Cloning and Expressional Characterization of a Novel Galanin Receptor
1997; Elsevier BV; Volume: 272; Issue: 51 Linguagem: Inglês
10.1074/jbc.272.51.31949
ISSN1083-351X
AutoresSuke Wang, Chaogang He, Tanaz Hashemi, Marvin Bayne,
Tópico(s)Cardiovascular, Neuropeptides, and Oxidative Stress Research
ResumoGalanin, a 29–30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors.125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (K d = 0.6 nm) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin. Galanin, a 29–30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors.125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (K d = 0.6 nm) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin. Galanin is a 29–30 amino acid neuropeptide with no significant homology to any known family of biologically active peptides (1Tatemoto K. Rokaeus A. Jornwall H. McDonald T.J. Mutt V. FEBS Lett. 1983; 164: 124-128Crossref PubMed Scopus (1345) Google Scholar). Galanin is widely distributed in the central and peripheral nervous systems and is highly expressed in various regions of the brain. Many physiological processes are modulated by galanin, including neurotransmitter and hormone release (2Dunning B.E. Ahren B. Veith R.C. Bottcher G. Sundler F. Taborsky Jr., G.J. Am. J. Physiol. 1986; 251: E127-E133PubMed Google Scholar), spinal reflexes, nociception (3Verge V.M.K. Xu X.-J. Langel U. Hokfelt T. Wiesenfeld Z. Bartfai T. Neurosci. Lett. 1993; 149: 193-197Crossref PubMed Scopus (76) Google Scholar), firing of noradrenergic neurons, and contraction of gastrointestinal smooth muscle (4Ekblad E. hakanson R. Sundler F. Wahlestedt C. Br. J. Pharmacol. 1985; 86: 241-246Crossref PubMed Scopus (245) Google Scholar, 5Crawley J.N. Regul. Pept. 1995; 59: 1-16Crossref PubMed Scopus (134) Google Scholar). Like neuropeptide Y, centrally administered galanin potently stimulates food intake in animals (6Crawley J.N. Austin M.C. Fiski S.M. Martin B. Consolo S. Berthold M. Langel U. Fisone G. Bartfai T. J. Neurosci. 1990; 10: 3695-3700Crossref PubMed Google Scholar), suggesting a role for galanin in control of body weight.A large body of evidence suggests that galanin mediates the various physiological functions through interaction with distinct receptor subtypes. Pharmacological studies with several peptidic agonists and antagonists of galanin receptor suggest the existence of multiple receptor subtypes (7Rossowski W.J. Rossowski T.M. Zacharia S. Ertan A. Coy H.H. Peptides. 1990; 11: 333-338Crossref PubMed Scopus (61) Google Scholar, 8Fisone G. Berthold M. Bedecs K. Unden A. Bartfai T. Bertorelli R. Consolo S. Crawley J. Martin B. Nilsson S. Hokfelt T. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9589-9591Crossref Scopus (121) Google Scholar, 9Wynick D. Smith D. Ghatei M. Akinsanya K. Bhogal R. Purkiss P. Yanaihara N. Bloom E.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4231-4235Crossref PubMed Scopus (122) Google Scholar). The first of these receptors (GalR1) has been cloned from several species (10Habert-Ortoli E. Amiranoff B. Loquet I. Laburthe M. Mayaux J.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9780-9783Crossref PubMed Scopus (323) Google Scholar, 11Burgevin C.M. Loquet I. Quarteronet D. Habert-Ortoli E. J. Mol. Neurosci. 1995; 6: 33-41Crossref PubMed Scopus (170) Google Scholar, 12Parker E.M. Izzarelli D.G. Nowak H.P. Mahle C.D. Iben L.G. