Molecular Cloning of a Functional Human Galanin Receptor

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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 9780-9783, October 1994 Neurobiology Molecular cloning of a functional human galanin receptor (Bowes melanoma cei line/guanine nucleotide binding protein-coupled receptor/cAMP) ESTELLE HABERT-ORTOLI*t, BRIGITTE AMIRANOFFt, ISABELLE LOQUET*, MARC LABURTHEt, AND JEAN-FRAN4OIS MAYAUX* *Rh6ne-Poulenc Rorer SA, Centre de Recherche de Vitry-Alfortville, DE6partement Biotechnologie, 13 quai Jules Guesde, 94400 Vitry sur Seine, France; and *Laboratoire de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Sante et de la Recherche MWdicale, U 410, 16 rue Henri Huchard, 75018 Paris, France Communicated by Tomas Hokfelt, June 21, 1994

ABSTRACT The ubiquitous neuropeptide galanin controls In this context, we describe here the expression cloning of numerous functions such as endocrine secretions, intestinal a cDNA encoding a galanin receptor from the human Bowes motility, and behavioral activities. These regulatory effects of melanoma cell line (18) as a source of mRNAs.§ galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive MATERIALS AND METHODS guanine nucleotide binding proteins Gj/G0 as transducing elements. We report here the isolation of a cDNA coding for a Expression Cloning of a Galanin Receptor cDNA. A cDNA human galanin receptor from a Bowes melanoma cell line library constructed with poly(A)+ from the human Bowes cDNA expression library, by using a radioligand binding melanoma cell line (ATCC CRL 9607) was purchased from strategy. The nucleotide sequence ofthe cloned receptor reveals Invitrogen. Briefly, the amplified mixed oligo(dT)/random- an open reading frame encoding a 349-amino acid protein with primed size-selected cDNA library (106 primary recombi- seven putative hydrophobic transmembrane domains and sig- nants) was constructed in the mammalian cell expression nificant homology with members of the guanine nucleotide vector pcDNAI (19). Plasmid DNA prepared from pools of binding protein-coupled neuropeptide receptor family. The 8000 bacterial clones was transfected into COS cells by cloned receptor expressed in COS cells specifically binds hu- electroporation (35 pig per 107 cells). The transfected cells man, porcine, and rat galanin with high affinity (Kd in the were then grown on 15-cm diameter dishes (Nunc) in Dul- nanomolar range) and mediates the galanin inhibition of ade- becco's modified Eagle's medium (DMEM) supplemented nylate cyclase. A 2.8-kb galanin receptor transcript was iden- with 10%6 (vol/vol) fetal calf serum, 6 mM glutamine, peni- tified in several human tissues. Cloning of this galanin receptor cillin (100 units/ml), and streptomycin (100 ,ug/ml) at 370C in should enhance our knowledge of its distribution, structure, 5% C02/95% air. Three days after transfection, the plated and function in human physiology and pathophysiology. cells were screened for binding. Cells, preincubated in DMEM for 1 h, were then incubated for 5 h at 20'C in 20 ml Galanin, a 29-amino acid neuropeptide (30 amino acids in of 25 mM Tris-HCl (pH 7.4) containing 0.1 nM 125I-labeled humans), was originally isolated from pig intestine (1) and porcine galanin (2200 Ci/mmol; 1 Ci = 37 GBq; NEN), 10 distributed in the central and mM MgCl2, 2% (wt/vol) bovine serum albumin, aprotinin (10 later reported to be widely pg/ml), bacitracin (1 mg/ml), pepstatin A (10 ,ug/ml), and 10 peripheral nervous systems of numerous species (2). Galanin ,uM phenylmethylsulfonyl fluoride (buffer 1). After five is unrelated to the other known families of