CDB15:0000676 GAL — GALR1

ABSTRACT: In this work, we studied a novel chimeric peptide, M242, galanin(1-13)-[D-Trp(32)]-neuropeptide Y(25-36)amide, and examined its properties in comparison with its parent peptide, M32, galanin(1-13)-neuropeptide Y(25-36)amide, a previously known high-affinity ligand for galanin receptors, and galanin itself. Binding assays performed in Bowes cells known to express human galanin receptor type 1 (hGalR1) and in Chinese hamster ovary cells overexpressing human galanin receptor type 2 (hGalR2) revealed that all three ligands had comparable affinities: at hGalR1<1 nM and at hGalR2<10 nM. However, in rat hippocampal membranes M242 had a 24-fold lower affinity than galanin (9.4 vs. 0.4 nM) and 134-fold lower affinity than M32 (9.4 vs. 0.07 nM). In the same tissue, we also examined the effects of these peptides on adenylate cyclase activity. M32 showed a weak antagonistic behaviour but M242 acted as a potent biphasic regulator of adenylate cyclase. In conclusion, we present and characterise a new peptide M242, which could be a useful tool in studies of galaninergic signalling.

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: The ubiquitous neuropeptide galanin controls numerous functions such as endocrine secretions, intestinal motility, and behavioral activities. These regulatory effects of galanin are mediated through the interaction with specific membrane receptors and involve the pertussis toxin-sensitive guanine nucleotide binding proteins Gi/Go as transducing elements. We report here the isolation of a cDNA coding for a human galanin receptor from a Bowes melanoma cell line cDNA expression library, by using a radioligand binding strategy. The nucleotide sequence of the cloned receptor reveals an open reading frame encoding a 349-amino acid protein with seven putative hydrophobic transmembrane domains and significant homology with members of the guanine nucleotide binding protein-coupled neuropeptide receptor family. The cloned receptor expressed in COS cells specifically binds human, porcine, and rat galanin with high affinity (Kd in the nanomolar range) and mediates the galanin inhibition of adenylate cyclase. A 2.8-kb galanin receptor transcript was identified in several human tissues. Cloning of this galanin receptor should enhance our knowledge of its distribution, structure, and function in human physiology and pathophysiology.

ABSTRACT: Galanin, a neuroendocrine peptide of 29 amino acids, binds to G/G.-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the highaffinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the Nterminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of the receptor.

ABSTRACT: The diverse biological functions of galanin are mediated via membrane bound high-affinity receptors. In order to identify and characterize potential galanin receptor subtypes, we have examined the specific 125I-galanin binding to the CHP-212 human neuroblastoma cell line. The galanin receptors expressed in CHP-212 cells, like GALR1 have high affinity for galanin (Kd = 0.07 nM) and the potency for inhibition of 125I-galanin binding by galanin peptides parallels that of hGALR1 expressed in a stable CHO cell line. We confirmed that GALR1 is expressed in these cells by RT-PCR. We further determined the tissue expression patterns of hGALR1 which is expressed in a variety of human tissues at a very low level, with the highest levels seen in heart, small intestine and prostate. A species of approximately 70 kDa is recognized by antisera specific for hGALR1 by Western blot analysis and should allow future measurements of receptor protein expression.

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: The galanin neuropeptide system is widely distributed throughout the brain and periphery and is thought to play a role in feeding, pain and reproduction. To evaluate the human galanin receptor 1 as a potential therapeutic target, we fully characterized its interaction with several galanin-like peptides. The human galanin receptor 1 receptor was stably expressed using an episomal system in human embryonic kidney 293E cells. Saturation isotherms using 125I-human galanin revealed two distinct populations of receptor affinity states in membranes and whole cells with picomolar and nanomolar affinities at the high- and low affinity states, respectively. A scintillation proximity assay revealed that 125I-human galanin binding in membranes reached steady-state within 2 to 2.5 hr; however, only 50% of galanin radiolabel dissociated from the receptors by excess galanin or guanosine 5'-O-3-thiotriphosphate even after 20 hr. In contrast, galanin binding in whole cells was completely reversible within 1 hr. Competition binding assays showed that galanin-like peptides bound with picomolar affinities in membranes and whole cells. These peptides behaved as full agonists as determined by the inhibition of forskolin-stimulated cyclic 3'5'-adenosine monophosphate production and the stimulation of guanosine 5'-O-(3-[35S]thiotriphosphate binding. The agonist profile of M40, a representative chimeric peptide, was found not to be the result of receptor reserve because receptor inactivation by partial alkylation experiments confirmed its full intrinsic efficacy under conditions of a "zero" reserve state. These observations suggest that the antagonist effects in vivo of M40, and perhaps other chimeric peptides, are not mediated via direct interactions with the galanin receptor 1 receptor.