OBJECTIVE:To identify and functionally characterize single-nucleotide polymorphisms (SNPs) in melanin-concentrating hormone (MCH)-R1 and -R2. RESEARCH METHODS AND PROCEDURES:The entire coding regions and intron/exon splice junction regions of MCH-R1 and MCH-R2 were sequenced from anonymous white (n=45) and African-American (n=46) individuals. DNA was analyzed, and SNPs were identified using Phred, Phrap, and Consed software. DNA constructs containing MCH-R1 and MCH-R2 SNPs were generated and expressed in CHO cells. The effect of the SNPs in MCH-R1 and MCH-R2 were assessed in receptor binding assays and functional assays measuring changes in intracellular cAMP and Ca2+ levels. RESULTS:We identified 12 SNPs in the MCH-R1 gene. Two of these SNPs are in coding regions, and one produces an arginine-for-glycine substitution at residue 34 in the MCH-R1 sequence. This SNP is present at a minor allele frequency of 15% in the African-American population tested in this study. We identified eight SNPs in the MCH-R2 gene. Four of these SNPs are in coding regions, and two produce amino acid substitutions. Lysine substitutes for arginine at residue 63 of the African-American population, and glutamine substitutes for arginine at residue 152 in whites (minor allele frequency of 2% for both SNPs). No changes in receptor binding or functional signaling were observed with the SNP mutations in MCH-R1 or MCH-R2. DISCUSSION:These data indicate that potential therapeutics designed to act at the MCH receptor are unlikely to have altered effects in subpopulations that express variant forms of MCH-R1 or MCH-R2.

Rhesus monkey MCH-R1 and MCH-R2 receptors were cloned. Amino acid homology is 98.8% between monkey and human MCH-R1, while monkey and human MCH-R2 are 98% homologous. Binding and intracellular signaling characteristics of the monkey receptors were compared with the human homologues. The results demonstrate that MCH binds to the monkey MCH-R1 receptor with a K(d) of 6.5 nM and monkey MCH-R2 with a K(d) of 2.2 nM similar to K(d) values for human MCH-R1 and MCH-R2. Additionally, monkey MCH-R1 couples through G(i)/G(o) and G(q)-type G proteins similar to human MCH-R1 whereas monkey and human MCH-R2 utilize the G(q) signaling pathway.

Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.

The receptor for melanin-concentrating hormone (MCH) was recently identified as the orphan G protein-coupled receptor SLC-1. In this study, a CHO cell line expressing the MCH receptor (Kd = 1.3 nM; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to Gi, Go, and Gq proteins. The results demonstrate that MCH inhibits forskolin-stimulated cAMP production in a pertussis toxin- (PTX)-sensitive manner in CHO-MCHR cells (EC50 = 100 pM), indicating that the MCH receptor couples to one or more members of the Gi subfamily of G proteins. In addition, MCH stimulates increases in phosphoinositide metabolism (EC50 = 50 nM) and in intracellular free Ca2+ levels (EC50 = 10 nM). MCH-stimulated inositol phosphate production and increases in intracellular free Ca2+ are partially inhibited (60% and 40%, respectively) by PTX pretreatment, demonstrating that there are at least two components of each of these signaling pathways. One component is PTX sensitive and therefore mediated through a Gi/Go protein. A distinct G protein-coupled (probably Gq type) mediates the PTX-insensitive component. To distinguish Gi vs. Go coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. Gi and Go use separate signaling pathways to mediate MAP kinase activation in CHOcells. Protein kinase C (PKC) activity is essential in the Go-dependent MAP kinase signaling pathway, but is not required in the GC-dependent MAP kinase signaling pathway. MCH stimulated MAP kinase activity is decreased (50%), but not abolished, by inhibition of PKC activity or depletion of cellular PKC, indicating that MCH-stimulated MAP kinase activity is mediated through both Gi- and Go-dependent signaling mechanisms. The results of this study are the first to clearly demonstrate that the MCH receptor couples to multiple G proteins to mediate several diverse intracellular signaling pathways.

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

Annals of the New York Academy of SciencesVolume 863, Issue 1 p. 457-458 Differential G-Protein-Coupling Profiles of the GalR1 and GalR2 Galanin Receptors SUKE WANG, SUKE WANG Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorTANAZ HASHEMI, TANAZ HASHEMI Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorSTEVEN FRIED, STEVEN FRIED Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorANTHONY L. CLEMMONS, ANTHONY L. CLEMMONS Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorBRIAN E. HAWES, BRIAN E. HAWES Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this author SUKE WANG, SUKE WANG Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorTANAZ HASHEMI, TANAZ HASHEMI Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorSTEVEN FRIED, STEVEN FRIED Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorANTHONY L. CLEMMONS, ANTHONY L. CLEMMONS Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this authorBRIAN E. HAWES, BRIAN E. HAWES Department of CNS/CV Biological Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USASearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10723.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume863, Issue1GALANIN: BASIC RESEARCH DISCOVERIES AND THERAPEUTIC IMPLICATIONSa December 1998Pages 457-458 RelatedInformation