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar, 13Wang S. He C. Maguire M. Clemmons A. Burrier R. Guzzi M. Strader C. Parker E. Bayne M. FEBS Lett. 1997; 411: 225-230Crossref PubMed Scopus (67) Google Scholar). More recently, a second galanin receptor subtype (GalR2) was cloned. GalR2 is markedly dissimilar to the GalR1 receptor, sharing only 40% sequence homology (14Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weiger D.H. Feighner S.D. Cascieri 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, 15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar). Hydropathy analysis suggests that both GalR1 and GalR2 receptors have seven hydrophobic transmembrane domains, typical of members of the G protein-coupled receptor superfamily (16Probst W.C. Snyder L.A. Schuster D.I. Brosius J. Sealfon S.C. DNA Cell Biol. 1992; 11: 1-20Crossref PubMed Scopus (677) Google Scholar). The GalR2 receptor is distinguished pharmacologically from the GalR1 receptor by its high affinity for ligand galanin-(2–29). In addition to the pharmacological differences, the GalR2 transcript is widely distributed in both central and peripheral tissues (14Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weiger D.H. Feighner S.D. Cascieri 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, 15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar), whereas the expression of GalR1 is more restricted to brain and spinal cord (12Parker E.M. Izzarelli D.G. Nowak H.P. Mahle C.D. Iben L.G. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar, 13Wang S. He C. Maguire M. Clemmons A. Burrier R. Guzzi M. Strader C. Parker E. Bayne M. FEBS Lett. 1997; 411: 225-230Crossref PubMed Scopus (67) Google Scholar).Given that a large number of physiological actions are modulated by galanin, it is unlikely that the two cloned galanin receptors mediate all the functions of galanin. A complete understanding of the roles of galanin requires identification and characterization of all the galanin receptor subtypes. In this report, we describe the cloning of a novel rat galanin receptor subtype by polymerase chain reaction (PCR), 1The abbreviations used are: PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; GalR, galanin receptor; RACE, rapid amplification of cDNA ends; DAGO, [d-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin; GMAP, galanin message-associated peptide; kb, kilobase pair(s). 1The abbreviations used are: PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; GalR, galanin receptor; RACE, rapid amplification of cDNA ends; DAGO, [d-Ala2,N-methyl-Phe4,Gly5-ol]enkephalin; GMAP, galanin message-associated peptide; kb, kilobase pair(s). sib selection and rapid amplification of cDNA ends (RACE). The receptor shares highest homology with GalR1 and GalR2 and is capable of binding galanin and galanin analogs. We thus designate this new receptor as the GalR3 galanin receptor.MATERIALS AND METHODS125I-Porcine galanin (2200 Ci/mmol) and [α-32P]dATP (5000 Ci/mmol) were purchased from NEN Life Science Products. LipofectAMINE transfection agent and oligonucleotides used in this study were purchased or custom-synthesized by Life Technologies, Inc. and the sequences of the primers are: oligo93C, gctggcagtgctcctgcagcctggc; oligo120B, aagcggccgtaccagaagcacaggatg; oligo164, ccaagtgcctggcaggagccaagcag; oligo167, gcgggttaaggcangagttggcgtaggc; oligo172, tgcgggccccagcagagcgcgtagag, oligo177, catccagtgtgtagatggctgcct; oligo184, cgcgtagagcgcggccactgccagcatg; and oligo185, caagggctgaatcaanaagctccagc. Rat multiple tissue Northern blots were obtained from CLONTECH. Rat galanin, rat galanin-(1–16), M40, C7, preprogalanin-(1–30), and galantide (M15) were purchased from Peninsula Laboratories (Belmont, CA). Rat galanin-(2–29), rat galanin-(1–19), rat galanin-(10–29), and rat galanin-(3–29) were custom-synthesized by Bio-synthesis, Inc. (Lewisville, TX). Galanin-(1–13)-bradykinin-(2–9) (M35), DAGO, somatostatin, and galanin messenger-associated peptides GMAP-(1–41) and GMAP-(44–59) were from Sigma. [Ala6,d-Trp8]galanin-(1–15)-ol and [d-trp6,d-trp8,9]galanin-(1–15)-ol were custom-synthesized by AnaSpec, Inc. (San Jose, CA).PCR Sib SelectionPCR with galanin receptor-specific primers was used to screen pools of a rat hypothalamus cDNA