed using the DEAE-dextranmethod. The EcoRI N terminus ~ 5 ' - A T G A G ~ C C C ~ G A ~ T C C C A ~ A T CCOS-7 ~ ~ C ~ Cand -the 3C terminus ' ~ ( 5 ' - G ~ T ~ G ~ G T C fragment ~ T " coding forthe entireAVP receptor was inserted in the pECE C~C~TCAT~TATC~AG~ATCC as ~we11 - 3 as ' ) the , first expression vector (10,ll), and this construct was mixed with serum( 5 " ~ ~ G ~ T ~ C T G T G C T G ~ C ~ T A G C A G T G T G free C TDMEM ~ T ~ -and D33AE-dextran, then applied to lo6 COS-7 cells in a T GCC-3'), second( 5 " A ~ C ~ A G A C C ~ ~ T C ~ C ~75-cmz C ~ flask C CforA90~min. h r exposure to chloroquine for4 h, Me2S0 at room temperature for 5 min. Expression of A G T A ~ A C C ~ A Gsix& - ~ ~ (, 5 ~ - C C A C A ~ ~ A C ~ treatment ~ ~ G was ~ Gperformed o receptors n ~ in thetransferrted cells was assessed 48 h later G C ~ C C A ~ ~ ~ A ~ and T seventh A ~ ~ . 5~ ' -) G, ~ C A T ~ - n ~AVP experiments and inositol phosphate production G ~ A T A T C C A C ~ ~ A ~ A ~ transmem~ T C ~ by~ radioligand ~ ~ 3 binding ' ) as described below. brane segments. They were synthesized with a Applied Biosystems Stable Expression in CHO Cells-Stable transfection of DNA into o~igonucl~tide synthesizer and purified on a 15%~lyacrylamidegel. FoIlowing ~ ~ r o p h o r e s i s , o l i ~ n u c l ~ twere i d e s visualized by short CHO cells was perfomed using the CaP04prec~pitationmethod (12). wave W illumination, excised from the gel, and l~philized.For hy- The express~onplasmid pECE c o n t a ~ i n gthe AVP receptor clone was b r i ~ ~ a t i opurposes, n the o l i ~ o n u c l ~ t i dwere e s labeled with "ZPa t the mixed with the resistance plasmid pSV2Neo and 250 m~ CaCIZin HBS 5 ' - h y ~ x y l pusing u p [-psZPIATP and T4 p o l ~ u c ~ e o t i kinase, de and buffer tin m: Hepes 25, NaCll40, N a m 4 1.5, NaHZP041.5, pH 7.4) for 30 min at room ~ m p e r then a ~applied ~ ~ to lo6 CHC, cells in a T then purified over Nensorb 20 cartridges to screen the libraries. n 4h at 37 OC, 12.5%glycerol treatment A gtl0 Library ~ r e e n i ~ - ~ m b i n aphages nt (lo6)fmm an ampii- 75-cm2 flask.f i r i n ~ b a t i o for d t e m p e r a ~ r efor 2 min. Pure clones were sefied A gtl0 human liver cDNA library purchased from Clonetech (HL was ~ r f o ~ aet Mom 1115a)were incubated with E. coli C600 cells for 20 min at 37 "C in SM lected for their resistance to the antibiotic Geneticin and purified by the medium, mixed with 0.7% LB agarose, and plated on 1.5% LB agar in l ~ i t i n gdilution techn~que.Clones expressing the greatest receptor in binding experiments were further explored by fluorescent 150-mmPetri dishes at a density of 50,000 p l a q u e - f o ~ i n g u n i ~ d i s