Volume 292, n u m b e r 1,2, 243-248 FEBS 10364 © 1991 Federation of European Biochemical So=ieties 00145793/91/$3.50 ADONIS 001457939101079F

November 1991

Cloning, sequencing and tissue distribution of a candidate G protein-coupled receptor from rat pituitary gland Karin A. Eidne, Jelka Zabavnik, Tracey Peters, Shigeru Yoshida', Lorraine Anderson and Philip L. Taylor M R C Reproductive Biology Unit, Centre f o r Reprodttctive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK Received 2 September 1991 A new member of the family of G protein-coupled receptors has been isolated from a rat pituitary eDNA library by the polymerase chain reaction (PCR) using degenerate oligonucleotide primers. The corresponding protein sequence shows seven transmembrane domains and contains conserved regions of homology characteristic of the G protein-coupled class of receptors. The novel receptor mRNA is expressed in the brain, pituitary gland and testis, and has been localized by in situ hybridization in discrete regions of the brain. Expression of the receptor mRNA in Xenopus ooeytes and in transfected mammalian cells has not yet permitted identification of the corresponding ligand for this receptor. Polymerase chain reaction; G-protein receptor; Guanine nucleotide-bindingregulatory protein; Homology probing

1. INTRODUCTION Recent developments in receptor biology have shown that a number of the receptors which interact with G protein signalling systems have sequence homologies and may be a part of a receptor superfamily [1]. Specific features o f this family include seven stretches of 20-26 residues of hydrophobic amino acids which include several invariant amino acids; potential N-linked glycosylation sites near the amino terminus and phosphorylation sites near the carboxy terminus. The presence of partially conserved regions of sequence in the structures o f the receptor superfamily permits the use of homology screening in order to isolate novel receptors. We used PCR amplification of e D N A with degenerate oligonucleotide primers corresponding to conserved sequences of various transmembrane domains and have isolated a novel receptor-coding clone from a rat pituitary library. This paper describes the cloning, sequence analysis and tissue expression and localization of this receptor molecule, one of a growing number of G protein- coupled receptors for which ligands have not yet been identified [2-5].

The G-protein coupled receptor cDNA sequence reported here has been assigned the accession number X61496 by the EMBL Data Library.

"Present address: Department of Physiology, Nagasaki University, School of Medicine, Nagasaki 852, Japan. Corre.vmmlence address: K.A. Eidne. MRC Reproductive Biology Unit. Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK, Fax: (44) (31) 228 5571.

Publ/shed IU' Elsevier Sch,m'e Publ/slmrs I3. V.

2. MATERIALS AND METHODS 2.1. Rat pituitary cDNA librao, screens Library screens of a size-selected lambda ZAP I1 eDNA library (107 independentclones) prepared from rat pituitary tissue were performed using standard techniques [6]. Bluescript plasmids were rescued from plaque purified clones by M I3 excision (Stratagene). 2.2. Oligonucleotide~ Oligonucleotides were synthesized on an Applied Biosystems PCRMate 391A DNA synthesizer according to the manufacturer's instructions. Primer TM3 was based on conserved sequences found in the third transmembrane region [2]; (TM3, 5'GTCGACCTGTG(TC)G (TC)(GC)AT(TC) GCIIT(TG)GA (TC)(AC) G(GC)TAT3'), primer TM6 on those in the sixth transmembrane region; (TM6, 5"AAGCTTA(TG)G(AT)AG(AT)AG GGCAGCCAG CAGAI (GC)(AG)(TC)AAA3'). Primers for analytical PCR were: (matching at 869) 5'TGTAAGAT TGTGATGAGGCACG3'(matching at 1223) 5' GAGAACGCTGCTACACATCG 3" 2.3. PCR amplification of pituitary, cDNA attd tissue RN,4s eDNA reverse transcribed from rat tissue mRNA was submitted to 30 cycles of PCR with Taq polymerase [7]. Cycle conditions were 1.5 rain at 93°C, 1 rain at 50°C and 2 rain at 72°C. PCR was carried out in the presence of [32P]dCTP (2 pCi) for the tissue distribution experiment and the reaction products electrophoresed on a sequencing gel with size standards. Positive and negative controls were run in parallel to check amplification of known sequences and to detect possible template contamination. 2.4. DNA sequenc#tg attd comput#tg DNA sequencing was carried out using Sequenase (USB) and analysis was provided by means of a program (GeneJockey) running on Apple Macintosh microcomputers (P.L. Taylor, Biosoft, Cambridge, UK). General database searching and access to the AMT digital array processor (DAP) was obtained via SEQNET (SERC Daresbury Laboratory). 2.5. hl situ h),bridi=atiot~ Riboprobe preparation from R334 eDNA and in situ hybridization were carried out according to previously described methods [8].