library. Positive pools so identified were subdivided and screened further by the PCR screening until the final round of screening, at which time individual colonies were selected by analysis of plasmid mini-preps. The rat cDNA library was constructed as described previously (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar).PCR and RACE Amplification of cDNA FragmentUnless otherwise specified, PCR was performed using KlenTaq polymerase, which possesses proof reading activity (CLONTECH) and a cycling profile of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for 2 min (40 cycles). Approximately 1 μl of overnight Escherichia coli culture was used in the PCR for sib selection. For RACE, nested primers specific to rat GalR3 cDNA and nested adaptor primers were used in the primary and secondary PCRs. A thermal cycling profile of 94 °C for 30 s, 65 °C for 30 s, and 72 °C for 90 s (25 cycles) and rat brain cDNA as a template were used in primary PCR. A cycling profile of 94 °C for 1 min and 70 °C for 4 min (30 cycles) and 5 μl of the primary PCR product (diluted 1:50) as a template were used in the secondary PCR. The RACE product was cloned into vector pCR 2.1. A GC melt reagent (CLONTECH) at 10–20% (v/v) of original stock (5 m) was always used in both PCR and RACE reactions.DNA Sequencing and AnalysisThe DNA sequences of clones were determined on both strands using Applied Biosystems Prism dye termination DNA sequencing reagents and an Applied Biosystems 373 automated sequencing apparatus (Perkin-Elmer). DNA and protein sequence comparisons were performed with the DNA* software (DNAstar Inc., Madison, WI).Transfection of COS-7 CellsRat GalR3 cDNA was introduced into COS-7 cells by electroporation as described previously (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar) or by the use of LipofectAMINE method (Life Technologies, Inc.) according to the manufacturer's instructions. The two methods gave comparable levels of expression.Receptor Membrane PreparationThree days following the transfection of the COS-7 cells, receptor membrane was prepared as previously described (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar).125I-Galanin Binding AssayBinding of125I-porcine galanin to the membrane preparations was performed as previously described (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar) except that 20 μg of membrane protein was used in the saturation and competition binding assays. All data were analyzed by nonlinear regression (Prism, GraphPad, San Diego, CA) and the K i calculated according to the method of Cheng and Prusoff (17Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12185) Google Scholar).Northern Blot Analysis of Rat GalR3A rat multitissue Northern blot (CLONTECH) was hybridized for 15 h at 55 °C in an ExpressHyb solution (CLONTECH) using 32P-labeled rat GalR3 cDNA as a probe (labeled with a random priming kit, Life Technologies, Inc., specific activity = 3 × 109 cpm/μg). After hybridization, the blot was washed with wash solution I (2 × SSC, 0.05% SDS) for 30 min at room temperature then with wash solution II (0.1 × SSC, 0.1% SDS) for 30 min at room temperature, 1 h at 48 °C, 1 h at 52 °C, and 30 min at 54 °C. The blot was then wrapped with Saran Wrap and exposed to Kodak BioMax films at −80 °C for 1 week. The same blot was stripped and hybridized in a similar manner with a 32P-labeled actin cDNA to ensure loading of poly(A)+ mRNA from the tissues onto the blot.RESULTS AND DISCUSSIONBLAST search (18Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69088) Google Scholar) of the GenBank™ data base with the human GalR1 receptor amino acid sequence (10Habert-Ortoli E. Amiranoff B. Loquet I. Laburthe M. Mayaux J.