hdensity . Plates were incubated a t 37 "C until lytic plaques began to appear (8-9 labeling and radioligand binding experiments, as well as measurement h). GRer coaling at 4 "C for 1h, phages were li&d onto duplicate BA 85 of tCa2+limobilization and inositol phosphate production, as described n i t ~ l ~ ofilters, s e which were denatured, neutralized, and heatedat below. ~ a ~ ~ o B~ i d~i ngg aAssays-We n ~ prepared the radioiodinated 80 "C for 2 h. Replicate fiiters were prehybridized for 12 h a t 42 "C in a compound p h e n y l a c e t y ~ - ~ - ~ ~ t ~ P h e - V ~ - ~ n - L y s - ~ [ ~ ~ ~ solution containing 6 x SSC, 0.1% SDS, 5 x Denhardt's, and 100 &ml ) the Iodogen technique and HPLC purification as sheared salrnon sperm DNA. The p~hybrid~zation solution was dis- ( E 1 2 s I ~ P h a aby described previously(9).Control and transfected COS-7 and CHO cells carded and replaced by the same hybndizat~onsolution, which contained labeled first and seventh TMS nucleotides (5 x loe cpdfilter) for were grown to confluence in 24-well dishes and washed twice with further incubation at 42 "C for 24 h. Filters were washed with 6 x SSC DMEM .t 25 RIM Hepes + 0.25% bovine semm albumin, pH 7.4. Com+ 0.1%SDS a t roam temperature for 1h, thenwith 2 x SSC + 0.1%SDS petition binding experiments were performedin duplicate by incubating at 42 OC and 1x SSC + 0.1% SDS a t 55 "C until b a ~ radioac~ ~ thed cells (final volume 250 $) in the same medium with one fixed tivity monitored with a hand-held Geiger counter had decreased to concentration of [12bI]TyrPhaa (0.30nrd and increasing concentrations acceptable levels. Subsequentl~ the filters were dried and subjected to of unlabeled AVP or the linear V1,AVP antagonists phenylacetyl-w a u t o r a d i ~ a p h yon Kodak XAR-5 films with one Du Pont Quanta I11 Tyr(Et)-Phe-Val-Asn-Lys-~o-Tyr-~~ or phenylacetyi-~-~Et)-Phescreen for 12-24 h at -70 "C. The positive clones were isolated and G I n - ~ n - ~ y ~ - P r othe - ~ n~o-n~~ ~p t, ~ dV,,e AW antagonist SR VP, plaque-purified by secondary and tertiary screening procedures using 49059, the V2 AW antagonist d f C H 2 ) 6 [ ~ - n e 2 , n e q ~ a - ~ 2or~oxythe labeled second and sixth TMS oligonucleotides. tocin for 30 min at 30 "C. The cells were washed thme times with n ~ clone ~ for the human VI, AVP ice-cold PBS and lysed with 0.5 ml of 0.2 N NaOH, 1% sodium dcdecyi In order to isolate a ~ - 1 e cDNA receptor, a second A gtl0 human liver cDNA library (a generous giR sulfate. C e l l - ~ [lZsIl'QrPhaa ~d was counted in a y counter. IC, valfromDr. Savio Woo, BayIor College of Medicine, Houston, TX) was ues were derived fmm nonlinear least square analysis, and K,values

