Takuya Watanabe

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.

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.

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.

Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline

Pituitary adenylate cyclase activating polypeptide (PACAP) provoked the rat chromaffin cells to secrete adrenaline. Within 20 min, the amount of adrenaline secreted by PACAP (10(-8) M) was as much as that caused by acetylcholine (10(-4) M). PACAP, but not acetylcholine, induced a long-term (over 120 min) increase in secretion of adrenaline. PACAP also activated adenylate cyclase and elevated cytosolic Ca2+ concentration. Furthermore, we found immunoreactive PACAP and PACAP binding sites in the rat adrenal medulla. These results suggest that PACAP has an important role in stimulating secretion of adrenaline in the adrenal medulla.

Galanin-like peptide stimulates food intake in the rat

We have isolated a novel hypothalamic peptide, Galanin-like peptide (GALP), as a ligand for galanin receptor subtype GalR2. To investigate the physiological role of GALP, we examined the effect of the intracerebroventricular administration of GALP and found that GALP induced food intakes. GALP had ten-fold the orexigenic activity of galanin. We also observed the anxiogenic-like behavior after the administration of 1 nmol GALP. These results suggest that GALP is a novel orexigenic and anxiogenic peptide.

Molecular identification of receptor for pituitary adenylate cyclase activating polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide with 38 (PACAP38) or 27 (PACAP27) amino acid residues, structurally related to vasoactive intestinal peptide (VIP). Bovine brain membrane has a PACAP specific receptor interacting with both PACAP27 and PACAP38. Affinity-labeling of the receptor with [125I]PACAP27 identified a dominant band of Mr = 60 k. The labeling density of the 60 k band decreased in the presence of unlabeled PACAP27 or PACAP38, whereas the 60 k band remained in the presence of unlabeled VIP. Binding of [125I]PACAP27 to the membrane decreased in the presence of GTP and the labeling density of the 60 k band decreased concomitantly. The results indicate that bovine brain has a specific PACAP receptor, whose apparent molecular weight is 57 k (substracting the molecular weight of [125I]PACAP27 from 60 k).

Adrenal pheochromocytoma PC12H cell respond to pituitary adenylate cyclase activating polypeptide

An adrenal pheochromocytoma cell line, PC12h, was found to respond to a novel hypothalamic neuropeptide, Pituitary Adenylate Cyclase Activating Polypeptide (PACAP). The cells elevated both intracellular and extracellular cAMP levels on stimulation by PACAP, whereas they showed little response to VIP which is structurally related to PACAP. Using [125I]PACAP27 (a shorter form of the peptide) and [125I]VIP, we found large amounts of specific binding sites for PACAP but few binding sites for VIP in PC12h cells. These results indicate that PC12h cells respond to PACAP via a specific PACAP receptor.

Intracellular degradation of recombinant proteins in relation to their location in Escherichia coli cells

Abstract Recombinant interferon-αA (rIFN-αA) accumulated in Escherichia coli 294 was rapidly degraded in vivo when glucose or oxygen in the medium was limited, whereas recombinant interferon-γ (rIFN-γ) and recombinant interleukin-2 (rIL-2) were stable under the same conditions. The degradation in vitro of rIL-2 by E. coli proteases was as rapid as that of rIFN-αA. On the other hand, rIFN-γ maintained the same activity after proteolysis in vitro. Western blot analysis of the reaction mixture, however, showed that the rIFN-γ molecule was converted into a smaller protein of about 15 kDa by losing the COOH-terminal portion of the peptide. Electron microscopic observations showed that rIFN-γ and rIL-2 accumulated and formed inclusion bodies. No clear inclusion bodies were found in the cells accumulating rIFN-αA, which is rapidly degraded in vivo. These results indicate that intracellular location and protein structure are related to the stability in vivo of recombinant proteins. rIFN-αA was also degraded rapidly in a lon− mutant of the same organism, suggesting that protease La does not participate in this degradation process.

Intracerebroventricular administration of urotensin II promotes anxiogenic-like behaviors in rodents

We identified urotensin II (U-II) as the endogenous ligand for the orphan G-protein-coupled receptor GPR14 or SENR. Both U-II and GPR14 are expressed not only in peripheral tissues but also in the brain of rodents, suggesting that U-II plays a physiological role in the central nervous system. In the present study, we investigated the central effects of U-II in rodents. Intracerebroventricular administration of U-II induced anxiogenic-like behaviors in the elevated plus maze test and the hole-board test in mice in a dose-dependent manner, as did corticotropin releasing factor (CRF). The effective doses of U-II were 10-100-fold higher than these of CRF in these tests. Our results suggest that U-II is a candidate for the mediator of some aspect of stress or anxiety in the central nervous system.

Expression of human pituitary adenylate cyclase activating polypeptide (PACAP) cDNA in CHO cells and characterization of the products

cDNA encoding human PACAP precursor was expressed in non‐neuroendocrine Chinese hamster ovary cells, CHO‐K1. The cells were transfected with expression vector (pTS705) containing the human PACAP cDNA by electroporation. A cell line which produced more than 80 ng/ml of immunoreactive PACAP (ir‐PACAP) into the conditioned medium was established. RP‐HPLC analysis of culture medium of this established cell line exhibited the presence of two types of PACAP, i.e. PACAP38 and PACAP27. At the same time, it was also revealed that immunoreactive PACAP‐related peptide (ir‐PRP) was secreted into the cultured medium. The ir‐PACAPs were confirmed to have biological activities such as induction of cAMP and neurite outgrowth in rat pheochromocytoma PC12h cells.