Molecular characterization of chicken galanin and galanin receptor family

Abstract



Galanin is a multi-functional neuropeptide with widespread distribution in the central and peripheral nervous systems of different species. It exerts a broad spectrum of physiological functions through the interactions with three galanin receptor subtypes namely the GalR1, GalR2 and GalR3, which have only been identified in mammals. In contrast to the extensive studies in mammals, little is known about the structures and functions of galanin and its receptors in avian species. In the present study, a total of nine chicken cDNAs including four galanin prepropeptide transcript variants (cGal -v1 to -v4), the three known galanin receptor subtypes (cGalR1, cGalR2 and cGalR3), and two novel galanin receptor subtypes designated the GalR1-like (cGalR1-L) and GalR2-like (cGalR2-L), were cloned from the brain and intestine tissues, respectively. The four cGal transcript variants encode precursor peptides of 117, 141, 88 and 150 residues, respectively, which eventually produce two isoforms of mature chicken galanin peptide, the cGal (1-29) and a novel cGal (1-53), through proteolytic processing of the galanin prepropeptide. The five isolated cGalR cDNAs encode receptor proteins of 357-to 405-residue-long, which share amino acid sequence identities ranged from 50% to 86% with their corresponding mammalian counterparts.

From RT-PCR analysis, the cGal and cGalRs (except for cGalR3) showed diverse mRNA expression profiles among the adult chicken tissues examined including different regions of the oviduct. Using luciferase reporter systems, it was demonstrated that all the five cGalRs, transiently transfected in Chinese hamster ovary (CHO) cells, were functionally and differentially coupled to Gs, Gi or Gq protein signalling pathways upon the activations of chicken galanin peptides cGal (1-29), cGal (1-53) and human galanin-like peptide hGALP (1-60), with distinct potencies. The newly identified cGalR1-L and cGalR2-L share similarities with cGalR1 and cGalR2, respectively, in terms of the sequence homology, exon/intron organization and signalling pathways, thus suggesting an early gene duplication event in vertebrate evolution in the galanin receptor family. On the other hand, the failure of identifying GalR1-L and GalR2-L genes in mammalian species suggests their deletion events in the mammalian lineage, which are supported by the comparative synteny analyses of galanin receptor family between the human, chicken, zebrafish and Xenopus genomes. Together, these findings establish a molecular basis to elucidate the physiological roles of galanin and its receptors in birds.