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

3306

Human V I ,AVP Receptor Clone

were calculated by the equation of Cheng and plusoff: Ki = ICsd(l + LJKd). Additionally,saturation binding experiments ofAVP receptors of control and transfected CHO cells were done in 24-well dishes in duplicate as described abbvebut 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 scintillation spectrometry (Beckman counter LS 5801, yield = 64%). Affinity ( K d ) 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 aninternal standard. Neutralite Fluorescein-AvidinLabeling-We coupled phenylacetyl-oTyr(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 columnas previously described (9).VI,AVP receptors of CHOcells were visualized by PhaaGln-dodecabiotinmade 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-dodecabiotin aloneor in thepresence of 5 p~ PhaaGln for 30 minat 37 "C. Thereafter, the coverslips were washed five times with 1 ml of buffer and incubated with 25 pg of Neutralite fluorescein-avidinfor 2 h in the dark. The coverslips were washed again five times, and furation was performed for15 min a t 4 "C in 3% paraformaldehyde.The preparations were examined with the microscope/computersystem described previously (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-welldishes, washed with inositol-freeDMEM, and labeled with myo[2-3Hlinositol( 2 3 pCi/ml) in inositol-free DMEM for 20 to 24 h (14). Thereafter, the cells werepreincubated for 1h in DMEM, followed bya 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 1ml 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 removed and loaded ontoAG1-X8 Dowexcolumns (chlorideform, 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 werewashed with a theoric upper phase solution containing 1 m~ inositol and counted. The amount of inositol phosphates released was expressed as dpdwellor as a fraction of total labeled phosphoinositides. Typicalincorporation into phosphoinositides amounted to approximately 3.5-5 x lo' 3H-labeled dpdwell. Inaddition, 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 phosphate production to pertussis toxin (15). CAMP Production-Subconfluent monolayer cultures of control and transfected CHO cells were grown for 48 h in 12-welldishes to measure adenylyl cyclase activity in the presence and absence ofAVP as described before (16). Measurements of Intracellular Calcium-Free intracellular calcium concentration was measured as described previouslyin serum-starved monolayer cultures of control and transfected CHO cells loadedwith 1 p~ fura-2 AM (3,9).Autofluorescenceby 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 V I ,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 EcoRIfragment indicated a 79% identity of the coding sequencewith the nucleotide sequenceof the rat liver V1, AVP receptor (data not shown). Sequencing revealed that this1.7-kb EcoRI fragment encompassed twothirds 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 human liver V1, AVP receptor, we screened another human liver cDNA library (developed and kindly provided byDr. Savio Woo). This library was screened with the 32P-labeled 1.7-kb fragment isolated above as well as the radiolabeled oligonucleotides derived fromthe N terminus and the first transmembrane segment of the ratVI, AVP receptor sequence. Subsequent subcloning and sequencing identified a 1.5-kb insert, the composition of which is shown in Fig. 1. The 1472nucleotide sequence of the human V1, AVP receptor cDNA enthe codes a 418-amino acidprotein (M,= 46,745) deduced from open reading frame of 1254 nucleotides spanning from nucleotide 62-1315 of the cloned cDNA. The ATG of GACAGCATGCis assigned as codon 1 on the basis of its close match to the GCC(A/G)CCATGG Kozakconsensus sequence forinitiation of translation in vertebrates (17) and by homology with the sequences of the other members of the AVPloxytocin family of receptors. However, another in-frame ATG is present a t position 44 of the cloned sequence, and the definite answer regarding the truetranslation initiation site will beobtained 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 ratV2 AVP receptor nucleotide sequences is 83,68,62,and 65, respectively. Homology with other families of seven-transmembrane domain receptors is less than 50%. Hydropathicity analysis done accordingto Eisenberg using a window of 20 residues (18) shows that the translated protein has thetypical 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 precedingthe first putative transmembrane domain contains two potential N-linked glycosylationsites (N-X-(S/T))at Asn-14 and Asn-27. An additional N-linked glycosylation site ispresent at the level of the second extracellular loop (Asn-196). There are 8 threonine 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, therefore suggesting that the functions of the human VI, AVP receptor are regulated by protein kinases. Indeed, there are consensus sequences specificfor 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 involvedin the tertiary structure of the receptor and ligand binding. As a matter of fact, we reported that [3HIAVPbinding to V1, AVP receptors 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 Cys366) are present in the C-terminal region of the receptor sequence and may anchor this partof 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 97of the human V1,AVP receptor sequence (12,21). By the same token, an alanine residue at the carboxyl end of the third intracytoplasmic loop instrumental in G protein coupling is present in position 285 of the human VI, AVP receptor sequence (22). Moreover, the canonical sequence Asp-Arg-Tyrpresent at the end of the third transmembrane domain of the substance P, substance K, dopamine D2, dopamine DS, and endothelin B receptor genes is also present at theend of the third transmembrane domain of the

Human V,, AVP Receptor Clone ggcgcgaqggctqgagctCcgaagagggccgagtaggagctgcatggacagc

-52 ATG CGTCTCTCCGCCGGTCCCGAC

GCGGGG

M e t a r g l e u ser a l a g l y pro asp

ala gly pro

CTGGCCACC

CCACCG

CCC TCG GGC ARC TCC AGC CCA TGG TGG CCT

GGC GCT GGC AAC ACA AGCCGG

l e u a l a t h r gly ala gly

ser gfyasn

ser ser pro trp trp pro

GAG GCC GAA GCC CTC GGG GAG

GGC AAC GGC

asn t h r ser arg glu a l a glu a l a l e u 9ly glu g l y asn gly

I""""""""

AGG GAC GTG CGC AAC GAGGAG

CTG GCC AAA CTG GAG ATC GCC GTGCTG

GCG GTG

l e u ala l y s l e u glu i l e a l a val l e u a l a Val

pro pro arg asp val arg asn glu glu

I

-""""""""""""""1"""

ACT TTC GCG GTG GCCGTG

3307

CTG GGC ARC AGC AGC GTA CTGCTGGCTCTG

CAC CGG ACG CCG

t h r phe ala Val a l a v a l l e u gly asn s e r ser v a l l e u l e u a l a l e u h i s a r g t h r pro

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

-1 60 20 I20 40 190 60

240 80

arg lys thr

ATC CGA CAC CTC AGC CTG GCC GAC CTG GCC GTG ser arg m e t h i s l e u phe i l e arg h i s l e u ser l e u ala asp l e u a l a Val