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9780-9783Crossref PubMed Scopus (323) Google Scholar) as query identified a portion of a human genomic clone (accession number Z82241) that possessed high homology with the amino acids 37–132 of human GalR1 (third reading frame of the positive strand, 55% identity). The homology was greater than that between rGalR1 and rGalR2 (40%), suggesting that this human clone may contain a portion of a new galanin receptor.Several pairs of PCR primers were generated according to the human genomic sequence and used in PCR with cDNA reverse-translated from rat liver RNA as a template to obtain rat GalR3 cDNA (Fig.1 A). A PCR cycling paradigm employing low annealing temperature with two PCR primers, oligonucleotide 93C and oligonucleotide 120B, produced a PCR product. The approximately 700-base pair fragment was cloned into vector pcR3.1 and sequenced. Comparison of the DNA sequence with the nucleotide sequences in GenBank™ revealed 86, 65, and 63% identities with the human genomic clone (Z82241), rat GalR2, and human GalR1, respectively. Therefore, the rat clone appeared to be a portion of a novel rat galanin receptor (Fig. 1 A).RACE and PCR sib selection were performed to extend the cDNA sequence toward the 5′ and 3′ directions. In RACE, primers oligo172 and AP1 (outer adaptor primer) were used in the primary PCR with a rat brain cDNA library as a template and primers oligo177 and AP2 (inner adaptor primer) were used in the secondary PCR with product of the primary PCR as a template. The final RACE product, ∼1.8-kb, overlapped with the 5′ portion of the rat 0.7-kb GalR3 cDNA and the upstream 5′-untranslated region (Fig. 1 A). For PCR sib selection, two primers, oligo164 and oligo167, were used to screen a cDNA library constructed from rat hypothalamus (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar). Pool A28 gave a positive band and was subdivided and screened until a single clone (clone A28-1, 1.3 kb) was obtained (Fig. 1 A).A full-length clone of GalR3 cDNA was obtained by performing further sib selection on the hypothalamus cDNA library using primer oligo185, designed based on the sequence of the 5′-RACE product, and primer oligo184, designed based on the sequence of clone A28-1. A single clone A5-3, selected from library pool A5, was obtained and sequenced. The clone was 2.2 kb long and contained all the sequence of clone A28-1 and a portion of the 5′-RACE product (Fig. 1 A). A complete open reading frame was identified corresponding to a protein of 370 amino acids with a calculated molecular mass of 40.3 kDa (Fig.1 B). The clone was termed GalR3 receptor. Hydrophathy analysis revealed seven putative transmembrane-spanning domains (TMs) typical of G protein-coupled receptors. The GalR3 receptor also contains a single potential N-linked glycosylation site in the N-terminal region, two Cys residues in the first and second extracellular loops that form a putative disulfide bond in these receptors, and two Cys residues in the C-terminal region that may be involved in palmitoylation (Fig. 1 C).The amino acid sequence of the rat GalR3 receptor is significantly different from those of rat GalR1 and GalR2. The overall homology is 36% to rat GalR1 and 54% to rat GalR2 as analyzed by the Jutun Hein method (19Hein J. Methods Enzymol. 1990; 183: 626-645Crossref PubMed Scopus (348) Google Scholar) (Fig. 1 C). A search of the SwissProt data bank revealed that rat GalR3 has high homology to the rat somatostatin type 4 receptor (31%) (20Bruno J.F. Xu Y. Song J. Berelowitz M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11151-11155Crossref PubMed Scopus (314) Google Scholar) and the rat μ-type opioid receptor (27%) (21Fukuda K. Kato S. Mori K. Nishi M. Takeshima H. FEBS Lett. 1993; 327: 311-314Crossref PubMed Scopus (286) Google Scholar). Sequence alignment of rGalR3 with rGalR1 shows the greatest similarity in TM2, TM7, and TM1, being 62, 