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• W1986608344 abstract "The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of125I-pGAL to transiently expressed GALR2 receptors was saturable (K D = 0.15 nm) and displaceable by galanin peptides and analogues in rank order: porcine galanin ≃ M32 ≃ M35 ≃ M40 ≥ galanin-(1–16) ≃ M15 ≃ [d-Trp2]galanin-(1–29) > C7 ≫ galanin-(3–29). This profile resembles that of the rat GALR1 receptor with the notable exception that [d-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C. The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of125I-pGAL to transiently expressed GALR2 receptors was saturable (K D = 0.15 nm) and displaceable by galanin peptides and analogues in rank order: porcine galanin ≃ M32 ≃ M35 ≃ M40 ≥ galanin-(1–16) ≃ M15 ≃ [d-Trp2]galanin-(1–29) > C7 ≫ galanin-(3–29). This profile resembles that of the rat GALR1 receptor with the notable exception that [d-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C. Galanin is a neuropeptide with a broad distribution in both central and peripheral nervous systems. Within the central nervous system, high levels of galanin are found in the hypothalamus, hippocampus, amygdala, basal forebrain, brainstem nuclei, and spinal cord (1Skofitsch G. Jacobowitz D.M. Peptides. 1985; 6: 509-546Crossref PubMed Scopus (604) Google Scholar, 2Bennet W.M. Hill S.F. Ghatei M.A. Bloom S.R. J. Endocrinol. 1991; 130: 463-467Crossref PubMed Scopus (20) Google Scholar, 3Merchenthaler I. López F.J. Negro-Vilar A. Prog. Neurobiol. 1993; 40: 711-769Crossref PubMed Scopus (332) Google Scholar). In accord with its distribution pattern, galanin has been found to participate in the regulation of feeding behavior, cognition, neuroendocrine regulation, and nociception (for reviews, see Refs. 4Bartfai T. Hokfelt T. Langel U. Crit. Rev. Neurobiol. 1993; 7: 229-274PubMed Google Scholarand 5Kask K. Langel U. Bartfai T. Cell. Mol. Neurobiol. 1995; 15: 653-673Crossref PubMed Scopus (89) Google Scholar). In the periphery, galanin is found in the gastrointestinal tract, adrenal medulla, dorsal root ganglia, and sympathetic neurons (6Bauer F.E. Adrian T.E. Christofides N.D. Ferri G.L. Yanaihara N. Polak J.M. Bloom S.R. Gastroenterology. 1986; 91: 877-883Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 7Villar M.J. Cortes R. Theodorsson E. Wiesenfeld-Hallin Z. Schalling M. Fahrenkrug J. Emson P.C. Hokfelt T. Neuroscience. 1989; 33: 587-604Crossref PubMed Scopus (431) Google Scholar, 8Elfvin L.G. Hokfelt T. Bartfai T. Bedecs K. Microsc. Res. Tech. 1994; 29: 131-142Crossref PubMed Scopus (17) Google Scholar). Galanin released from sympathetic nerve terminals in the pancreas is a potent regulator of insulin release in several species (9Ahrén B. Lindskog S. Int. J. Pancreatol. 1992; 11: 147-160PubMed Google Scholar, 10Boyle M.R. Verchere C.B. McKnight G. Mathews S. Walker K. Taborsky Jr., G.J. Regul. Pept. 1994; 50: 1-11Crossref PubMed Scopus (25) Google Scholar). High levels of galanin are also found in the pituitary where its mRNA levels may be up-regulated by ovarian steroids (11Vrontakis M.E. Peden L.M. Duckworth M.L. Friesen H.G. J. Biol. Chem. 1987; 262: 16755-16758Abstract Full Text PDF PubMed Google Scholar, 12Kaplan L.M. Gabriel S.M. Koenig J.I. Sunday M.E. Spindel E.R. Martin J.B. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7408-7412Crossref PubMed Scopus (260) Google Scholar). The observation that galanin is also up-regulated in a variety of conditions of neuronal injury has led to the suggestion that galanin may play a role in the pathophysiology of chronic pain and Alzheimer's disease (13Chan-Palay V. Adv. Neurol. 1990; 51: 253-255PubMed Google Scholar, 14Wiesenfeld-Hallin Z. Xu X.J. Langel U. Bedecs K. Hokfelt T. Bartfai T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3334-3337Crossref PubMed Scopus (200) Google Scholar). Multiple receptor subtypes are proposed to underlie the physiological effects of galanin (15Bartfai T. Bedecs K. Land T. Langel Ü. Bertorelli R. Girotti P. Consolo S. Yu Y.