300 100

GCA TTCTTC

CAG GTGCTGCCG

CAA ATG TGC TGG GAC ATC ACC TAC CGC TTC CGC GGC CCC

360

CGC AAG ACG

TCCCGC

ATG CAC CTCTTC

I

"""""1"

a l a phe phe gln v a l l e u pro gln met cys t r p asp i l e t h r t y r a r g phe arg gly pro

120

GAC TGG CTG TGC CGC GTG GTG AAG CACCTG CAG GTG TTC GGC ATG TTT GCG TCGGCCTAC asp t r p l e u cys arg val val l y s his leu gln val phe g l y met phe ala ser a l a t y r

620 140

ATG CTGGTAGTC

ATG ACA GCC GAC CGC TAC ATC GCG GTG TGC CAC CCG CTC AAG ACTCTG met leu v a l val met t h r ala asp arg tyr i l e a l a v a l cys h i s pro leu lys thr leu

480

CAA CAG CCC GCG CGC CGC TCG CGC CTC ATG ATC GCG GCC GCC TGG GTG CTG AGC TTC GTG

540

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

180

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

".. """1

i

"""""""1

gln gln pro

"""""VI""

I

CTG AGC ACG CCG CAG TACTTCGTCTTCTCC ATG ATC GAG GTG AAC AAT l e u set t h T pro gln t y r phe v a l phe s e r met i i e glu v a l asnasn

160

GTC ACC RAG GCC

600

val t h r lys a l a

2 00

1 FIG.1. Primary nucleotide stmcture of the hamum V E - V ~AVP C ~ ~CGC GAC TGC TGG GCC ACC TTC ATC CAG ccc TGG GGT TCT CGT GCC TAC GTG ACC TGG ATG receptor cDNA. Positions of the pu&arg asp cys trp ala t h r phe i l e glnpro t r p g l y ser arg a l a t y r v a l t h r t r p met tive T&fS 1-mI are indicated by iiws v I above thenucleotide sequence. ACG GGC GGC ATC ?TT GTG GCG CCC GTG GTC ATC TTG GGT ACC TGC TAC GGC TTC ATC TGC

" " " "

."---."-----

thr gly gly ile

-----_-------

-----."____

660 220

__---_-_-".-I

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 GGGAAGACGGCG

TCG CGC CAG AGCAAG

t y r asn i l e t r p cys asn Val a r g g l y l y s t h r a l a s e r a r g CAA GCG GGT GTG GCC TTC CAR AAG GGG

TTC CTG CTC GCA CCCTGTGTC

gln ala gly

Val

phe leu leu

TCC ATTTCC

CGG GCC AAG ATC CGC ACG GTG AAG ATG

ala phe gln lys gly

GGT GCA GAG

gln ser l y s g l y a l a g l u AGCAGC

GTG AAG

ser v a l l y s

ala pro cys val ser

ACT TTT GTG ATC GTG ACG GCTTAC

720 240 780 2 60 840 280 900

s e r ile s e r arg a l a l y s i l e 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

300

ATC GTCTGC

960 320

I

""""""""-IV"""-"

TGG GCG CCTTTCTTC

ATC ATC CAG ATG TGG TCTGTC

TGG GAT CCC ATG TCC

i l e va? cys t r p ala pro phe phe i l e i l e g l n met t r p s e r val t r p asp pro met ser

1""""""*

GTC TGG ACC GAA TCG GAR AAC CCT ACC ATC ACC ATC ACT GCA TTA CTG GGTTCCTTGART Val t r p t h r q l u ser gluasnpro t h r i l e t h r ile thr ala leu l e u gly ser l e u asn

1020

AGC TGCTGT

AAT CCC TGG ATA TAC ATG TTTTTT

ser cyscys

asn pro t r p i l e t y r metphe

1080 360

""~"""-vI"-"""""~""

1

CAA AGC

TTC CCA TGC TGC CAA AAC

gln ser

phe procyscys

ATGAGC

AGA AGA

AGT GGC CAT CTCCTT

phe ser g l y h i s leuleu

ATG AAG GAA AAA

TTC AAC AAA GAA

CAA GAC

TGTGTT

g l n asp cys v a l GAT ACT GAC AG?