46, and 46% identical, respectively. Alignment with rGalR2 displayed a generally higher homology in the TMs than rGalR1, with the highest in TM2, TM3, and TM4, being 92, 83, and 71% identical, respectively. The N terminus and the C terminus possess least homology with both rGalR1 and rGalR2 receptors (Fig. 1 C).Northern blot analysis was performed to examine the tissue distribution of GalR3 mRNA. A single band of ∼3.5 and ∼3 kb was detected in heart and testis, respectively (Fig. 2). There was a strong, broad band at higher molecular weight (5–8 kb) in spleen and testis, indicating a heterogeneous population of the transcript in these tissues. There was no significant expression in kidney, skeletal muscle, liver, brain, or lung. The low abundance of GalR3 in the brain is contrasted to the distribution of GalR1, which is significantly expressed only in brain and spinal cord (12Parker E.M. Izzarelli D.G. Nowak H.P. Mahle C.D. Iben L.G. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar, 13Wang S. He C. Maguire M. Clemmons A. Burrier R. Guzzi M. Strader C. Parker E. Bayne M. FEBS Lett. 1997; 411: 225-230Crossref PubMed Scopus (67) Google Scholar), and to that of GalR2, which is expressed in both central and peripheral tissues (14Howard A.D. Tan C. Shiao L.-L. Palyha O.C. McKee K.K. Weiger D.H. Feighner S.D. Cascieri 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, 15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar). Given that the GalR3 cDNA was cloned from a hypothalamus library, it seems likely that there is a low level of expression of GalR3 in this part of the brain. The overall expression pattern of GalR3 suggests that the receptor may mediate galanin actions in the cardiovascular system (22Ulman L.G. Evans H.F. Iismaa T.P. Potter E.K. McCloskey D.I. Shine J. Neurosci. Lett. 1992; 136: 105-108Crossref PubMed Scopus (17) Google Scholar, 23Ulman L.G. Potter E.K. McCloskey D.I. Regul. Pept. 1994; 51: 17-23Crossref PubMed Scopus (13) Google Scholar) and in sexual behavior (24Poggioli R. Rasori E. Bertolini A. Eur. J. Pharmacol. 1992; 213: 87-90Crossref PubMed Scopus (35) Google Scholar, 25Benelli A. Arletti R. Bertolini A. Menozzi B. Basaglia R. Poggioli R. Eur. J. Pharmacol. 1994; 260: 279-282Crossref PubMed Scopus (27) Google Scholar).Figure 2Tissue distribution of rat GalR3 by Northern blot analysis. Northern blot containing rat poly(A)+ mRNA was hybridized with a 32P-labeled the 700-base pair rat GalR3 cDNA sequence (the cloned PCR fragment with primers 93C and 120B, Fig. 1 A). Lane 1, testis;lane 2, kidney; lane 3, skeletal muscle;lane 4, liver; lane 5, lung; lane 6, spleen; lane 7, brain; and lane 8, heart. The blot was reprobed with 32P-labeled β-actin cDNA to ensure comparable loading of mRNA from the tissues (bottom).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To characterize the pharmacology of the rat GalR3 receptor, the GalR3 plasmid (the 2.2-kb clone A5-3) was expressed in COS-7 cells. Binding of 125I-porcine galanin to membranes prepared from transfected COS-7 cells demonstrated a low but reproducible level of specific binding, while binding of the radioligand to membranes from COS-7 cells transfected with vector alone was negligible. The specific binding was saturable at high affinity, showing a K d value for 125I-galanin of 0.55 ± 0.15 nmand a B max of 28.1 ± 1.1 fmol/mg of membrane protein (mean ± S.D. from three independent transfections). A representative binding curve is illustrated in Fig.3. No specific binding was observed when125I-labeled somatostatin was used in the binding assays.Figure 3Saturation analysis of125I-porcine galanin (125I-pGal) binding to membranes prepared from transfected COS-7 cells with pcDNA3-rGalR3 (clone A5-3). Data shown represent the specific binding (total binding minus nonspecific binding). The