-J. Weisenfeld-Hallin Z. Nilsson S. Pieribone V. Hökfelt T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10961-10965Crossref PubMed Scopus (162) Google Scholar, 16Hedlund P.B. Yanaihara N. Fuxe K. Eur. J. Pharmacol. 1992; 224: 203-205Crossref PubMed Scopus (81) Google Scholar, 17Wynick D. Smith D.M. Ghatei M. Akinsanya K. Bhogal R. Purkiss P. Byfield P. Yanaihara N. Bloom S.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4231-4245Crossref PubMed Scopus (123) Google Scholar, 18Gu Z.-F. Pradhan T.K. Coy D.H. Jensen R.T. J. Pharmacol. Exp. Ther. 1995; 272: 371-378PubMed Google Scholar), but until recently only one cloned galanin receptor subtype (GALR1) had been described (19Habert-Ortoli E. Amiranoff B. Loquet I. Laburthe M. Mayaux J.-F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9780-9783Crossref PubMed Scopus (325) Google Scholar, 20Parker E.M. Izzarelli D. Nowak H. Mahle C. Iben L. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar, 21Gustafson E.L. Smith K.E. Durkin M.M. Gerald C. Branchek T.A. Neuroreport. 1996; 7: 953-957Crossref PubMed Scopus (128) Google Scholar). We report here the isolation by expression cloning and the pharmacological characterization of a novel galanin receptor from a rat hypothalamic cDNA library. The new galanin receptor subtype, termed GALR2, has the same predicted amino acid sequence as the galanin receptor cDNA recently described by Howard et al. (22Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weinberg D.H. Feighner S.D. Cascierie M.A. Smith R.G. Van Der Ploeg L.H.T. Sullivan K.A. FEBS Lett. 1997; 405: 285-290Crossref PubMed Scopus (192) Google Scholar) which was reported to have a pharmacological profile similar to the GALR1 receptor. We provide evidence here that GALR2 is distinct from GALR1 in both its pharmacological profile and its primary signal transduction pathway. A plasmid cDNA library was constructed as described previously (23Gerald C. Walker M.W. Vaysse P.J-J. He C. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1995; 270: 26758-26761Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) from 5 μg of rat hypothalamus poly(A)+ RNA. A total of 2.6 × 106 independent clones with a mean insert size of 2.2 kb 1The abbreviations used are: kb, kilobase(s);125I-pGAL, 125I-porcine galanin; DMEM, Dulbecco's modified Eagle's medium; CHO, Chinese hamster ovary; GPCR, G protein-coupled receptor; TM, transmembrane domain; Gpp(NH)p, guanosine 5'-(β,γ-imido)triphosphate; IP3, inositol 1,4,5-trisphosphate. were generated. The library was plated on agar plates (ampicillin selection) in 584 primary pools of ∼4,500 independent clones. Preparation and transfection of library pool DNA were carried out as described (23Gerald C. Walker M.W. Vaysse P.J-J. He C. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1995; 270: 26758-26761Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Forty-eight h after transfection, the galanin binding assay was carried out with 1 nm125I-porcine galanin (125I-pGAL; specific activity ∼2200 Ci/mmol; NEN Life Science Products) in 20 mm HEPES-NaOH, pH 7.4, containing 1.26 mmCaCl2, 0.81 mm MgSO4, 0.44 mm KH2PO4, 5.4 mm KCl, 10 mm NaCl, 0.1% bovine serum albumin, and 0.1% bacitracin for 1 h at room temperature. Slides were examined for galanin binding as described previously (23Gerald C. Walker M.W. Vaysse P.J-J. He C. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1995; 270: 26758-26761Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Pools with positive cells were subdivided and rescreened until a single cDNA was isolated. Sequencing was carried out on both strands using Sequenase (U. S. Biochemical Corp., Cleveland, OH) according to the manufacturer protocols. Nucleotide and peptide sequence analyses were performed using the Wisconsin Package (GCG, Genetics Computer Group, Madison, WI). A Northern blot (Rat MTN Blot,CLONTECH) containing mRNA purified from various rat tissues was hybridized at high