regulatory pep- washes with the same ice-cold buffer, the cell culture plates tides and, to date, remains the only known member ofits own were dried and autoradiographed for 15 days at -80'C family. It exerts multiple regulatory functions such as (i) (Amersham Hyperscreen MP films). No binding was de- control of endocrine and exocrine pancreatic secretions, (ii) tected in mock-transfected cells. One pool, out of 90 tested, regulation of intestinal motility, and (iii) modulation of be- reproducibly gave rise to a specific binding signal. This pool havioral, cognitive, and sensory functions such as feeding, was successively subdivided until a single positive clone, learning, memory, and nociception (for reviews, see refs. 2 designated GALR1, was isolated. and 3). Galanin exerts its actions via binding to specific Binding of Radiolabeled Galanin to Transfected COS Cell membrane receptors (4). Biochemical and molecular studies, Membranes. Three days after transfection, cells were dis- performed in brain and pancreas, indicate that the galanin rupted in an ice-cold 5 mM Hepes (pH 7.4) and centrifuged receptor is a glycoprotein of 54 kDa (5-7) coupled to the (13,000 x g for 15 min), and the final membrane pellet was inhibitory guanine nucleotide binding (G) protein Gi, identi- stored at -80'C until use. 125I-labeled galanin binding assays fied as Gil, Gi2, and Gi3 in pancreatic 83 cells (8, 9). Depending were performed essentially as described (5). Briefly, mem- on the target tissue, different pathways for intracellular branes (10 ,g of protein) were incubated in 250 Al of buffer signaling by galanin are involved: inhibition of adenylate 1, containing 0.05 nM 1251-labeled galanin without or with cyclase (10), blockage of voltage-dependent Ca2+ channels galanin (human and rat, Bachem; porcine, Sigma), galanin- (11), and activation of ATP-sensitive K+ channels (12). (1-16) (Nova Biochem), or galanin-(3-29) (Rh6ne-Poulenc Structure-activity studies, with galanin fragments (13-15) Rorer Biochemistry Department) for 30 min at 20'C. The and chimeric peptides (16, 17), generally emphasize the reaction was stopped by centrifugation and the radioactivity importance of the N-terminal fragment of galanin for the in the pelleted membranes was measured. Nonspecific bind- interaction with the peptide receptor. These studies also raise ing defined in the presence of 100 nM porcine galanin was the possibility of the existence of galanin receptor subtypes, <5% oftotal about 15% an issue that may be properly addressed with the molecular binding. Specific binding represented cloning of the galanin receptor. Abbreviations: G protein, guanine nucleotide binding protein; TM, transmembrane. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L34339). 9780 Downloaded by guest on September 28, 2021 Neurobiology: Habert-Ortoli et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9781 of total radioactivity. Each experiment was performed at 50%6 (vol/vol) formamide/1 M NaCl/0.05 M sodium phos- least three times. Data were analyzed using the EDBA/ phate, pH 7.5/5 mM EDTA/0.5% SDS/10%o (wt/vol) dextran LIGAND software (20). sulfate/salmon sperm DNA (0.5 mg/ml)/5x Denhardt's so- cAMP Assay. The transfected COS cells were subcultured lution. After washing, the filters were autoradiographed for in 24-well dishes (105 cells per well) for 3 days. After a 1-h 10 days with an intensifying screen. preincubation in DMEM, cells were incubated in DMEM containing 2% bovine serum albumin, 0.2 mM 3-isobutyl-1- RESULTS AND DISCUSSION methylxanthine, and 0.1 mM forskolin with or without the indicated peptides, for 30 min at 37°C. Intracellular cAMP