counts for nonspecific binding, determined in the presence of 5 μm rat galanin, were approximately 40% of the counts for total binding. The curve shows the fit of one-site hyperbolic binding by nonlinear regression analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ability of several galanin antagonists and agonists to bind GalR3 were examined in radioligand competition binding assays.125I-porcine galanin binding to membranes prepared from COS-7 cells transfected with pcDNA3-rGalR3 cDNA could be displaced with galanin fragments and galanin-related chimeric peptides (Table I). Galanin, C7, galanin-(1–19), M35, and galanin-(2–29) bound GalR3 with high affinity (within 9-fold K i of galanin), while M40, galanin-(1–16), and M15 bound rGalR3 with relatively lower affinities (33–60 times K i of galanin) (Table I). No binding was detected in the assays with galanin-(3–29), galanin-(10–29), preprogalanin-(1–30), GMAP-(1–41), GMAP-(44–59), and [d-Thr6,d-Trp8,9]galanin-(1–15)-ol (Table I). [Ala6,Trp8]Galanin-(1–15)-ol, an antagonist shown to inhibit the effect of galanin on forskolin-induced insulin release from RINm5F cells (26Kakuyama H. Mochizuki T. Iguchi K. Yamabe K. Hosoe H. Hoshino M. Yanaihara N. Biomed. Res. 1997; 1: 49-56Crossref Scopus (11) Google Scholar), bound the GalR3 receptor with high affinity (Table I). When tested in the same competition binding assays, somatostatin, dynorphin (κ-opioid receptor-specific ligand) and DAGO (μ-opioid receptor-specific ligand) did not cause detectable displacement of the radioligand. The high affinity of galanin-(2–29) for GalR3 (Table I) revealed that, like GalR2, Gly1 of galanin is not critical for galanin to bind GalR3, whereas this residue is important for binding of galanin to GalR1 (15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar). In contrast, galanin-(3–29) did not bind either GalR1, GalR2, or GalR3 (Table I), indicating a crucial role of Trp2 of galanin in binding of galanin to all three receptor subtypes.Table ILigand binding profile of the cloned rat GalR3 receptorLigandKi nmKi(ligand)/K i(galanin)Rat GalR3 receptor Rat galanin-(1–29)1.47 ± 0.42 (7)1 C7 (galanin-(1–13)-spantide I)0.75 ± 0.06 (2)0.51 Rat galanin-(1–19)6.25 ± 0.81 (2)4.25 M35 (galanin-(1–13)-bradykinin(2–9))7.50 ± 2.95 (4)5.10 Rat galanin-(2–29)12.6 ± 6.69 (3)8.57 [Ala6,d-Trp8]Galanin-(1–15)-ol17.9 ± 8.6 (4)12.0 Rat galanin-(1–16)49.6 ± 15.3 (3)33.7 M4054 ± 23.7 (2)36.7 M15 (galantide or gal-(1–13)-SP-(5–11))85 ± 52 (4)57.8 Rat galanin-(3–29)>1000 (3)>650 Rat galanin-(10–29)>1000 (2)>650 Preprogalanin-(1–30)>1000 (2)>650 [d-Thr6,d-Trp8,9]Galanin-(1–15)-ol>1000 (2)>650 GMAP-(1–41)>1000 (2)>650 GMAP-(44–59)>1000 (3)>650Rat GalR2 receptor Rat galanin-(1–29)1.48 ± 0.59 (5)1 M151.11 ± 0.16 (4)0.75 Rat galanin-(2–29)1.92 ± 0.92 (4)1.30 M402.88 ± 1.48 (4)1.95 Rat galanin-(1–19)4.7 ± 2.0 (3)3.18 Rat galanin-(1–16)5.66 ± 3.7 (2)3.85 Rat galanin-(3–29)>1000 (4)>650 GMAP-(44–59)>1000 (2)>650Rat GalR1 receptor Rat galanin-(1–29)0.98 ± 0.22 (3)1 Rat galanin-(1–16)4.8 ± 1.5 (2)4.9 Rat galanin-(2–29)85 ± 12 (3)87 Rat galanin-(3–29)>1000 (2)>1020 Open table in a new tab Pharmacological characterization revealed some striking differences between the profile of GalR3 and those of GAlR1 and GalR2. Galanin-(1–16), and two galanin-related chimeric peptides, M40 and M15, displayed significantly lower affinities for GalR3 than GalR2 and GalR1 (Table I and see Refs. 12Parker E.M. Izzarelli D.G. Nowak H.P. Mahle C.D. Iben L.G. Wang J. Goldstein M.E. Mol. Brain Res. 1995; 34: 179-189Crossref PubMed Scopus (210) Google Scholar and 15Wang S. Hashemi T. He C. Strader C. Bayne M. Mol. Pharmacol. 