Haruo Onda

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1–21)NH2, bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3–7 form a unique β-coil structure that is preceded by an N-terminal extended tail. This β-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8–21) forms an α-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named ‘metastin’. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

A prolactin-releasing peptide in the brain

Hypothalamic peptide hormones regulate the secretion of most ofthe anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin,. These peptides do not regulate the secretion of prolactin,, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis,, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an ‘orphan’ receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1–3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

Cloning and sequence analysis of cDNA encoding the precursor of a human endothelium-derived vasoconstrictor peptide, endothelin: Identity of human and porcine endothelin

A cDNA encoding a human endothelium‐derived vasoconstrictor peptide, endothelin, was isolated from a human placenta cDNA library. The nucleotide sequence of this cDNA clone showed that the primary structure of the human preproendothelin has 212 amino acid residues and is highly homologous to porcine preproendothelin, and that human endothelin is identical with porcine endothelin.

Abundance of endothelin-3 in rat intestine, pituitary gland and brain

We established a highly sensitive and specific sandwich-enzyme immunoassay (EIA) for endothelin-3 (ET-3), which showed no crossreactivity with endothelin-1 (ET-1), endothelin-2 (ET-2) and big-endothelin-1 (big-ET-1). We had previously established a sensitive sandwich-EIA for ET-1, which fully crossreacted with ET-2, but not with ET-3 or big-ET-1. These EIAs were used to examine the tissue distribution of immunoreactive (ir-) ET-3 and compare them with those of ir-ET-1 (including ir-ET-2) in Sprague-Dawley rats. High concentrations of ir-ET-3 were found in the intestine, lung, pituitary gland and brain (greater than 100 pg/g wet tissue), ir-ET-1(ET-2) showed widespread distribution, with large amounts in the lung and colon (greater than 1000 pg/g wet tissue). The pituitary gland was the only organ containing higher amounts of ir-ET-3 than ir-ET-1 (ET-2). In reverse phase-high performance liquid chromatography coupled with EIAs, the ir-ET-3 was exclusively eluted at the position of synthetic ET-3, indicating that the ir-ET-3 was identical to ET-3. The abundance of ET-3 in the intestine, pituitary gland and brain indicates that ET-3 is a new brain-gut peptide which may have a physiological function in nervous and endocrine systems.

Molecular properties of apelin: tissue distribution and receptor binding

We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [<Glu(65)]apelin-13) forms were detected in the mammary gland. In Scatchard analyses, the radioiodinated apelin-36 analogue bound to the receptor, APJ, with high affinity. In competitive binding assays, apelin-36 and apelin-19 far more efficiently inhibited the binding of the labeled apelin-36 analogue with APJ than [<Glu(65)]apelin-13. In analyses for the dissociation of apelin from APJ, unlabeled apelin-36 replaced more rapidly the labeled apelin-36 analogue bound with APJ than [<Glu(65)]apelin-13. Our results demonstrate that the long and short forms of apelin differently interact with APJ.

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Isolation and cDNA Cloning of a Novel Galanin-like Peptide (GALP) from Porcine Hypothalamus

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [35S]GTPγS binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9–21) was completely identical to that of galanin-(1–13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1–60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1–24 and 41–53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1–60) had a high affinity for the GALR2 receptor (IC50 = 0.24 nm) and a lower affinity for the GALR1 receptor (IC50 = 4.3 nm). In contrast, galanin showed high affinity for the GALR1 (IC50= 0.097 nm) and GALR2 receptors (IC50 = 0.48 nm). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.