stringency according to the manufacturer specifications using a probe representing the full coding region of GALR2. The probe was labeled with 32P by random priming to a specific activity greater than 109 dpm/μg. The Northern blot was apposed to Kodak Biomax film with one Biomax intensifying screen for 4 days at −80 °C. The genomic Southern blot (Rat Genoblot, CLONTECH) was hybridized at high stringency using the same GALR2 probe and apposed to film for 6 days without intensifying screens. COS-7 cells were grown on 150-mm plates in DMEM supplemented with 10% bovine calf serum, 4 mm glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin) at 37 °C, 5% CO2. Stock plates of COS-7 cells were trypsinized and split 1:6 every 3–4 days. Expression vectors containing GALR2 and GALR1 receptor cDNAs were transiently transfected into COS-7 cells by the DEAE-dextran method (24Warden D. Thorne H.V. J. Gen. Virol. 1968; 3: 371Crossref PubMed Scopus (35) Google Scholar) using 1 μg of DNA/106 cells. To create a cell line stably expressing the GALR2 receptor, an expression vector containing the GALR2 receptor cDNA was co-transfected with a G-418 resistance gene into CHO cells using the calcium phosphate transfection method (25Cullen B. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar). Transfected CHO cells were selected with G-418 and screened as membrane preparations for specific binding of 125I-pGAL. Membranes from transiently transfected COS-7 cells were prepared as described (26Wetzel J.M. Salon J.A. Tamm J.A. Forray C. Craig D. Nakanishi H. Cui W. Vaysse P.J.-J. Chiu G. Weinshank R.L. Hartig P.R. Branchek T.A. Gluchowski C. Recept. Chan. 1996; 4: 165-177PubMed Google Scholar) and suspended in binding buffer (50 mm Tris-HCl, 5 mmMgSO4, 1 mm EDTA, pH 7.5, supplemented with 0.1% bovine serum albumin, 2 μg/ml aprotinin, 0.5 mg/ml leupeptin, and 10 μg/ml phosphoramidon). In equilibrium saturation binding assays, membrane preparations were incubated with increasing concentrations (0.1–4 nm) of 125I-pGAL in a final volume of 0.25 ml. Nonspecific binding was determined with 10 μm unlabeled porcine galanin. The binding affinities of galanin analogs were determined in equilibrium competition binding assays using 0.3 nm125I-pGAL in the presence of 12 different concentrations of the displacing ligands. Binding reaction mixtures were incubated at 30 °C for 40 min, and the reaction was stopped by filtration through GF/B filters treated with 0.5% polyethyleneimine using a cell harvester. Radioactivity was measured by scintillation counting and data were analyzed using a computerized non-linear regression program (Prism, GraphPAD Software for Sci.). Protein concentration was measured by the method of Bradford (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216412) Google Scholar) with the Bio-Rad reagent using bovine serum albumin as a standard. Changes in phosphatidylinositol turnover were measured in intact cells as described previously (26Wetzel J.M. Salon J.A. Tamm J.A. Forray C. Craig D. Nakanishi H. Cui W. Vaysse P.J.-J. Chiu G. Weinshank R.L. Hartig P.R. Branchek T.A. Gluchowski C. Recept. Chan. 1996; 4: 165-177PubMed Google Scholar). Briefly, intact CHO cells were incubated for 18 h in DMEM supplemented with 1% fetal bovine serum and 5 μCi/ml [3H]d-myo-inositol (18.3 Ci/mmol). Prelabeled cells were incubated with porcine galanin or analogs in phosphate-buffered saline containing 10 mm LiCl at 37 °C in 5% CO2 for 60 min. The [3H]inositol phosphates produced by the cells were isolated by ion-exchange chromatography on Dowex 1-X8 (200–400 mesh). EC50 and maximum response values from concentration-response curves were estimated by fitting the data to a four-parameter logistic model using a computerized non-linear regression analysis program (Prism, GraphPAD Software for Sci.). To measure intracellular calcium levels, CHO cells stably expressing the GALR2 receptor were seeded into 35-mm plates with glass cover slip inserts. Intracellular free [Ca+2] was measured by microspectrofluorometry using Fura-2/AM as described previously (23Gerald C. Walker M.W. Vaysse P.J-J. He C. