title: Molecular cloning, regulation, and functional analysis of two GHS-R genes in zebrafish.
abstract: Mammalian ghrelin is derived from stomach and regulates growth hormone release and appetite by modulating GHS-R (Growth hormone secretagogue receptor) activity. Zebrafish has been developed as a forward genetic screening model system and previous screening identified a number of genes involved in multiple signaling pathways. In this system, ghrelin has been identified and its function and regulation have been shown to be highly conserved to that of mammals. Here, we identified three isoforms of zGHS-R1 and one of zGHS-R2 (zGHS-R2a), and characterized their expression, regulation and function. Three isoforms of zGHS-R1, which we named zGHS-R1a, zGHS-R1b, and zGHS-R1c, are generated by alternative splicing. The expression of zGHS-R1 is highly enriched in brain, intestine tissue, and skin tissues. Compared to zGHS-R1, the expression pattern of zGHS-R2a is rather evenly distributed. A 15 day fasting elevated expression of zGHS-R1 and zGHS-R2 transcripts in anterior intestine tissues, but not in brain. Whereas zGHS-R1a, zGHS-R1c, and zGHS-R2a appear to be presented on the plasma membrane, the localization of zGHS-R1b seems to be restricted in the intracellular region. Treatment of ghrelin agonist, L692,585 or goldfish ghrelin peptides but not rat ghrelin, elevated intracellular Ca(2+) level and phosphorylation of ERK in HEK-293 cells expressing zGHS-R1a, but not zGHS-R1b, zGHS-R1c, or zGHS-R2a. It appears that besides core ghrelin peptide sequence of GS/TSF additional amino acids are required for the activation of zGHS-R1a, as rat ghrelin induces neither intracellular Ca(2+) mobilization nor ERK phosphrylation. These results suggest that ghrelin system in zebrafish is highly conserved to that of mammals, and thus is an ideal in vivo model for dissecting ghrelin system.
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea.
authors.forename: Ji
authors.initials: J
authors.lastname: Eom
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea.
authors.forename: Areum
authors.initials: A
authors.lastname: Hong
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Kang
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea.
authors.forename: Han-Ju
authors.initials: HJ
authors.lastname: Yoo
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea; Department of Anatomy and Cell Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Eun-Ju
authors.initials: EJ
authors.lastname: Chang
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea; Department of Anatomy and Cell Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Sang-Wook
authors.initials: SW
authors.lastname: Kang
authors.affiliation: Department of Anatomy and Cell Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Seung-Yong
authors.initials: SY
authors.lastname: Yoon
authors.affiliation: Asan Institute for Life Sciences, Asan Medical Center, Seoul 138-736, Republic of Korea; Department of medicine, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea.
authors.forename: Sang-Yeob
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Department of Biomedical Sciences, Cell Dysfunction Research Center (CDRC), University of Ulsan, College of Medicine, Olympic-ro 43-gil 88, SongPa-Gu, Seoul 138-736, Republic of Korea; Department of Anatomy and Cell Biology, University of Ulsan, College of Medicine, Seoul 138-736, Republic of Korea. Electronic address: ysong@amc.seoul.kr.
authors.forename: Youngsup
authors.initials: Y
authors.lastname: Song
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects*; D054439:Ghrelin / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D054440:Receptors, Ghrelin / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D015027:Zebrafish
chemical_list: D054439:Ghrelin; D020033:Protein Isoforms; D012333:RNA, Messenger; D054440:Receptors, Ghrelin; D002118:Calcium
keywords: Ca(2+) oscillation; GHS-R; Ghrelin; Zebrafish