Analysis of glucagon receptor by a novel functional cloning technique

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1.A chimeric gene containing the 5'-flanking region of the human proopiomelanocortin gene and E.coli lacZ gene was stably introduced into various cell lines including ACTH-producing mouse AtT-20 cells. The chimeric gene was expressed in AtT-20 cells in a cell-specific manner. A cell-spcific enhancer of the proopiomelanocortin gene was identified.2.PC12-VG cells, which are PC12 rat pheochromocytoma cells stably transformed with a chimeric gene containing the 5'-flanking region of the human vasoactive intestinal peptide (VIP) gene, was developed. PC12-VG cells had minimal beta-galactosidase activities but showed high beta-galactosidase activity after treatment with reagents that activate protein kinase A.The PC12-VG cells were further transfected with a plasmid expressing the human luteinizing hormone receptor and stimulated with human chorionic gonadotropin showed high beta-galactosidase activity whereas no beta-galactosidase activity was detected in neither non-transfected nor unstimulated PC12-VG cells. Thus, PC12-VG cells were shown to be useful to detect exogenously introduced cDNA of membrane receptors that are positively coupled with adenylate cyclase.3.VIP gene expression in PC12 cells were analyzed. Forskolin, an adenylate cyckase activating agent and TPA increased VIP mRNA content in PC12 cells as well as VIP release from PC12 cells. It was also shown that VIP induced VIP mRNA in PC12 cells.