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1995; 270: 26758-26761Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Fluorescence measurements were converted to [Ca+2] using a standard curve and reported as mean ± S.E. Female Xenopus laevis (Xenopus-1, Ann Arbor, MI) were anesthetized in 0.2% 3-aminobenzoic acid ethyl ester, and a portion of ovary was removed using aseptic technique (28Quick M.W. Lester H.A. Methods Neurosci. 1994; 19: 261-279Crossref Scopus (100) Google Scholar). Oocytes were defolliculated using 3 mg/ml collagenase (Worthington Biochemical Corp., Freehold, NJ) in a solution containing 87.5 mm NaCl, 2 mm KCl, 2 mm MgCl2, and 5 mm HEPES, pH 7.5. Oocytes were injected (Nanoject, Drummond Scientific, Broomall, PA) with 50 nl of rat GALR2 mRNA or other mRNA for use as a negative control. RNA was prepared by linearization of a plasmid containing the entire coding region of the GALR2 cDNA, followed byin vitro transcription using T7 polymerase (MessageMachine, Ambion, Austin, TX). Oocytes were incubated at 17 °C on a rotating platform for 3–8 days postinjection. Dual electrode voltage clamp (GeneClamp, Axon Instruments Inc., Foster City, CA) was performed using 3 m KCl-filled glass microelectrodes having resistances of 1–3 megohms. Unless otherwise specified, oocytes were clamped at a holding potential of −80 mV. During recordings, oocytes were bathed in continuously flowing (2–5 ml/min) medium containing 96 mmNaCl, 2 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 5 mm HEPES, pH 7.5 (ND96). Drugs were applied by switching from a series of gravity-fed perfusion lines. To clone additional members of the galanin receptor family, we used an expression cloning strategy based on the hypothesis that there were multiple galanin receptors present in hypothalamus (17Wynick D. Smith D.M. Ghatei M. Akinsanya K. Bhogal R. Purkiss P. Byfield P. Yanaihara N. Bloom S.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4231-4245Crossref PubMed Scopus (123) Google Scholar, 29Bartfai T. Langel Ü. Bedecs K. Andell S. Land T. Gregersen S. Ahren B. Girotti P. Consolo S. Corwin R. Crawley J. Xu X. Weisenfeld-Hallin Z. Hökfelt T. Proc. Natl. Acad. Sci. U. S. A. 1993; 88: 11287-11291Crossref Scopus (127) Google Scholar). A randomly primed cDNA expression library was constructed from rat hypothalamus and screened in pools by radioligand binding/photoemulsion detection using 125I-pGAL. Slides were inspected for positive cells by direct microscopic examination. One positive pool, J126, was subdivided and rescreened until an individual cDNA clone was isolated (K985) that conferred galanin binding. The isolated GALR2 plasmid contained a 3.8-kb insert. Sequence analysis of this cDNA revealed a complete coding region for a receptor protein similar to G protein-coupled receptors (GPCRs). Searches of GenBankTM/EBI Data Banks indicated that the sequence was unique and that the most similar sequence was that of the galanin receptor GALR1. The nucleotide sequence of the coding region was 56% identical to rat GALR1 and encoded a 372 amino acid protein with 38% amino acid identity to rat GALR1. The deduced amino acid sequence of GALR2 compared with GALR1 is shown in Fig.1. Hydropathy plots of the predicted amino acid sequence reveal seven hydrophobic regions that may represent transmembrane domains (TMs; data not shown), typical of the G protein-coupled receptor superfamily. In the putative TM domains, GALR2 exhibits 48–49% amino acid identity with rat and human GALR1. Like most GPCRs, the GALR2 receptor contains consensus sequences forN-linked glycosylation in the N terminus (positions 2 and 11) and in the predicted extracellular loop between TMs IV and V. The GALR2 receptor also contains two highly conserved cysteine residues in the first two extracellular loops that are believed to form a disulfide bond stabilizing the functional