Production of recombinant human relaxin 3 in AtT20 cells.

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8.

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.

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.

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.

Immunocytochemical localization of a galanin-like peptide (GALP) in pituicytes of the rat posterior pituitary gland.

A galanin-like peptide (GALP) was recently isolated as a ligand of GalR2, a galanin receptor subtype. The GALP mRNA is expressed in the arcuate nucleus of the hypothalamus and the posterior pituitary (PP). In this study, we demonstrated the localization of GALP-immunoreactive (-ir) cells in the rat PP. In normal conditions, a few GALP-ir cells were detected in the PP, and these cells increased on dehydration for 4 days. The GALP-immunopositive reaction was dramatically enhanced by the intraperitoneal injection of colchicine. For the identification of GALP-ir cells in the PP, we performed electron microscopic observation, and also double immunocytochemical staining for GALP and S-100 protein. Both studies clearly indicated that the GALP-ir cells in the PP are pituicytes.

[Drug discovery based on the research on orphan receptor/ligand].

The present world-wide leading products are mostly receptor antagonists and agonists and enzyme inhibitors when classified by the mechanism of action. We consider there will be less possibilities of taking the initiative in successful finding of a target for an innovative drug winning a worldwide contest only by taking up known receptors and enzymes. Therefore, we have used the results of current genetic researches to utilize orphan G-protein-coupled receptors (GPERs) whose ligands are not known yet as targets for drug discovery, and have conducted various examinations about how to implement it. The results obtained here can be combined with the conventional methods for drug discovery. Hence, this technology is considered to be the main stream of future drug discovery research.