CDB15:0000677 GAL — GALR2

ABSTRACT: The novel neuropeptide spexin (SPX) was discovered using bioinformatics. The function of this peptide is currently under investigation. Here, we identified SPX along with a second SPX gene (SPX2) in vertebrate genomes. Syntenic analysis and relocating SPXs and their neighbor genes on reconstructed vertebrate ancestral chromosomes revealed that SPXs reside in the near vicinity of the kisspeptin (KISS) and galanin (GAL) family genes on the chromosomes. Alignment of mature peptide sequences showed some extent of sequence similarity among the 3 peptide groups. Gene structure analysis indicated that SPX is more closely related to GAL than KISS. These results suggest that the SPX, GAL, and KISS genes arose through local duplications before 2 rounds (2R) of whole-genome duplication. Receptors of KISS and GAL (GAL receptor [GALR]) are phylogenetically closest among rhodopsin-like G protein-coupled receptors, and synteny revealed the presence of 3 distinct receptor families KISS receptor, GALR1, and GALR2/3 before 2R. A ligand-receptor interaction study showed that SPXs activate human, Xenopus, and zebrafish GALR2/3 family receptors but not GALR1, suggesting that SPXs are natural ligands for GALR2/3. Particularly, SPXs exhibited much higher potency toward GALR3 than GAL. Together, these results identify the coevolution of SPX/GAL/KISS ligand genes with their receptor genes. This study demonstrates the advantage of evolutionary genomics to explore the evolutionary relationship of a peptide gene family that arose before 2R by local duplications.

ABSTRACT: Galanin is a peptide hormone which modulates a wide variety of physiological processes, including secretion, muscle contraction, cognitive function, the reproductive axis, and feeding. Two galanin receptor subtypes, GalR1 and GalR2, have been cloned; however, for GalR2 only the rat sequence has been reported in the literature. Our cloning of human GalR2 reveals its amino acid sequence to be 85% identical to rat GalR2 and 39% identical to human GalR1. Binding of [125I]galanin to the human GalR2 receptor transiently expressed in COS-7 cells was saturable (Kd = 0.24 nM +/- 0.06 nM) with a receptor density of 383 +/- 66 fmol/mg protein. Human galanin(1-30) bound with high affinity to the human GalR2 receptor, with a Ki value of 0.86 +/- 0.12 nM. With the identification of a second galanin receptor subtype, the specific functions of human galanin receptor subtypes can now begin to be addressed.

ABSTRACT: The neuropeptide galanin mediates a diverse spectrum of biological activities by interacting with specific G protein-coupled receptors. We have used homology genomic library screening and polymerase chain reaction (PCR) techniques to isolate both genomic and cDNA clones encoding the human homolog of the recently cloned rat GALR2 galanin receptor. By fluorescence in situ hybridization, the gene encoding human GALR2 (GALNR2) has been localized to chromosome 17q25.3. The two coding exons of the human GALNR2 gene, interrupted by an intron positioned at the end of transmembrane domain III, encode a 387 amino acid G protein-coupled receptor with 87% overall amino acid identity with rat GALR2. In HEK-293 cells stably expressing human GALR2, binding of [125I]porcine galanin is saturable and can be displaced by galanin, amino-terminal galanin fragments and chimeric galanin peptides but not by carboxy-terminal galanin fragments. In HEK-293 cells, human GALR2 couples both to Galphaq/11 to stimulate phospholipase C and increase intracellular calcium levels and to Galphai/o to inhibit forskolin-stimulated intracellular cAMP accumulation. A wide tissue distribution is observed by reverse transcriptase (RT)-PCR analysis, with human GALR2 mRNA being detected in many areas of the human central nervous system as well as in peripheral tissues.

ABSTRACT: The neuropeptide galanin has been implicated in the regulation of processes such as nociception, cognition, feeding behavior, and hormone secretion. Multiple galanin receptors are predicted to mediate its effects, but only two functionally coupled receptors have been reported. We now report the cloning of a third galanin receptor distinct from GALR1 and GALR2. The receptor, termed GALR3, was isolated from a rat hypothalamus cDNA library by both expression and homology cloning approaches. The rat GALR3 receptor cDNA can encode a protein of 370 amino acids with 35% and 52% identity to GALR1 and GALR2, respectively. Localization of mRNA by solution hybridization/RNase protection demonstrates that the GALR3 transcript is widely distributed, but expressed at low abundance, with the highest levels in the hypothalamus and pituitary. We also isolated the gene encoding the human homologue of GALR3. The human GALR3 receptor is 90% identical to rat GALR3 and contains 368 amino acids. Binding of porcine 125I-galanin to stably expressed rat and human GALR3 receptors is saturable (rat KD = 0.98 nM and human KD = 2.23 nM) and displaceable by galanin peptides and analogues in the following rank order: rat galanin, porcine galanin approximately M32, M35 approximately porcine galanin-(-7 to +29), galantide, human galanin > M40, galanin-(1-16) > [D-Trp2]galanin-(1-29), galanin-(3-29). This profile resembles that of the rat GALR1 and GALR2 receptors with the notable exception that human galanin, galanin-(1-16), and M40 show lower affinity at GALR3. In Xenopus oocytes, activation of rat and human GALR3 receptors co-expressed with potassium channel subunits GIRK1 and GIRK4 resulted in inward K+ currents characteristic of Gi/Go-coupled receptors. These data confirm the functional efficacy of GALR3 receptors and further suggest that GALR3 signaling pathways resemble those of GALR1 in that both can activate potassium channels linked to the regulation of neurotransmitter release.

ABSTRACT: Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.