protein structure (30Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (684) Google Scholar). The coding region of the GALR2 cDNA was interrupted by an intron of ∼1 kb; splicing of the intron recreated an open reading frame within a highly conserved region of the GPCR family, at the end of TMIII (LD(R/Y); see Fig. 1). The presence of an intron within the cDNA isolated from rat hypothalamus indicates that the mRNA from which the cDNA was reverse transcribed was incompletely spliced. It is of interest to note that several GPCRs have previously been reported to contain introns at this location, including the human dopamine D3, D4, and D5 receptors, the rat substance P receptor, and the human substance K receptor (30Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (684) Google Scholar). In particular, the rat 5-HT7 receptor (31Shen Y. Monsma Jr., F.J. Metcalf M.A. Jose P.A. Hamblin M.W. Sibley D.R. J. Biol. Chem. 1993; 268: 18200-18204Abstract Full Text PDF PubMed Google Scholar,32Bard J.A. Zgombick J. Adham N. Vaysse P. Branchek T.A. Weinshank R.L. J. Biol. Chem. 1993; 268: 23422-23426Abstract Full Text PDF PubMed Google Scholar) contains an intron in the same location we now report for GALR2, within the A(G/G) codon for the highly conserved amino acid arginine at the end of TMIII. Recently, the cloning of a galanin receptor with the same predicted amino acid sequence was described by Howard et al. (22Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weinberg D.H. Feighner S.D. Cascierie M.A. Smith R.G. Van Der Ploeg L.H.T. Sullivan K.A. FEBS Lett. 1997; 405: 285-290Crossref PubMed Scopus (192) Google Scholar), who isolated both intron-containing and correctly spliced cDNAs. Interestingly, the nucleotide sequence of the 490-base pair intron reported by Howard et al. (22Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weinberg D.H. Feighner S.D. Cascierie M.A. Smith R.G. Van Der Ploeg L.H.T. Sullivan K.A. FEBS Lett. 1997; 405: 285-290Crossref PubMed Scopus (192) Google Scholar) is contained entirely within the ∼1-kb intron described here, suggesting that the intron itself can be alternatively or incompletely spliced. Northern blot analysis revealed a transcript of ∼1.8–2.0 kb after a 4-day exposure of the autoradiogram (Fig.2 A). The GALR2 transcript is widely but unevenly distributed: GALR2 mRNA was observed in brain, lung, heart, spleen, and kidney, with lighter bands in skeletal muscle, liver, and testis (Fig. 2 A). By reprobing the blot with 1B15 (Fig. 2 A) (33Danielson P.E. Forss-Petter S. Brow M.A. Calavetta L. Douglass J. Milner R.J. Sutcliffe J.G. DNA ( N. Y. ). 1988; 7: 261-267Crossref PubMed Scopus (1034) Google Scholar) and β-actin (not shown), we confirmed that similar amounts of RNA were present in each sample. GALR2 transcripts containing the ∼1-kb intron in the coding region are undetectable by Northern blot but can be amplified by reverse transcribed-polymerase chain reaction (data not shown), suggesting that the proportion of unspliced mRNA in native tissues is low. Conversely, the intronless form of GALR2 is the predominant species amplified by reverse transcribed-polymerase chain reaction. The mismatch between the size of the GALR2 transcript (∼1.8–2.0 kb) and the isolated GALR2 cDNA (∼3.8 kb) suggests that the 5′-untranslated sequence in the cDNA may contain another intron. Hybridization of a rat genomic Southern blot at high stringency revealed band patterns consistent with the presence of a single copy of the GALR2 gene (Fig. 2 B). Multiple bands observed in BamHI and PstI lanes are predicted by the presence of these restriction enzyme sites within the ∼1-kb intron described above. The pharmacology of GALR2 was studied in COS-7 cells transiently transfected with the plasmid containing the GALR2 cDNA (K985). Membrane preparations of COS-7 cells transfected with K985 displayed specific and saturable binding to 125I-pGAL with aK d of 150 pm and aB max of 250 fmol/mg protein. Peptide analogs of galanin were used to generate a comparative pharmacological profile for GALR2 versus GALR1 receptors. As shown in TableI, both GALR1 and GALR2 receptors bound the full-length peptide porcine galanin-(1–29) with high affinity, as well as the truncated analogs galanin-(1–16) and galanin-(1–15). Both receptor subtypes also displayed high affinity for the putative galanin antagonists M15, M35, M40, and C7 (15Bartfai T. Bedecs K. Land T. Langel Ü. Bertorelli R. Girotti P. Consolo S. Yu Y.