243

Volume 292, number 1,2

November 1991

FEBS LETTERS

I CTGCGACCTGCGGGGGCGCGCGCAGCCTCGTGGGGT 37 T C T c G ~ G G A T G G ~ G ~ C G G G ~ G G G G A G ~ G ~ C A G G G c G G A G A G ~ c G G G c G C ~ A G C G A ~ G ~ A G C T ~ A c ~ T G ~ C G c G G G c G C c A C C A C G 124 G A C G T ~ C C A C G ~ G G G T G G c ~ C T c ~ c T A T T c ~ G c A G c A c ~ G A A G G A G c C c C ~ C ~ T ~ G G T ~ T G G ~ C G T G ~ A A G A G G A ~ A G G G G T T A A A V MeH Ash Glu Asp Pro Lys val Ash Leu Set o l y Leu PEO AEg A s p Cys Ile GIu Ala Gly Thr Pro

22

211 ATG AAC G A A GAC CCG A A G GTC A A T TTA AGC G G G C T G CCT COG GAC TGT A T A G A A O C T G G T A C T CCG V Glu Ash Ile SeE A l a A l a Val Pro Sot Gln G l y S e t Val Val Glu Set Glu P r o GIu Leu Val Val

44

279 GAG AAC A T C T C A G C C O C T GTC CCC TCC CAG G G C T C T GTT GTG GAG T C A GAA C C C GAG C T C GTT GTC

I ASh 344 A A C Lou

Pro T r p Asp

Ile Val

Cvs GIu Ash

Ala Val val Val

CCC T G G GAC A T T G T C TTG T G C AGC TCA G G A A C C CTC ATe TGC TGT GAA A A T

GCC G T C GTG GTC

Ii~ Ile Phe

His

Leu Cys Set Set G i y T h r Leu ~i.o Cys

Set Pro S e t Leu Arg A l a P r o M@H Phe L~u

Leu Ile Glv

Ser Leu Ala

Leu

410 CTT ATe A T C TTC C A C A G C CCC A G C CTG CGA G C A C C C ATG TTC CTG

CTG A T A GGC

AGC C T G OCT CTT

G I 7 Leu G l y Leu 11o Ile A S h Ph@ Val Phe Ala Tyr Leu

Leu G l n Set Glu

66

88

H ALa

Asp Leu Leu A 1 a

476 G C A GAC C T G CTG G C T G O T CTG G G A CTC ATC A T C A A T TTT GTT TTT

GCC TAC CTG

CTT CAG TCA G A A

Ser Ph@ Set Ala Sot Val

Cys Set L e u Leu

542 OCt ACC A A G CTG G T C A C A ATT G G A CTC ATT GTC G C C TCT TTC TCT GCC TCT GTC

TGC A G T TTG CTG

110

m Ala

Ala

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Tyr His

Set Gly Gin Asp A r g

608 OCT ATC A C T GTG G A C

C G C TAC CTC TCG CTG T A T T A C GCC CTG ACG

TAC CAC TCC

G G A GAG GAC C O T

Cys Leu GI~

L@u Leu Leu Tyr

132

154

IV His 674 CAC Gly

Leu T y r Leu C y s M o t

Lou Val M~H Lou T r p G 1 7 Thr Sot Thr

CTT T A C CTA T O T A T G CTA GTG ATG CTC TOG G G A A C T TCC ACC

TGC CTG GGG CTG CTG CTG TAT

Lou G I u Lou P E o

Thr L@u ThE

G l u Gly A r g Val His Leu G i n A r g Gly Gln

Lys Ash Ash Ala

17~

198

740 GGG CTG G A A CTG C C T G A G GGA C G A GTC CAC CTG C A G CGT GOT CAG A C C GtC A C T A A G AAC AAC GCC

V Ala

If@ Leu Set Ile

806 GCC A T T C T C TCC A T C

S e t Ph@ Leu Ph~ Met Ph~ A l a

Lou Met Leu Gin Leu Tyr

T C C TTC CTC TTC ATG TTC G C A CTG ATG CTC

Zle Gin

Ile Cys

C A A CTC TAC A T C CAG ATT T G T

Lys

If@ V a l Met A r g

His Ala His Gln Ii~ Ala Leu Gln His His Phe Leu Ala

87~ AAG

ATT G T G ATG A G G

C A C GCC CAT GAG ATA GCC G T G CAG CAC CAC

TTC CTG OCT A C G TCG CAC TAC

Val

Thr T h E A r g L y s

Gly

Thr Phe Ala A l a Cys Trp M @ t

938 GTG ACT A C C COG A A A

Ile Set Thr Lou Ala Leu

Ile Lsu Gly

220

T h r Ser His Tyr

242

264