gln asn met l y s 9lu l y s phe asn l y s g l u a s p t h r a s p s e r

340

1140 380

CAG ACT?TTTAT TCT AAC AAT CGA AGC CCA ACA AAC AGT ACG GGT ATG gln t h r phe t y r ser asn asn arg ser pro t h r asn s e r t h r g l y met

1200 400

ACT TGA gcC

i l e l y s phe i l e pro Val ser t h r OPA

1260 418

ttg cat tca tgc aac t t g a t t c t t g t g a t t gac t t t t t q g c t c a t t a g c t g a a t t g a g c t aga aat cac dag dacaaa t a c a c t t t a t t a a t a t a d cca t a a a t c a a t t c a t t g t g t a t g aga c t g t q t t t c t a q t t q c a t t t c a t a t t q e t a cca aac c

1320 1360 1420

met ser arg arg TGG AAG

GAC TCG CCT AAA TCTTCC

t r p l y s asp ser pro

AAG TCCATC

l y s ser ser lys ser

AAA TTCATTCCTGTTTCA

human VlaAW receptor sequence (23). are identical for the human VI, AVP receptor and the other Alignment of the amino acid sequences of the human VI, members of the AW-oxytocin receptors'famils Among the five members of the AW/oxytocin family of reAVP receptor, the rat VI, A W receptor, the human oxytocin receptor, the human V2 AVP,and the ratV, AVP is presented in ceptors isolated so far, several amino acid sequences are reFig. 2. The amino acid sequence identity of the human VlaA W markably conserved(Fig. 2). AH receptors share the sequence receptor with the rat liver VI, A W receptor, the humanoxyto- FQVLPQ present at the 3' end of the second t r a n s m e m b ~ ~ e cin receptor, the human V, A W receptor, and the rat V2 AW domain. This sequence is thought to play a major role in ligand receptor sequences is 72, 45, 36, and 37%, resp~ively.This bindingand signal transduct~on.Thesequence FXGPRXLdegree ofidentity is comparable to those observed between the CRXW present at the 3' end of the first extracellular loop and members of other 7-Th([S receptor groups like the adrenergic the sequence R C W ~ located ~ Win the ~ second extraeeland muscarinic receptor families.Amino acids enclosed in boxes Iular loop are present in all AVP/ox+in receptor sequences.

3308 HvIa RatVfs Hv2

mv2 Ho*Y

HVla RatVlo

Hv2

Ratvz

HOxy

Hvla RatVia Hv2

Ralvz

HOxy FIG.2. Alignment of the amino acid sequences of 7-TMS receptor prob i n s of the AW-oxytoein family. Deduced amino acid sequences of the human V1.AVP receptor (HVla),the rat liver Vi. 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 throughout the family of A V P - 0 receptors ~ ~ family are bored, and positions of the putative TMS I-VI1 are indicated by lines above the amino acid sequences.

Hvh RatVfa HvZ Ratv2 H*Y

HVlo RatVla HV2 Ram HOxy

Hvto RatVla

Hv2

Ratv2 HOxy HVI a Ratvia Hv2

Ralw H*Y

Hvla RatVla Hv2 Ratv2 €foxy

XKFXPVST

These two extracellular sequences are not found in other members of the superfamily of 7-TMS receptors and are plausible elements of the ligand r ~ ~ i site. ~ Additiona~ly, o n 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 cysteine residues are present in allC termini and, through palmitoylation, 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, whichplays a key rolein G protein coupling, differs drastically between the Vx and Vz sequences (Fig.2). It also comes as no surprise that theN- and C-terminal regions differ widely between the five sequences. ~ a ~ i # l i g a nBinding d Characteristics of the Expressed Haman VI, AVP Receptor in COS7 and CHO Cells-The radioligand binding c h a r a c ~ ~ s t iof c sthe cloned human VI,AVP receptor werefirst explored in the transientlytransfected COS-7

cells.Competition binding experiments confirmed that the cloned receptorhas thetypical pharmacologic profileof the VI, subtype (Fig. 3a).The most potent competi~rof [12511QrPhaaspecific binding was the linear V1, antagonist phenylacetyl-D~ E t ~ - P h e - ~ l n - A ~ n - L y (Xi s-= ~ o1.24 - ~*- 0.16 ~ ~ ma), followed bythe linearVI,non-peptide a n ~ g o n i sSR t 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-LysPro-Tyr-NHi%(Ki = 2.96 = 0.52nM), and the Vz antagonist d ( C H z ) 6 [ ~ I l e 2 , n e 4 , ~ a - ~ ~(Ki l A =W67.61 2 16.91 m). Finally, 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 p h ~ a c o l o g i c a characteristics l of the cloned human V1, AVP receptor, we used CHO cells stably transfected with the full-length 1.5-kb clonewe isolated. We verified in competition experiments with [12611TyrPhaathat control CHO cells did not have endogenous VI, AW receptors