-J. Weisenfeld-Hallin Z. Nilsson S. Pieribone V. Hökfelt T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10961-10965Crossref PubMed Scopus (162) Google Scholar, 29Bartfai T. Langel Ü. Bedecs K. Andell S. Land T. Gregersen S. Ahren B. Girotti P. Consolo S. Corwin R. Crawley J. Xu X. Weisenfeld-Hallin Z. Hökfelt T. Proc. Natl. Acad. Sci. U. S. A. 1993; 88: 11287-11291Crossref Scopus (127) Google Scholar, 34Langel U. Land T. Bartfai T. Int. J. Pept. Protein Res. 1992; 39: 516-522Crossref PubMed Scopus (96) Google Scholar, 35Gregersen S. Lindskog S. Land T. Langel U. Bartfai T. Ahren B. Eur. J. Pharmacol. 1993; 232: 35-39Crossref PubMed Scopus (29) Google Scholar), all of which share a common galanin-(1–13) N terminus. Furthermore, binding affinity was lost for either receptor subtype when the first two N-terminal residues were deleted, as in the case of galanin-(3–29). A common feature of GALR1 and GALR2 therefore appears to be a preferential interaction with the N-terminal residues of the parent galanin peptide. While there were many similarities in the pharmacology profiles thus derived, GALR2 was readily distinguished from GALR1 by its high affinity for the modified peptide [d-Trp2]galanin-(1–29) (see TableI), demonstrating a greater tolerance of GALR2 for modifications in position two of galanin. [d-Trp2]galanin-(1–29) thus represents the first known peptide selective for GALR2 versus GALR1.Table IPharmacological profile of the cloned rat GALR2 receptor and comparison with the cloned rat GALR1 receptorPeptiderGALR1rGALR2K iK iEC50nmnmPorcine galanin-(1–29)0.5 ± 0.10.5 ± 0.118 ± 3Galanin-(1–15)3 ± 0.30.7 ± 0.0865 ± 9Galanin-(1–16)2 ± 0.052 ± 0.594 ± 12Porcine galanin-(3–29)>1,000>1,000>1,0001-an = 1.Galantide (M15)10 ± 22 ± 0.33001-an = 1.[D-Trp2]galanin-(1–29)>1,0007 ± 2150 ± 361-bn = 2.C-716 ± 0.419 ± 43 ± 2M320.6 ± 0.10.6 ± 0.0613 ± 4M350.3 ± 0.050.6 ± 0.129 ± 6M404 ± 0.70.7 ± 0.328 ± 8Binding data were determined from competitive displacement of125I-pGAL binding to membranes from COS-7 cells transiently transfected with the rat GALR2 or rat GALR1 receptor cDNA. Data are shown as the mean K i ± S.E. (n ≥ 3). Receptor-mediated activation of phosphatidylinositol turnover was measured in CHO cells stably expressing the rat GALR2 receptor. All the peptides tested elicited maximal responses within 10% of those measured for porcine galanin. Data are shown as EC50 mean ± S.E. (n ≥ 3 unless stated otherwise).1-a n = 1.1-b n = 2. Open table in a new tab Binding data were determined from competitive displacement of125I-pGAL binding to membranes from COS-7 cells transiently transfected with the rat GALR2 or rat GALR1 receptor cDNA. Data are shown as the mean K i ± S.E. (n ≥ 3). Receptor-mediated activation of phosphatidylinositol turnover was measured in CHO cells stably expressing the rat GALR2 receptor. All the peptides tested elicited maximal responses within 10% of those measured for porcine galanin. Data are shown as EC50 mean ± S.E. (n ≥ 3 unless stated otherwise). To assess the functional coupling of GALR2, we examined the second-messenger pathways linked to receptor activation. In CHO cells stably transfected with the GALR2 receptor cDNA, porcine galanin-(1–29) stimulated the formation of [3H]inositol phosphates by 8-fold with an EC50 of 18 ± 3 nm (Table I). Galanin-(1–15), galanin-(1–16), and the putative antagonist peptides C7, M32, M35, and M40 were also found to be full agonists in the stimulation of phosphoinositide hydrolysis in CHO cells expressing the GALR2 receptor, as was [D-Trp2]galanin-(1–29); these chimeric peptides also behaved as agonists at the cloned rat GALR1 receptor (36Forray C. Smith K. Gerald C. Vaysse P. Weinshank R. Branchek T. Soc. Neurosci. Abstr. 