Isolation and Structural Characterization of the GalRl was assayed with a RIA kit (Amersham) according to the Clone. We have isolated a human cDNA (GALR1) encoding instructions of the supplier. In those conditions, basal and a galanin receptor by using an expression cloning strategy in forskolin-stimulated cAMP production levels were estimated COS cells (21). The cDNA library was synthesized from the at 4 and 210 pmol per 106 cells, respectively. human Bowes melanoma cell line, where galanin receptors Northern Blot Analysis. Poly(A)+ RNA (5 ,g) from human have been described (18). Nucleotide sequencing of the tissues (purchased from Clontech) and from Bowes and HT29 GALR1 cDNA clone revealed a single open reading frame of cells was electrophoresed on a denaturing formaldehyde/1% 1053 bp encoding a protein of 349 amino acids with a agarose gel and transferred to nitrocellulose filter (Hy- predicted molecular mass of 38.9 kDa (Fig. 1). A Kyte- bondC+, Amersham). Hybridization was performed with a Doolittle hydropathy analysis (26) indicates that the protein 32P-labeled randomly primed cDNA probe (spanning from the contains seven regions ofhydrophobic amino acids that could second intracellular loop to the C-terminal fragment of the generate transmembrane (TM) spanning domains with an galanin receptor) at 42°C for 72 h at the following stringency: extracellular N terminus and a cytoplasmic C terminus,

"p "p GALR1 ------VILS------GRSCXPPAPZPGPLFGIG----VENFVTLVV 39 GRPR MIJiLDcFUVDRN -c-----CS ISSUDDUSNPG---- IL-YVIPAV 46 SSR4 M---- SAPSLPG3L SAANAS GEDU)kASAL---VAIQCI 53 DOR1 NZLVPSARIZLQSSOLVNLSDAFPS -P------ASGSPGJ MRSASSILALAIATAL 55 * ** TX IT_ II GALR1 LIFALGVLGISLVITVLARSKPGKPRSTTNLFILNLSIADLAYLLFCIPFQATVYALP 99 GRPR YGVUI XGLIGNITLXKIFCTVXS---MRNVPNLrISSLALGDLLLLTXCAPVDASRYLAD 104 SSR4 YALVCLVGLVGNVI--MILRYADGWAWTNIYLNADLrIUS-VPFVMSSAALR 110 DOR1 YSAVCAVGLLVLVM--FGIVRYTKLKTATNIYIFILALADAILTST-IPFQSAKYLME 112 * * * .-** % *- * . -- *-- . $ ______TXuIII_ GALIl TIVLGAFCxir z?TI1vsxIFrTLMMSVRYVAISRRSSSLtVSRNALLGVGCI 159 GRPR RMLFGRIxGCxLIprQLT VTLTLSADRYKLZV"PTRQUMUMICLXAFI 164 SSR4 MIPFGSVLCAVLSVDGL1WTSVCLTVLSVDRYVAVVUPLRAATYRRPSVAKLINLGV 170 DOR1 TwPFG AVSIDYYNOUTSIFTLTHLKSVRYIAvcBPVKALDrRTPAKAKLINICI 172 ****. .. . . *.* *...*-**.* .*.** TX IV _T4 V GALR1 WALsIA F SPvAYIQGL--FHPR&SNQTF--CWZQWPDPR--HKKtAYVVCTFm GYLLPL 213 GRPR wIISIIPZA vrsDLBPFH XSTNQTFISC-APYPESNUILPKINSMASFLVrYVIPL 223 SSR4 iLhSLLVTLPIAIrADTRPARGGQA---V-ICNl.QNPPAN--SAVFVVYTFLLGFLLPV 224 DOR1 WVLASGVGVPIMVhVTQP-RDG-A----VVcILQFPSPSWYWDTVTKICVFLFAFVVPI 226 * ... * . . * ..* . . *. ..*.

_TX VI GALIR1 LLXcFCYAKVLNKIJML------ImS S KV 266 GRPR sixI YYYIAKNLIQAYNLPVWhINV5QZSR [RIAKTVLVFVGLFAFCWLNNVI 283 FIG. 1. Comparison of the 8SR4 LAIY LIV----GRAVALR R--SKKTRLV VVVFVLC FYVV 278 amino acid sequence of the gala- DOR1 LIITVCYGL -----LRLRSVLLSGSKZKDR--SLRRITRVLVVVGAFVVCWAPIHIF 280 nin receptor GALR1, as deter- * . * .. .**.* * * *.-. * mined by DNA sequencing ofboth _TM VII strands of the GALR1 clone, with GALR1 LVAZFGVFPLTPASFLFRITAC--- LYSNSSVNPIIYAFLSNFRK&AYK-QV----- 317 the sequence of the human gas- GRPR YLYRSYB-YSZVDTSNHFVTSIC LFTNSCVPFALYLLSKSZRKQFNTQL ----- 337 trin-releasing peptide receptor SSR4 QLLNL--VVT-----SSLD ATVNMVSLILSYANSCANPILYGFLSDNrRSFQRVLCLRCC 331 (GRPR, ref. 22), human somato- DOR1 VVWT--LVDINRRDPLVVAALE IALGYANSSLNPVLYAFLDZNFKRCF-RQLCRTPC 337 statin receptor 4 (SSR4, ref. 23), and mouse * ~~* * *..