Human V I ,AVP Receptor Clone

3309

a

0 0

Competitor Concentration (M)

5

10

15

20

25

Free [3MAVP (nM)

FIG.4. Characteristics ofAVPbinding sites in CHO cells transfected with the human VI, receptor clone, Transfected CHU cetls prepared as described under “Experimental Procedures” were grown to

b

confluence in24-well dishes. Saturation binding experiments were performed with increasing concentrations of[SHIAVf? Specific binding is shown as open circles, and nonspecific binding was deterrnined in the presenceof 1 AW (open squures). The inset is aScatchardlinear 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 comparedto the control CHO cells. The fluorescence was I . ,o.12 IO"^ 1g* 10.* 1 0 ” lo5 speci~cal~y displaced by an excess of unlabeled PhaaGln (data Competitor Concent~ationfM) not shown). AVP-~n~uced ~nosjto~ ~ ~ o s p ~ a t e inPCOS7 r ~ uand ~ ~ w ~ FIG.3.Specific binding ofA W to COS? and CHO cells transfected with thehuman V,, receptor clone. Transfected COS-7 and CHO Ce€ls--The signal transduction of the cloned human V,I CHO cells prepared as described under~ ~ ~ r i mProcedures” e n ~ l sec- AW receptor was explored in the transiently transfected tion were grown to confluence in 24weE dishes. The figures show the COS-7 cells by measuring AW-jnduced inositol p h o s p h a ~promean of compet~tionbinding experiments performedwith one conceno n30 min induced a dosetration of [laaIj”haa and increasing Con~ntTationsof A W OT its duction (Fig. 56s). A W s t ~ ~ a t i for of IPI (2-fold, 3607 dpnt, analogs (n s 3-5 for each anafog)in COS-7 cells (panel a ) or CHU cells dependent increase in thef o ~ a t i o n (panel b). IPS(Bfold, 802 d p ~ w e l l ) well), fP2 (7-fold, 4375dpmlwell~, and fmm respective base lines of 1805,645, and309 d p ~ w e l(ln = (data notshown). Competitionbinding experiments confirmed 6). These data suggest that the cloned receptor is indeed that the cloned receptor belongs to the VI, subtype (Fig. 3b). coupled to phospholipase C, No s t i m ~ a t i o nof IP production The most potent competitor of [12511~Phaa-specificbinding 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 was the linear V1,a n ~ g o n i sphenylacetyl-D-~Et)-Phe-Gin~ t A s n - L y s - ~ o - ~ g - N H(Ki 2 = 0.79 rt 0.23 m), followed by the conditions). linear Vla non-peptide antagonist SR 49059 (Xi = 1.12 rt 0.08 A ~ ~ ~ ninositol d ~ cphosphate e ~ p~uction was also studied m),then A W itself (Ki = 1.77 rt 0.12 m),then the linear VI, in control and transfected CHO cells. No stimulation of IP antagonist p h e ~ y l a c e t y l ” D - ~ E t ) - P h e - V a l - A s n - L y s - ~pmduction ~was observed in control CHO cells (data not shown). NH, (Ki= 2.37 * 0.07 m), and the V, antagonist d ~ C H 2 ) 5 [ ~As shown in Fig. 5b,AW induced a dose-de~ndentincrease in n e z , n e ~ * ~ a - ~ 2 (Ki 1 A=W52.94 t 7.60 DM).Finally, o ~ i nthe f o ~ a t i o nof IP1(20-fold, 16,787 d p ~ w e l l ) IP, , (50-fold, was the weakest c o m ~ t i t oCKi r = 87.14 * 22 mal. This order of 2,053 d p ~ w e l l )and , IPS( 3 ~ f o l d1,026 , d p d w e l l ~from respecaanities is therefore identical to that observed in COS-7 cells tive base lines of 834, 393, and 422 dpdwell f n = 6). The and confirms the VI, subtype of the cloned receptor. s t ~ u l a taction i ~ of A W was blocked by the non-~ptide VI, In addition, saturat~onbinding experiments with f3H.@SW a n ~ g o n i s tSR 49059. AW stimulation of inositol phosphate were performed in CHO cells transfected with the cloned hu- p ~ u c t i o nwas greater in stably transfected CHO cells than in man V1, A W receptor cDNA (Fig. 4). A single class of b ~ n d ~ transiently g t r ~ s f e c t e dCOS-7 cells, because of the selection of sites was identified with a ~ssociationconstant, K d = 1.21 rt the best CEO clones in terms of AW receptor expression. In 0.06 m and a total capacity B,, = 780 t 39 fmol/mg of protein addition, the profile of st~mulationwas different between the f n = 24). N o n s ~ i f i binding c was negli~blein transfected CHO two celllines (CNO cells:IP1 > IP2> IPSuersus COS7 cells: IP2 cells. No specific binding was observed in nontransfected cells > IPS> IF’& p r e s ~ a b l because y of different phospha~seconand in cells transfected with the pECE vector alone (data not tents in thetwo cell lines. These data suggest that thecloned shown). Ki and & values derived from c o m ~ t i t i o nand satu- receptor is indeed coupled to phospholipase C. Ib test if AVP ration binding experiments are well in agreement with those act~vationof inositol phosphate product~on int r a n s f e ~ dcells for the human platelet VLaAW receptor we extensively char- was pertussis toxi~-sensitive or not, t r ~ s f e c t e dCHO cells acterized (2,9,13,24). The ra~oligandbinding characteristics were pretreated with 0.1 d m 1 pertussis toxin prior to AVP of the cloned human VI,AW receptor in CHO cells we present stimulation. The dramatic increase of inositol phosphate prohere are similar to those reported by Morel et al. ( 5 ) for the duction induced by A W was not altered after pretreatment cloned rat V1, A W receptor in transfected CHO cells (& = 0.67 with pertussis toxin (three i n d e ~ n d e nsets t of triplicate exma, = 584 fmo2lmg of protein). p e ~ e n t s This ~ . pertussis toxin ~ n s e n s i t i ~of t ythe cloned reFluorescent Labeling ofthe Human V I ,A ~ P ~ c e p t oinFCHO ceptor is in agreement with our previous obse~ation that the Cells-As we reported recently, linear V1-vascular A W antago- VI, receptor of human platelets iscoupled to a pertussis toxinnists can be coupled to dodecabiotin to visualize AW receptors insensitive G protein belonging to the G,11 family (19). d i n(9).Fluorescent labeling by addition of a ~ u o r e s c ~ n - a ~tag AVP Eflect on A ~ ~ yCyclase l y ~ ~ c ~ ~inu CHO i ~ yCeZls-Ib of CHO cells with PhaaGln-dodecabiotin-~uorescein-avidin re- confirm that the cloned human VI, AVP receptor cRNA is

