权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of glucagon receptor by a novel functional cloning technique

通过新型功能克隆技术分析胰高血糖素受体

基本信息

批准号：
05807087
负责人：
TSUKADA Toshihiko
金额：
$ 1.22万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05807087/
关键词：
beta-Galactosidase Menbrane Receptor Vasoactive Intestinal Peptide Proopiomelanocoertin Gene Expression Cyclic AMP AtT-20 PC12 CAMP POMC

项目摘要

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.

1.将含有人促黑素原基因5′侧区和大肠杆菌lacZ基因的嵌合基因稳定导入多种细胞系，包括产生acth的小鼠at -20细胞。嵌合基因以细胞特异性方式在at -20细胞中表达。发现了一种细胞特异性的促黑色素皮质素基因增强子。利用含有人血管活性肠肽（VIP）基因5′-侧翼的嵌合基因稳定转化PC12大鼠嗜铬细胞瘤细胞PC12- vg细胞。PC12-VG细胞具有最低的-半乳糖苷酶活性，但在激活蛋白激酶a的试剂处理后显示出较高的-半乳糖苷酶活性。用表达人黄体生成素受体的质粒转染PC12-VG细胞，用人绒毛膜促性腺激素刺激PC12-VG细胞，显示出较高的-半乳糖苷酶活性，而未转染和未刺激的PC12-VG细胞均未检测到-半乳糖苷酶活性。因此，PC12-VG细胞被证明可用于检测外源引入的与腺苷酸环化酶正偶联的膜受体cDNA。分析VIP基因在PC12细胞中的表达。腺苷酸环化酶活化剂Forskolin和TPA均增加了PC12细胞中VIP mRNA的含量以及VIP从PC12细胞中的释放。VIP在PC12细胞中诱导VIP mRNA表达。