title: Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES.
abstract: The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH2. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.
authors.affiliation: From the Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Housu-gun, Ishikawa 927-0553, Japan, t-sekiguchi@se.kanazawa-u.ac.jp.
authors.forename: Toshio
authors.initials: T
authors.lastname: Sekiguchi
authors.affiliation: the Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki 889-1692, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kuwasako
authors.affiliation: the Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Ogasawara
authors.affiliation: the Laboratory of Morphogenesis, National Institute for Basic Biology, 38 Nishigonaka Myodaiji, Okazaki 444-8585, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: the Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1, Seikadai, Seika-cho, Soraku-gun, Kyoto 619-0284, Japan, and.
authors.forename: Shin
authors.initials: S
authors.lastname: Matsubara
authors.affiliation: the Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1, Seikadai, Seika-cho, Soraku-gun, Kyoto 619-0284, Japan, and.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Osugi
authors.affiliation: the Department of Pharmacology, School of Medicine, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.
authors.forename: Ikunobu
authors.initials: I
authors.lastname: Muramatsu
authors.affiliation: From the Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Housu-gun, Ishikawa 927-0553, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sasayama
authors.affiliation: From the Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Housu-gun, Ishikawa 927-0553, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: the Bioorganic Research Institute, Suntory Foundation for Life Sciences, 8-1-1, Seikadai, Seika-cho, Soraku-gun, Kyoto 619-0284, Japan, and.
authors.forename: Honoo
authors.initials: H
authors.lastname: Satake
mesh_terms: D053607:Adrenomedullin / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002116:Calcitonin / Q000235:genetics* / Q000378:metabolism*; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002490:Central Nervous System / Q000378:metabolism; D002522:Chlorocebus aethiops; D043344:Chordata; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D019143:Evolution, Molecular; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D058228:Islet Amyloid Polypeptide / Q000378:metabolism; D064175:Lancelets / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D058261:Receptor Activity-Modifying Proteins / Q000378:metabolism; D018003:Receptors, Calcitonin / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D058286:Calcitonin Receptor-Like Protein; D058228:Islet Amyloid Polypeptide; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D053607:Adrenomedullin; D002116:Calcitonin; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide
keywords: G protein-coupled receptor (GPCR); amphioxus; calcitonin; chordate; molecular evolution; protein evolution; receptor activity-modifying protein; receptor modification; receptor regulation