G G G ATC ~CC ACC CTG OCT C T C ATC CTG GGG ACC T T T GCC G C C TGC TGG A T G

VII P r o Phe T h r Lou T y r 1004 CCT

s e t Leu Iio Ala Asp Tyr T h r Tyr Pro Set

TTT A C C CTC T A T T C C

Ilo Tyr Thr T y r Ala Thr Leu

286

TTG ATC GCC GAT TAC A C C TAC CCC TCC ATC TAC ACC T A C GCC ACC CTC

vn Lsu 1070 CTG

Pro A l a Thr T y r A S ~

If@ Asn Pro V a l

If@ Tyr Ala

Ph@ A r g Ash G i n Glu

Ile G l n

308

CCC q C C ~C~ T~.C ~ A T TCC ATC ATC AAC CCT G T C A T A TAT OCT TTC A G A AAC C A A GAG ATC C A G

Lys A1a P r o Leu P r o 1136 1210 1297 1384 1471

Set lie

His

Lou Leu Trp Val ~is P r o Stop

320

A A A GCC C C T CTG C C T C A T CTG CTG TOG GTG CAT C C C TAA C A C G C T G T C C A G A G A G C A C G C T C T C C C A G G G A T G T GTAG•AG•GTT•TcccCA•AGGA•GcTG••T•TAcTAAG•GAcCc•A•TG•••AGGGcAG••GGTGA•TT••TCcc•TTAAATT•TT TGcA•TGGATcTCACAGGcAGAAG•AA•GATATcTTTTAGA•TAGTGTTGGCAGTGGAAATcATGTTAcCAGTGTTTTAAAAAACTc AAcTT•T•GG•TTAG•ATTc•GTTGTTTGGTTTGGGAGTTAGGA•TTGTTTGTTTGTTTGTTTGTTTGGAGGGTGTAGTGG•A•CT• ATGTGGC C GTAA~ATTATACACAAGTCTCAGGATTTTTAACCTAGACTTGAA~ATAAATCnGTTTTAAAG GAAAAAAAAAAA

Fig. 1. eDNA and predicted amino acid sequence of the putative R334 receptor. Potcntial N-linked glycosylation sites in the NH=-terminal region (~), and phospho~lation sites in the COOH-tcrminal region ()) are indicated, The putative transmembrane domains I-VII are underlincd and are assigned on the basis of a Kyte and Doolittle [18] hydropathicity plot. The polyadenylation signal is underlined. 2.6. Eukaryotic e.,q~ression R334 was cloned into pcDNA 1neo vector (Invitrogen) and transfected using Lil.'cfcctin Reagent (BRL) into the 293 cell line. G418244

selected (Oeneticin, Gibco: 800 ~'g/ml) transfectants were isolated and

checked for integrationand expressionof pcDNAlneo R334 by PCR and Northern blotanalysisof cellularm R N A . Cellswere maintained

Ptv-WLPFCESSC--!iHPTLLGw IiwXQDNL---lPK&vY :T-l@dJWCDCW----1PPVLY

dopamine Fig. 2. Alignment of theamino acid sequ9#r encoded by R334 receptorwith the amino acid scquenccsof rcceptors(ral) for subsunos Pand K, teuahydr-bii,5HTIA, D2. acetylcho!ine(pig muscarinic Ml) and adrenaline(hamster /X2). The approximate positions of the hansmembrane regions are indicated by a broken he ahve the sequcoces and const~~~~Jresidues are boxed.

Ram 82

YFN RSP

R Ctdl

Subat

5HT 1A

---P CL!4

***

R 334 subst

A 334 Submt P tinab Subrt X Pg ARMI 4Hr IA %rn 82 02

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in DMEM media containing 10% (v/v) heat inactivated foetal calf serum, glutamine (0.3 mg/ml), penicillin (100 IU/ml) and streptomycin (100 ttg/ml) and incubated at 37°C in 5% CO.,. c A M P was measured [9. I0] as were total inositol phosphates (IP I, I P2 and IP3) after extraction and separation [I I]. Whole-cell current measurements in Xenopus oocytes injected with R334 m R N A transcripts were carried out using two-electrode voltage-clamp techniques [12].