- .

Human V I , AVP Receptor Clone

3310 a

l e -l 1e5 -0ei l9e-e.9-87

le-6

AVP Concentration (M)

le-10 l e - 9

Se-9

ie-8

le-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 CHOcells prepared as described under"Experi-

AVP-induced Calcium Mobilization in CHO Cells-% confirm that thecloned human VI, AVP receptor cDNA is coupled to phospholipase C in transfected CHO cells, AVP-induced mobilization 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 AVPoxytocin family, especiallyat thelevel of the seven transmembrane segments. We present several lines of evidence suggesting thatthe isolated clonebelongs 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 profileof a VI, AVP receptor. Moreover, saturation binding experiments in CHO cells characterize a single class of high affinity binding sites, like in human platelets we explored before. Results of inositol phosphate production and calcium mobilization experiments confirm that the cloned receptor is ~nctionallycoupled to phospholipase C in a pertussis to~n-independentfashion.

A c ~ n o w ~ ~ ~ ~ thank s - WAndrea e Bayer forskilled technical asmental Pmdures" were grown to confluence in 12-well dishes and sistance, Dr. Woo for granting us the permission to use his liver cDNA incubated in inosital-free DMEM buffer, pH 7.4, with my0-[2-~H]inosi- library, and Prof. Corvol for welcoming us in his research unit at the tal. Formation of [3H]inosital phosphates was measured after addition Coll&gede France, Paris, where this project was initialed. of increasing concentrationsof AVP for30 min in COS-7 cells (punel a) or CHO cells ( p a n e 2 b ) fn = 6 for each series). REFERENCES 400