*-***** ***--* S-opioid receptor (DOR1, refs. 24 and 25). Putative TM I-VII regions, delineating three extracellular and three intra- GALR1 ------FKCH----IRKDS------LSDTDKNXSRIDT---PPSTNCTH- 348 cellular loops, are overlined. The GRPR ------LCCQPGLIIRSBSTG---RSTTCHTSLKSTNPSVATrSLINGNICEER 382 potential glycosylation sites ( I ), SSR4 LZGLGGAZ ZPLDYYATALISKGGAGO PPIPCQQKLQPEZPGRKRIPLTRTTT---- 387 the phosphorylation site (0) for DOR1 ------GRQZP-----GSLRRPRQATTRZRVTACTPSD-GPGGG------Ak---- 371 cAMP/cGMP protein kinase, the farnesylation site (#), and cysteine residues (O) presumed to form a disulfide bond are indicated GALR1 -V 349 in the GALR1 sequence. Identical GRPR YV 384 and conserved amino acids are SSR4 -F 388 denoted by asterisks and by dots, DOR1 -A 372 respectively. Downloaded by guest on September 28, 2021 9782 Neurobiology: Habert-Ortoli et al. Proc. NatL Acad. Sci. USA 91 (1994) * tity), the five somatostatin receptor subtypes (31%), and the ______6-opioid receptor (30%6), allowing us to define the GALR1 receptor as a member ofthe G-protein-coupled neuropeptide receptor family. Alignment of the amino acid sequences of these four receptors (Fig. 1) delineates four clusters of high homology (identity plus similarity) in the second, third, sixth, and seventh TM domains, a general feature of G-protein- coupled neuropeptide receptors (27), the most pronounced sequence divergences being at the N and C termini. Binding and Functional Properties of the GaIRi Clone. We then characterized the galanin binding properties of the GALR1 clone. Membranes prepared from COS cells transfected with the GALR1 clone exhibited a concentration- dependent galanin binding (Fig. 2). Scatchard analysis indicates the presence of a single class of high-affinity binding sites with an affinity constant (Kd) of0.8 ± 0.2 nM (n = 4) and a maximal number of binding sites (B..) of 8.6 ± 1.2 pmol/mg of protein (n = 4). Competition experiments were performed with galanin from different species and various galanin fragments (Fig. 3A). Human, rat, and porcine galanins were able to displace 125I-labeled galanin with inhibition constant (K) values in the nanomolar range (Ki = 0.8 6 + 0.1, 0.3 ± 0.1, and 0.2 ± 0.1 nM, respectively; n = 3). The rotein Ki values for the porcine galanin fragments galanin-(1-16) and galanin-(3-29) were 5 ± 2 nM and >>10 j.M, respectively (n = 3). Moreover, no displacement was observed with struc- turally unrelated peptides (tested at 10 ,uM) such as angio- 0 5 10 15 20 25 tensin, bombesin, bradykinin, neuropeptide Y, neurotensin, Galanin, nM somatostatin, substance P, and substance K. To demonstrate whether the encoded GALR1 receptor FIG. 2. Binding of porcine galanin to GALR1-*transfected COS could transduce a physiological signal, we measured the cell membranes. (Inset) Scatchard analysis of binding data. Results effect of galanin and galanin fragments on the accumulation shown are from one representative experiment. ofcAMP in transfected cells (Fig. 3B). While human galanin characteristics of members of the super familIy of G-protein- did not affect basal cAMP production, it reduced the forsko- coupled receptors. The encoded protein cc)ftains, several lin-stimulated increase of cAMP in a dose-dependent man- in protein-coupled ner, the half-maximal effective concentration (ECs0) being amino acid residues present numerous G-p;+ *tein *Aupled 0.9 ± 0.1 nM (n = 3). At a m active dose (10 receptors (27). These include a pair of cys.teine residues, human galanin inhibited by 50% the cellular cAMP produc-0&), presumed to form a disulfide bond betweein the first and tion. Galanin-(1-16) also reduced the forskolin-stimulated second extracellular loop, and proline residue:s, in the fourth, increased cAMP level (ECso = 6 + 2 nM, n = 3), whereas fifth, and seventh TM domains, which are thiought to intro- galanin-(3-29) had