3. RESULTS A N D DISCUSSION PCR products generated from rat pituitary eDNA included a 400 bp band of PCR-amplified D N A which was purified by agarose gel electrophoresis and used to probe a rat pituitary e D N A library. Several independent cDNAs were isolated at low abundance and sequence analysis of these clones showed them to consist of identical stretches of sequence. The most complete eDNA clone, R334, of 1552 bp, contained a region of open reading frame corresponding to a peptide of 320 amino acids (Fig. l), possessing seven highly hydrophobic regions o f 20--25 amino acids. A search was performed on the OWL database for the protein sequence using the P R O S R C H program of Coulson et al. [13] running on the DAP. This yielded a list o f matching proteins, of which the best-fitting 50 included 47 which were 13 protein-coupled receptors. None of these proteins were identical with R334, or sufficiently close in structure to suggest membership of a known subfamily. The deduced amino acid sequence o f R334 and alignment with 7 other members of the G proteincoupled receptor family is illustrated in Fig. 2. The R334 sequence contains potential N-linked glycosylation sites Ash 8 and Asn -'4 in the amino terminal region and potential phosphorylation sites, Thr :4., Thr z4s and Ser TM in the carboxyl-terminal region. The Asp residue in TM3, shown to be essential for ligand binding in the fl-AR receptor [14], is absent in R334 (Fig. 2). O f all the cloned receptors aligned in Fig. 2, R334 contains the shortest third cytoplasmic loop. This loop has been shown to be important for G protein recognition [14]. R334 displays 22% homology across the TM domains with the cannabinoid receptor, 16% and 17% with rat substance P and substance K receptors, 17% with the human/~2 adrenergic receptor and 18% with the 5 H T I A receptor. The 3'-untranslated region contains a polyadenylation signal and a poly(A) tract (Fig. I). The tissue distribution of R334 m R N A in rat tissues was examined. Northern blotting showed a faint hybridizing band greater than 7 kb in brain and testis (not shown). Because Northern analysis was found to be too insensitive to satisfactorily detect the low-level expression of R334 m R N A in these tissues, P C R analysis of reverse-transcribed R N A prepared from pituitary, brain and peripheral tissues was carried out [16]. A PCR-amplified band exactly corresponding to the distance between the two primers as predicted from the R334 sequence (355 bp) was observed in the pituitary 246

November 1991

gland, brain and testis R N A (Fig. 3). No signal was observed in any of the other tissues examined (see Fig. 3). The localization and distribution of R334 m R N A in the pituitary gland and brain were studied by in situ hybridization. R334 m R N A was detected in pituitary tissue (Fig. 4A,B), in the piriform cortex of the brain (Fig. 4C,D) as well as in the lateral septal nuclei (not shown). Diffuse labelling of cells was seen in the anterior pituitary gland and posterior lobes (Fig. 4A), while in the brain, the signal was restricted to discrete areas only (Fig. 4C). Control sections probed with the sense riboprobe showed non-specific background hybridization only (Fig. 4B,D). We have investigated a number of possible ligands for :. -'. :...

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electrophoresed on a sequencinggel and autoradiographcd. From left to right', Lane i, t~oDNA; lane 2, R334eDNA; lanes 3-12, RNA fr,,m testis, lung. ovary, pituitary, brain, kidney, liver, spleen, adrenal and heart,

