Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.
We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) 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-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), 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-NH(2) (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.
Ukena
Kazuyoshi
K
Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
Koda
Aya
A
Yamamoto
Kazutoshi
K
Kobayashi
Tetsuya
T
Iwakoshi-Ukena
Eiko
E
Minakata
Hiroyuki
H
Kikuyama
Sakae
S
Tsutsui
Kazuyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D013006:Growth Hormone / Q000378:metabolism; D013007:Growth Hormone-Releasing Hormone / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000378:metabolism; D011892:Rana catesbeiana / Q000235:genetics*
D009479:Neuropeptides; C416987:RFamide peptide; D011388:Prolactin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone