Ghrelin receptor (GHS-R)-like receptor and its genomic organisation in rainbow trout, Oncorhynchus mykiss.
Ghrelin, a GH-releasing and appetite-regulating peptide that is released from the stomach is an endogenous ligand for growth hormone secretagogue-receptor (GHS-R). Two types of GHS-R are accepted to be present, a functional GHS-R1a and GHS-R1b with unknown function. In this study, we identified cDNA that encodes protein with close sequence similarity to GHS-R and exon-intron organization of the GHS-R genes in rainbow trout, Oncorhynchus mykiss. Two variants of GHS-R1a proteins with 387-amino acids, namely DQTA/LN-type and ERAT/IS-type, were identified. In 3'-RACE PCR and genomic PCR, we also identified three GHS-R1b orthologs that are consisted of 297- or 300-amino acids with different amino acid sequence at the C-terminus, in addition to the DQTA/LN-type and ERAT/IS-type variations. Genomic PCR revealed that the genes are composed of two exons separated by an intron, and that two GHS-R1a and three GHS-R1b variants are generated by three distinct genes. GHS-R1a and GHSR-1b mRNA were predominantly expressed in the pituitary, followed by the brain. Identified DQTA/LN-type or ERAT/IS-type GHS-R1a cDNA was transfected into mammalian cells, and intracellular calcium ion mobilization assay was carried out. However, we did not find any response to rat ghrelin and a homologous ligand, des-VRQ trout ghrelin, of either receptor in vitro. We found that unexpected mRNA splicing had occurred in the transfected cells, suggesting that the full-length, functional receptor protein might not be generated in the cells. Gene structure and characterization of protein sequence identified in this study were closely similar to other GHS-R, but to conclude that it is a GHS-R for rainbow trout, further study is required to confirm activation of GHS-R1a by ghrelin or GHS. Thus we designated the identified receptor proteins in this study as GHS-R-like receptor (GHSR-LR).
Kaiya
Hiroyuki
H
Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. kaiya@ri.ncvc.go.jp
Mori
Tsukasa
T
Miyazato
Mikiya
M
Kangawa
Kenji
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D005091:Exons; D016678:Genome; D007438:Introns; D017686:Oncorhynchus mykiss / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D054440:Receptors, Ghrelin / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D054440:Receptors, Ghrelin