1996; 22: 1304Google Scholar). Comparison of K i and EC50 values for a given peptide (see Table I) suggests that 125I-pGAL binds selectively to a high affinity conformation of the GALR2 receptor in membrane preparations under these experimental conditions in which the concentration of 125I-pGAL concentration is low (0.3 nm). Consistent with this proposal, specific binding of125I-pGAL to the GALR2 receptor was significantly reduced by 100 μm Gpp(NH)p (data not shown). Conversely, the low affinity conformation of the GALR2 receptor expressed in intact cells may be the primary route by which galanin induces a functional response. Heterologous expression of GPCRs in Xenopus oocytes has been widely used to determine the identity of signaling pathways activated by receptor agonists (37Gundersen C.B. Miledi R. Parker I. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1983; 219: 103-109Crossref PubMed Scopus (71) Google Scholar, 38Takahashi T. Neher E. Sakmann B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5063-5067Crossref PubMed Scopus (138) Google Scholar). We used the Xenopus oocyte system to further explore the functional coupling of GALR2 receptors. Application of porcine galanin (100–1000 nm) activated rapid inward currents in 36 of 46 oocytes injected with GALR2 mRNA (Fig. 3 A). Oocytes injected with buffer (ND96) alone did not exhibit detectable (<5 nA) responses to galanin (0 of 19 responded) nor did oocytes injected with GALR1 (0 of 20 responded). Current magnitudes in GALR2 mRNA-injected oocytes ranged from small fluctuations of less than 50 nA (excluded from analysis) to large, rapid currents (up to 3 μA) resembling those evoked by stimulation of other receptors (α1A receptors; data not shown) that are known to couple to IP3 release and activation of Cl− current from the resulting increase in intracellular free Ca2+ (38Takahashi T. Neher E. Sakmann B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5063-5067Crossref PubMed Scopus (138) Google Scholar). The currents stimulated by galanin in oocytes expressing GALR2 were most likely mediated by the endogenous calcium-activated Cl− channel (37Gundersen C.B. Miledi R. Parker I. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1983; 219: 103-109Crossref PubMed Scopus (71) Google Scholar) because they were blocked in oocytes injected with 50 nl of 10 mm EGTA (5 of 5 responded), and they displayed a current-voltage relation that exhibited outward rectification and a reversal potential of approximately −15 mV (data not shown). Consistent with these data, intracellular free [Ca+2] was also increased by 1 μm rat galanin in CHO cells stably transfected with GALR2 (Δ [Ca+2] = 310 ± 70 nm,n = 9) but not in untransfected cells (Fig.3 B). Increases in phosphatidylinositol release, chloride conductance, and calcium mobilization are all consistent with a primary signaling pathway for GALR2 receptors involving activation of phospholipase C via a Gq/G11-type mechanism. In this regard, the signal transduction pathway elucidated for the GALR2 receptor in this report differs from that described for the cloned GALR1 receptors, which couple to the inhibition of adenylate cyclase through members of the Gi family of G-proteins (19Habert-Ortoli E. Amiranoff B. Loquet I. Laburthe M. Mayaux J.-F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9780-9783Crossref PubMed Scopus (325) Google Scholar, 20Parker E.M. Izzarelli D. Nowak H. Mahle C. Iben L. