Characteristics and distribution of endogenous RFamide-related peptide-1.
We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.
Fukusumi
S
S
Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries Ltd., Tsukuba, Japan.
Habata
Y
Y
Yoshida
H
H
Iijima
N
N
Kawamata
Y
Y
Hosoya
M
M
Fujii
R
R
Hinuma
S
S
Kitada
C
C
Shintani
Y
Y
Suenaga
M
M
Onda
H
H
Nishimura
O
O
Tanaka
M
M
Ibata
Y
Y
Fujino
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D002417:Cattle; D002850:Chromatography, Gel; D006224:Cricetinae; D007031:Hypothalamus / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
C109334:GPR1 protein, S cerevisiae; D009479:Neuropeptides; C416987:RFamide peptide; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D029701:Saccharomyces cerevisiae Proteins