Tsutomu Kurokawa

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.

Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.

Glia-activating factor (GAF) is a novel heparin-binding growth factor purified from the culture supernatant of a human glioma cell line. It shows a spectrum of activity slightly different from those of other known growth factors. We have isolated the cDNA which encodes human GAF. A homology search revealed that GAF would be the ninth member of the FGF family, and we therefore call it FGF-9. The human FGF-9 cDNA cloned by using oligonucleotide probes encoded a polypeptide consisting of 208 amino acids. Sequence similarity to other members of the FGF family was estimated to be around 30%. Two cysteine residues and other consensus sequences in family members were also well conserved in the FGF-9 sequence. FGF-9 was found to have no typical signal sequence in its N terminus like those in acidic FGF and basic FGF. Acidic FGF and basic FGF are known not to be secreted from cells in a conventional manner. However, FGF-9 was found to be secreted from cells after synthesis despite its lack of a typical signal sequence. It could be detected exclusively in the culture medium of cDNA-transfected COS cells. The amino acid sequence of proteins purified from culture supernatant of the CHO cell line, which was cDNA transfected and selected as a high producer of FGF-9, showed that no peptides were cleaved from the N terminus except the initiation methionine. The rat FGF-9 cDNA was also cloned, and the structural analysis indicated that the PGF-9 gene is highly conserved. Expression of the FGF-9 gene could be detected in the brain and kidney of the adult rat. Restricted gene expression in organs and the unique secretion nature of the protein suggest that FGF-9 plays a physiological role which differs from those of well-characterized acidic FGF and basic FGF.

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.

Two types of complementary DNAs of rat brain protein kinase C. Heterogeneity determined by alternative splicing.

Two types of complementary DNA clones for rat brain protein kinase C were isolated. These clones encode 671 and 673 amino acid sequences, which differ from each other only in the carboxyl‐terminal regions of approx. 50 amino acid residues. This difference seems to result from alternative splicing. Elucidation of the sequences of these cDNA clones as well as some peptides from the purified rat brain enzyme suggests the existence of an additional species of protein kinase C in this tissue. It is attractive to imagine that the heterogeneity of protein kinase C may reflect diverse pathways of signal transduction into the cell.

Stabilizing basic fibroblast growth factor using protein engineering

Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.

Transformation of mouse BALB/c 3T3 cells with human basic fibroblast growth factor cDNA.

The expression of human basic fibroblast growth factor (bFGF) cDNA in mouse BALB/c 3T3 clone A31 cells induced morphological transformation. These transformed cells grew well and reached more than a sixfold-higher saturation density than parental A31 cells even in serum-free medium. They were able to form colonies in soft agar. The phenotypic alteration in the transformed cells was reversed by the addition of anti-human bFGF antibodies to the medium. These results suggest that the cellular transformation mediated by bFGF is caused by autocrine stimulation with secreted bFGF molecules.

Tissue distribution of prolactin-releasing peptide (PrRP) and its receptor.

Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.