no effect. duce a bend in the a-helical structure ofthe T.M domains and The binding and functional characteristics of the cloned to participate in the formation ofthe binding pocket (27). One receptor are consistent with those reported for the native potential site for phosphorylation is located in the second galanin receptor identified in Bowes cells (18), thereby dem- cytoplasmic domain, and the N-terminal regicrn contains two onstrating that GALR1 encodes a galanin receptor. When consensus N-glycosylation sites. Finally, one cysteine may compared to the galanin receptors identified to date, the attach the C-terminal region ofthe receptor to the membrane pharmacological profile ofthe GaiR1 galanin receptor closely via a farnesyl anchor. Search for amino acid sequence matches the properties of those described in pancreas, in- identities showed the GALR1 receptor to be homologous to testine, and brain (4, 15). Indeed, in those organs, activation the bombesin/gastrin-releasing peptide recer)tor (34%6 iden- of the galanin receptor requires the intact N-terminal part of 100

FIG. 3. (A) Displacement of 125I-labeled Q. 80- galanin binding to transfected COS cell 8 membranes by human (solid circles) and rat (4-4 0 (open circles) galanin, galanin-(3-29) (solid (4 e 60- triangles), and galanin-(1-16) (open squares) edO (for the clarity of the figure, porcine galanin C.) is not shown). Binding assays were per- +.A formed and results are expressed as percent- Cd i 40- age ofthe control values that were measured 0 in the presence of 1251-labeled glanin alone. (B) Human galanin and galanin fragments 20- reduced cAMP accumulation in COS cells 2 transfected with GALR1. Transfected cells were incubated in the presence of 0.1 mM B forskolin alone (control value) and with the indicated peptides. InA andB, results shown are from representative experiments per- log[peptide] log[peptide] formed in triplicate. Downloaded by guest on September 28, 2021 Neurobiology: Habert-Ortoli et al. Proc. Natl. Acad. Sci. USA 91 (1994) 9783 1. Tatemoto, K., Rokaeus, A., Jornvall, H., McDonald, T. J. & Mutt, V. (1983) FEBS Lett. 164, 124-128. 2. Merchenthaler, I., Lopez, F. J. & Negro-Vilar, A. (1993) Prog. Neurobiol. 40, 711-769. 3. Hdkfelt, T., Bartfai, T., Jacobowitz, D. & Ottoson, D. (1991) in Galanin: A New Multifunctional Peptide in the Neuro- Endocrine System, Wenner-Gren International Symposium Series (Macmillan, Cambridge, U.K.), Vol. 58. 28 S-- 4. Laburthe, M., Kitabgi, P., Couvineau, A. & Amiranoff, B. (1993) Handb. Exp. Pharmacol. 106, 133-176. 5. Lagny-Pournuir, I., Amiranoff, B., Lorinet, A. M., Tatemoto, 18 S.-e K. & Laburthe, M. (1989) Endocrinology 124, 2635-2641. 6. Amiranoff, B., Lorinet, A. M. & Laburthe, M. (1989) J. Biol. Chem. 264, 20714-20717. 7. Chen, Y., Fournier, A., Couvineau, A., Laburthe, M. & Amiranoff, B. (1993) Proc. Natl. Acad. Sci. USA 90, 3845- 3849. 8. Cormont, M., Le Marchand-Brustel, Y., Van Obbergen, E., Spiegel, A. M. & Sharp, G. W. G. (1991) Diabetes 40, 1170- 1176. 9. Gillison, S. L. & Sharp, G. W. G. (1994) Diabetes 43, 24-32. FIG. 4. Northern blot hybridization analysis ofthe humangalanin 10. Amiranoff, B., Lorinet, A. M., Lagny-Pourmir, I. & Laburthe, receptor. Poly(A)+ RNA (5 pg) from HT29 cells, Bowes melanoma M. (1988) Eur. J. Biochem. 177, 147-152. cells, small and fetal brain (lanes was 11. Homaidan, F. R., Sharp, G. W. G. & Nowak, L. M. (1991) intestine, 1-4, respectively) Proc. Nat!. Acad. Sci. USA 88, 8744-8748. used. Positions of 18S and 28S rRNAs are shown as size markers. 