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Novel Neuropeptides Related to Frog Growth Hormone-Releasing Peptide: Isolation, Sequence, and Functional Analysis

Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University (K.U., K.T.), Higashi-Hiroshima 739-8521, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. (K.U., K.T.), Tokyo 150-0002, Japan; Department of Biology, Waseda University School of Education (A.K., K.Y., S.K.), Nishiwaseda, Tokyo 169-8050, Japan; Department of Regulation Biology, Faculty of Science, Saitama University (T.K.), Shimo-Okubo, Saitama 338-8570, Japan; and Suntory Institute for Bioorganic Research (E.I.-U., H.M.), Mishima, Osaka 618-8503, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Ph.D., Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH₂) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H]²⁺), and their sequences, SIPNLPQRF-NH₂, YLSGKTKVQSMANLPQRF-NH₂, and AQYTNHFVHSLDTLPLRF-NH₂, were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH₂ (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10^-7 M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

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