1997; 52: 337-343Crossref PubMed Scopus (34) Google Scholar). Deletion of the C-terminal 10 amino acids (galanin-(1–19)) resulted in a 4-fold decrease in affinity for GalR3 (Table I). However, further truncation to galanin-(1–16) resulted in a further 8-fold decrease in affinity (Table I), indicating the importance of residues 17–19 of galanin in binding to GalR3. Examination of residues 17–19 of the high-affinity ligands reveals that a potential hydrogen bond donor is conserved at position 18 in all high affinity peptides, i.e. Asn in galanin, galanin-(1–19), and galanin-(2–29), Gln in C7, and Ser in M35 (Fig.4 A). Substitution of this position with Ala in M40 or Gly in M15 or deletion of this residue in galanin-(1–16) to remove the potential for hydrogen bond formation, significantly decreased the affinity of the ligands for the GalR3 receptor (Fig. 4 A). Delineation of the site of interaction of this residue with the GalR3 receptor will require a detailed receptor mutagenesis analysis.Figure 4Ligand specificity of the galanin receptor subtypes. A, specificity determined by residues 17–19 of galanin to the GalR3 receptor. Residues 17–19 of galanin and analogs are aligned and the putative hydrogen bond donor residues are indicated by boldface letters. Dashed Lines are used for galanin-(1–16) to indicate lack of the residues. B, ligand specificity of galanin to the three GalR subtypes. Residues of pharmacophores within galanin for interaction with the three receptor subtypes are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In summary, we have cloned and characterized a novel galanin receptor subtype termed GalR3. The receptor shares homology (36–54%) to the previously cloned GalR1 and GalR2 receptors. The distribution of GalR3 mRNA expression is strikingly different from those of GalR1 and GalR2 and appears to be restricted to the peripheral tissues. The pharmacology of the GalR3 receptor is distinguished from the other two receptors by the requirement of amino acids 17–19 of galanin (Fig. 4). The three galanin receptor subtypes show different pharmacological profiles with respect to galanin analogs, suggesting that they bind to distinct pharmacophores within the galanin peptide (Fig.4 B). This observation suggests the potential for physiological control of galanin receptor subtype activation by selective ligand modification such as differential proteolysis (27Bedecs K. Langel U. Bartfai T. Neuropeptides. 1995; 29: 137-143Crossref PubMed Scopus (33) Google Scholar,28.Jureus, A., Lindgren, M., Langel, U., Bartfai, T., Seventh Annual Summer Neoropeptide Conference, Key West, FL, June 22–26, 1997, 1997, International Neuropeptide Society.Google Scholar). The characterization of this new galanin receptor should aid in delineating specific physiological roles of the galanin receptor subtypes. Galanin is a 29–30 amino acid neuropeptide with no significant homology to any known family of biologically active peptides (1Tatemoto K. Rokaeus A. Jornwall H. McDonald T.J. Mutt V. FEBS Lett. 1983; 164: 124-128Crossref PubMed Scopus (1345) Google Scholar). Galanin is widely distributed in the central and peripheral nervous systems and is highly expressed in various regions of the brain. Many physiological processes are modulated by galanin, including neurotransmitter and hormone release (2Dunning B.E. Ahren B. Veith R.C. Bottcher G. Sundler F. Taborsky Jr., G.J. Am. J. Physiol. 1986; 251: E127-E133PubMed Google Scholar), spinal reflexes, nociception (3Verge V.M.K. Xu X.-J. Langel U. Hokfelt T. Wiesenfeld Z. Bartfai T. Neurosci. Lett. 1993; 149: 193-197Crossref PubMed Scopus (76) Google Scholar), firing of noradrenergic neurons, and contraction of gastrointestinal smooth muscle (4Ekblad E. hakanson R. Sundler F. Wahlestedt C. Br. J. Pharmacol. 1985; 86: 241-246Crossref PubMed Scopus (245) Google Scholar, 5Crawley J.N. Regul. Pept. 1995; 59: 1-16Crossref PubMed Scopus (134) Google Scholar). Like neuropeptide Y, centrally administered galani
Referência(s)