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar). To further evaluate this finding, we compared galanin-stimulated functional responses mediated by GALR1 and GALR2 in stably transfected CHO cells and the relative sensitivities of these responses to pertussis toxin (Fig.4). Galanin acting via GALR1 caused a reduction in cAMP levels in forskolin-treated cells but did not activate inositol phospholipid turnover. In contrast, galanin acting via GALR2 stimulated phosphatidylinositol turnover but did not decrease cAMP levels. Moreover, the phosphoinositide response in stably transfected CHO cells was insensitive to pertussis toxin under conditions in which the inhibition of adenylate cyclase was significantly reduced, supporting the conclusion that GALR2 is coupled to its effectors through a pathway independent of Gi/Go. Thus our functional data serve to distinguish GALR2 from GALR1 and also from the galanin receptors expressed in GH3 cells reported to couple to the inactivation of calcium channels (39Kalkbrenner F. Degtiar V.E. Schenker M. Brendel S. Zobel A. Heschler J. Wittig B. Schultz G. EMBO J. 1995; 14: 4728-4737Crossref PubMed Scopus (69) Google Scholar). Our data are also in contrast with a preliminary report in which a galanin receptor with the same amino acid sequence was reported to mediate inhibition of adenylate cyclase in HEK-293 cells (40Ahmad S. Shen S.H. Walker P. Wahlestedt C. Abstracts 8th World Congress on Pain. IASP Press, Vancouver, BC, Canada1996: 134Google Scholar); we studied cAMP regulation in 293 cells stably transfected with rat GALR2 (estimatedB max ∼0.15 pmol/mg protein) and found no evidence for galanin-dependent inhibition of cAMP levels. Previous reports of galanin-stimulated phosphoinositide turnover were limited to studies in small cell lung carcinoma cell lines (41Sethi T. Rozengurt E. Cancer Res. 1991; 51: 1674-1679PubMed Google Scholar) and mudpuppy heart (42Hardwick J.C. Parsons R.L. J. Auton. Nerv. Syst. 1992; 40: 87-90Abstract Full Text PDF PubMed Scopus (7) Google Scholar); recently, galanin-stimulated calcium mobilization was observed in RINm5F cells (43Ahren B. Biochem. Biophys. Res. Commun. 1996; 221: 89-94Crossref PubMed Scopus (7) Google Scholar). Within mammalian brain, however, galanin activation of phospholipase C has not been observed, while Gi/Go mechanisms are commonly reported (for review, see Ref. 5Kask K. Langel U. Bartfai T. Cell. Mol. Neurobiol. 1995; 15: 653-673Crossref PubMed Scopus (89) Google Scholar). Previous studies using [d-Trp2] analogues of galanin also failed to predict a [d-Trp2]-preferring receptor subtype within the brain. The existence of GALR2 was therefore not predicted by existing pharmacological tools. Within the family of neuropeptide receptors, the existence of two receptor subtypes (GALR1 and GALR2) with distinct primary signaling mechanisms is exceptional: the NPY, opioid, CCK, bradykinin, tachykinin, and somatostatin receptor families, as examples, all exhibit uniform coupling patterns among their respective subtypes. 2Trends in Pharmacological ScienceNomenclature Supplement (1996).In fact, GALR2 is the only receptor in the related opioid/somatostatin/galanin receptor group that does not readily signal through a Gi-type mechanism. In conclusion, we have isolated a cDNA clone encoding a galanin receptor from rat hypothalamus, termed GALR2, which exhibits pharmacological properties and a signaling pathway distinct from GALR1 receptors. The availability of the recombinant GALR2 receptor subtype should enable the design and development of selective agonists and antagonists that could be used as tools to study the physiological processes mediated by galanin receptors. We thank Steven Fried, Sherif Daouti, Michelle Smith, Nancy Shen, Yelena Shifman, Harvey Lichtblau, Raisa Nagorny, Zoya Shaposhnik, Rong Zhou, Melissa Yao, Tracy Taylor, Debbie Tambe, Janet Lee, and Tracy Johnson-Blake for superb technical assistance. We also thank Dr. Pierre Vaysse for cell biology support and George Moralishvilli, Adam Zolensky, and Ernest Lilley for preparing the illustrations." @default.
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• W1986608344 title "Expression Cloning of a Rat Hypothalamic Galanin Receptor Coupled to Phosphoinositide Turnover" @default.
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