title: Identification, tissue distribution and functional characterization of the ghrelin receptor in West African lungfish, Protopterus annectens.
abstract: We identified two ghrelin receptor isoforms, the ghrelin receptor type-1a (GHS-R1a) and its alternative splice form (GHS-R1b) for West African lungfish, Protopterus annectens. Lungfish GHS-R1a and 1b comprised 361 and 281 amino acids, respectively. Lungfish GHS-R1a showed the highest identity to coelacanth GHS-R1a (80.4%). The highest expression of GHS-R1a mRNAs was seen in the brain, liver, ovary, heart, intestine, and gills. GHS-R1b mRNAs were also detected in the same tissues with GHS-R1a, but their expression level was 1/20 that of GHS-R1a. In human embryonic kidney 293 cells transiently expressing lungfish GHS-R1a, rat and bullfrog ghrelin, and two GHS-R1a agonists, GHRP-6 and hexarelin, increased intracellular Ca(2+) concentrations. The intensity of the Ca(2+) increases induced by GHS-R1a agonists was twice when compared to that induced by ghrelin, although the median effective doses (ED50) were similar, suggesting a long-lasting effect of GHS-R1a agonists with similar affinity. We also examined changes in the GHS-R gene expression during an eight-week estivation. Body weight was slightly lowered, but plasma sodium and glucose concentrations decreased; plasma urea concentration increased significantly 4weeks after the start of estivation. Overall, expression of GHS-R1a mRNA decreased, but changes in GHS-R1b mRNA expression were inconsistent with those of GHS-R1a during estivation, suggesting an involvement of GHS-R in energy homeostasis, as seen in mammals. Our results suggest that the ghrelin-GHS-R1a system is present in this lungfish although ghrelin has not yet been found. The structure of GHS-R1a is closer to that of tetrapods than Actinopterygian fish, indicating a process of evolution that follows the Crossopterygii such as coelacanth.
authors.affiliation: Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Electronic address: kaiya@ncvc.go.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kaiya
authors.affiliation: Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan.
authors.forename: Norifumi
authors.initials: N
authors.lastname: Konno
authors.affiliation: Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
authors.affiliation: Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555, Japan.
authors.forename: Minoru
authors.initials: M
authors.lastname: Uchiyama
authors.affiliation: Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan.
authors.forename: Mikiya
authors.initials: M
authors.lastname: Miyazato
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D005399:Fishes / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D020033:Protein Isoforms / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
chemical_list: D018076:DNA, Complementary; D020033:Protein Isoforms; D012333:RNA, Messenger; D054440:Receptors, Ghrelin
keywords: Functional characterization; GHS-R1a; GHS-R1b; Ghrelin receptor; Lungfish; cDNA cloning