Volume 292, number 1,2

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November 1991

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Fig. 4. Autoradiographic detection of cells containing R334 mRNA in rat pituitary and brain tissue by in situ hybridization. Dark-field micrographs of emulsion dipped (A) pituitary ilorizontal sections (a, anterior; p, posterior), hybridized with R334 antisense riboprobe; (B) pituitary with sense riboprobe; (C) coronal sections through rat brain piriform cortex (pc) hybridized with antisense riboprobe; (D) sense riboprobe, this r e c e p t o r . Synthetic, c a p p e d R N A t r a n s c r i b e d f r o m R334 c D N A w a s injected into X e n o p u s laevis oocytes, a l o n g with R N A t r a n s c r i b e d f r o m the 5 H T l c r e c e p t o r c D N A c l o n e [17] as a positive control. 33 d i f f e r e n t potential l i g a n d s were applied to the b a t h i n g solution. A l t h o u g h definite electrophysiological r e s p o n s e s to ser o t o n i n w e r e d e t e c t e d (current = 500-1000 nA), no significant r e s p o n s e to any o f the d r u g s w a s a p p a r e n t . c A M P a n d total inositol p h o s p h a t e assays were also p e r f o r m e d on stable t r a n s f e c t e d cell lines expressing R334 m R N A following e x p o s u r e to these d r u g s . N o n e o f the s u b s t a n c e s tested s t i m u l a t e d c A M P p r o d u c t i o n ( o r inhibited c A M P p r o d u c t i o n in f o r s k o l i n - t r e a t e d cells). Similarly, m e a s u r e m e n t s o f total inositol phosp h a t e s s h o w e d n o m e a s u r a b l e effects (data not shown). A l t h o u g h we were unable to identify the ligand for the R 3 3 4 r e c e p t o r , the o b s e r v e d h o m o l o g i e s with k n o w n r e c e p t o r sequences a n d the characteristics o f the h y d r o p a t h i c i t y profile all suggest strongly that this sequence is t h a t o f a p r e v i o u s l y u n k n o w n r e c e p t o r belonging to the G p r o t e i n - c o u p l e d r e c e p t o r family. F u r t h e r studies a r e r e q u i r e d to d e m o n s t r a t e that this molecule functions as a biologically active receptor.

Acknowledgements: T h e a u t h o r s t h a n k Prof. D. Lincoln t b r e n c o u r a g e m e n t a n d s u p p o r t , D r J. Inglis for isolating the R334 clone, D r D, Julius for the gift o f the 5 H T I C clone a n d M. Millar for ,~reparing frozen sections, J, Z a b a v n i k is s u p p o r t e d by t h e Veterinary Faculty

in Ljubljana, Yugoslavia. REFERENCES [1] Dohlman, H.G., Caron, M,D. and Lefkowitz R.J. (1987) Biochemistry 26, 2657. [2] Libert, F., Parmentier, M.. Lefort, A., Dinsart. C., Van Sande, J., Maenhaut, C., Simons M.-J., Dumont, J. and Vassart, G. (1989) Science 244, 569-572. [3] Eva, C., Kein~nen, K., Monyer, H., Seeburg, P. and Sprengel, R. (1990) FEBS Lett, 271, 81-84. [4] O'Dowd, B.F., Nguyen, T., Tirpak, A., Jarvie. K,R., Israel. Y., Seeman. P. and Niznik, H.B. (1990) FEBS Lett. 262, 8-12. [5] Ross, P.C., Figler, R.A.. Corjay, M.H.. Barber, C.M,. Adam, N., Hareus, D.R. and Lynch. K.R. (1990) Proc. Natl. Acad. Sci. USA 87, 3052-3056. [6] Maniatis, T., Fritseh, E.F. and Sambrook, J. (1985) in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [7] Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Science 230, 1350-1354. [8] Peters. T., Taylor, P,L. and Eidne, K.A, (1991) in: Biological Signal Transduction, Nato ASI series H: Cell Biology, vol 52 247

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(Ross, E.M. and Wirtz, K.A. eds.) pp. 115-129, Springer-Verlag, Berlin. [9] Salomon, Y., Londos, C. and Rodbeil, M. (1974) Anal. Biochem. 58, 541-548. [10] Salomon, Y. (1979) in: Advances in Cyclic Nucleotide Research, vol 10 (Brooker, G., Greengard, P. and Robison, G.A. eds.) pp. 35-55, Raven Press, New York. [! 1] Dowries, C.P. and Miehell, R.H. (198 I) Biochem. J. 198, 133-140. [12J Yoshida, S., Plant, S., Taylor, P.L. and Eidne, K.A. (1989) Mol. Endocrinol. 3, 1953-1960.

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[13] Coulson A.F.W., Collins J.F. and Lyall A. (1987) Comput. J. 30, 420. [14] Strader, C.D., Sigal, I.S. and Dixon, A.F. (1989) Trends. Pharmacol. Sci. December Suppl., 26-30. [15] Kobilka, B.K., Kobilka, T.S., Daniel, K., Regan, J.W., Caron, M.G. and Lefkowitz, R.J. (1988) Science 240, 1310-1316. [16] Eidne K.A. (1991) Trends Endocrinol. Metab. 2, 170-176. [17] Julius D., MacDermott, A.B., Axel, R. and Jessell, T.M. (1988) Science 241,558-564. [18] Kyte, J. and Doolittle, R.F. (1982)J. Molec. Biol. 157, 105-132.