r

1. Michell, R. H., Kirk,C. J. & Billah M.M. (1979)Biochem. Sac. 'Dana.7, 861-865 2. Thibonnier, M. (1992) Regul. Pept. S S , 1-11 3. Thibonnier, M., Bayer,A. L., Simonson, M. S. & Kester, M. (1992)Edocrinofagy 129,2845-2856 4. Thibonnier, M. (1993) in N e u r ~ ~ a c r of~ the n oConcepts ~ ~ 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)Nafure866, 523-526 6. Lolait, S. d., O'Camll, A. M., McBride,0. W., Konig, M., Morel,A. & Brownstein, M. J. (1992)Nature Sa7,33fG339 7. Bimbaumer, M., Seibold,A., Gilbert, $.,Ishido, M., Barberis, C.,Anataramian, J A,, Brabet, P. & Rosenthd, W.(1992)Nature 557,333-335 8. Emma, T., Tanizawa, O., Man, K., Brownstein, M. J. & Okayama, H.(1922) 1 0 1 2 3 4 5 Nature SSS,526-529 Time (min) 9. Thibonnier, M., Bayer,A. L. & Madhun, Z. (1993)Am.J. Physiol. 266,in press 10. Ellis, L.,Clauser, E., Morgan, D., Edery, M., Roth,R. A. & Rutter, W.J. (1986) FIG.6. Am-induced [Cas+], mobilization in CHO cells transCell 45,721-732 fected with the human V,, receptor clone. Control andtransfected 11. Teutsch, B.. Bihoreau, C., Monnot, C., Bemstein, K E., Murphy, T. J., AlexCHO cells prepared as describedunder"ExperimentalProcedures" ander, R. W., Corvol, P. & Clauser, E. (1992) Biochem. Bwphys. Res. Comwere grown to confluence onA C W coverslips, loaded with fura-2, and mun. 187,1381-1388 incubated in Krebs-Henseleit-Hepesbuffer, pH 7.4. Basal fCa2+liwas 12. Bihoreau, C., Monnot, C., Davies, E., Teutsch, B., Bemstein, K, Corvol, P. & measuredfluorometrically after addition of 1 AVP in transfected Clauser, E.(1993)Pmc. Natl. Acad. Sci. U.S. A. SO, 5133-5137 cells (solid line) and control cells (dofted line). Figure shows represent- 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 ative tracings of four different experiments. 15. Dubvak G. R.. Cowen. D. S. & Meuller. L.(1988)J. Bid. Chem. 263,18108lilli coupled to phospholipase 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 AcidsRes. 15,81258148 D.,Weies, R. M. & Tedlliger, T.C. (1984)Proc. Nafl. Acad. Sci. (-THO In controlcells, luvr A~ did not significantlyalter 18. Eisenberg, U.S. A. 81, 140-144 CA" oroduction (23.563 uersus 15.261 fm01 of &b'fP/mE O f 19. Thibonnier. M..Govara T.. L. & Berti-MatteraL. M. (1993)Am.J. Phvsid. 284, . . C1336G13k protein, n = 6) whereas forskolin induced a 7-fold rise of CAMP 2 0 . ODowd, B. F., Hnabwich, M., Caron, M. G., Lefkowitz, R. J. & Bouvier, M. to 110,746 fmol of cAMP/mg ofprotein. Similarly,in transfected (1989) J. Biol. Chem. 264,7564-7569 cells, AVP did not alter d P production (25,350ue?-sus20,258 21. Homtman, D.A., Brandon, S., Wilson,A- L., Guyer, C.A, C r e e , E. J., Jr. & Limbird, L. E. (1990) J. Bid. Chem. 8ea, 21590-21595 fmol O f ~ P ' m gof protein*n 6) whereas forskolin induced a 22. melshrg, M. A., CoteccMa,s., OstrowsE, J., caron, M. G. LeRowitz, R. J. ?-fold rise of CAMP to 121,603 fmolof CAMP/m~of protein. (1992) J. BioZ. Chem. 287.1430-1433 Thus, the lack of &b'fP production decrease in thepresence of 23. Mizuno, T.,saito,Y., Itakura'M., 1% F., I%, T.. Moriyma, E., H&wma, H. & Hirose, S. (1992) Biochem. J. 287,306-309 in transfected suggests that the 'Ioned Aw receptor 24. Thibonnier, M., Hinko,A & Pearlmutter, A. F. (1987) J. Cardiouasc. Pitarmais not coupled to Gi. c d . 10,24-29

c

-

"