12. De Weille, J. H., Schmid-Antomarchi, H., Fosset, M. & Laz- dunski, M. (1988) Proc. Natl. Acad. Sci. USA 85, 1312-1316. the ligand (13-15), a feature that is not an absolute prereq- 13. Lagny-Pourmir, I., Lorinet, A. M., Yanaihara, N. & Laburthe, uisite in other tissues such as the rat anterior pituitary (16) M. (1989) Peptides 10, 757-761. 14. Fisone, G., Berthold, M., Bedecs, K., Unden, A., Bartfai, T., and gastric smooth muscle (15). Bertorelli, R., Consolo, S., Crawley, J., Martin, B., Nilsson, S. Tissue Expression of Galanin Receptor mRNA. A Northern & Hdkfelt, T. (1989) Proc. Nat!. Acad. Sci. USA 86,9588-9591. blot analysis of poly(A)+ mRNA from human tissues probed 15. Rossowski, W. J., Zacharia, S., Jiang, N. Y., Mungan, Z., with the GALR1 clone was performed (Fig. 4). A dominant Mills, M., Ertan, A. & Coy, D. H. (1993) Eur. J. Pharmacol. 2.7-kb GALR1 transcript was strongly detected in the Bowes 240, 259-267. 16. Wynick, D., Smith, D., Ghatei, M., Akinsanya, K., Bhogal, R., cell line, whereas the 2.7-kb transcript was detected to a Purkiss, P., Byfield, P., Yanaihara, N. & Bloom, S. R. (1993) much lower extent in human small intestine and fetal brain. Proc. Natl. Acad. Sci. USA 90, 4231-4235. An additional minor hybridization signal was observed at 17. Bartfai, T., Langel, U., Bedecs, K., Andell, S., Land, T., about 1.6 kb in small intestine and fetal brain. No hybridiza- Gregersen, S., Ahren, B., Girotti, P., Consolo, S., Corwin, R., tion signal was observed in the HT29 cell line, derived from Crawley, J., Xu, X., Wiesenfeld-Hallin, Z. & Hdkfelt, T. (1993) a human colon adenocarcinoma and in human Proc. Natl. Acad. Sci. USA 90, 11287-11291. (Fig. 4), liver 18. Heuillet, E., Bouaiche, Z., Menager, J., Dugay, P., Munoz, N., (data not shown), both devoid ofgalanin receptors (4). Thus, Dubois, H., Amiranoff, B., Crespo, A., Lavayre, J., Blan- the heterogeneity of hybridizing species in various tissues chard, J. C. & Doble, A. (1994) Eur. J. Pharmacol., in press. may reflect either multiple genes or different alternative RNA 19. Seed, B. & Aruffo, A. (1987) Proc. Natl. Acad. Sci. USA 84, splicing forms. 3365-3369. In conclusion, we have cloned a cDNA encoding a human 20. Munson, P. J. & Rodbard, D. (1980) Anal. Biochem. 197, receptor. This should allow a better un- 220-239. galanin ultimately 21. Lin, H. Y., Kaji, E. H., Winkel, G. K., Ives, H. E. & Lodish, derstanding of the role played by the galaninergic system in H. F. (1991) Proc. Natl. Acad. Sci. USA 88, 3185-3189. human physiological functions. Finally, a search for galanin 22. Coijay, M. H., Dobrzanski, D. J., Way, J. M., Viallet, J., receptor subtypes, as well as identification of the cellular Shapira, H., Worland, P., Sausville, E. A. & Battey, J. F. effector systems coupled to the GALR1 receptor, should aid (1991) J. Biol. Chem. 266, 18771-18779. in the development of pharmacological agents potentially 23. Xu, Y., Song, J., Bruno, J. F. & Berelowitz, M. (1993) Bio- useful for the treatment of diseases the chem. Biophys. Res. Commun. 193, 648-652. affecting gastrointes- 24. Kieffer, B. L., Befort, K., Gaveriaux-Ruff, C. & Hirth, C. G. tinal and central nervous systems. (1992) Proc. Nat!. Acad. Sci. USA 89, 12048-12052. 25. Evans, C. J., Keith, D. E., Jr., Morrison, H., Magendzo, K. & We acknowledge T. Ciora and N. Gosselet for oligonucleotide Edwards, R. H. (1992) Science 258, 1952-1955. synthesis and DNA sequencing and H. Dubois for galanin-(3-29) 26. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132. synthesis. We are grateful to Drs. E. Heuillet and L. Pradier for 27. Burbach, J. P. H. & Meijer, 0. C. (1992) Eur. J. Pharmacol. discussions during the course of this project. 227, 1-17. Downloaded by guest on September 28, 2021