CELLULAR TRANSPORT OF ACETYLCHOLINE RECEPTOR SUBUNITS

乙酰胆碱受体亚基的细胞转运

• 批准号: 2036625
• 负责人: STEVE KELLER
• 金额: $ 3.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2036625
• 关键词: Golgi apparatus; acetylcholine; cholinergic receptors; complementary DNA; confocal scanning microscopy; endoplasmic reticulum; glycosylation; intermolecular interaction; intracellular transport; membrane proteins; molecular chaperones; protein biosynthesis; protein transport; site directed mutagenesis; tissue /cell culture

The general aim of this project is to identify several cellular factors that contribute to the assembly and cell surface expression of the nicotinic acetylcholine receptor expressed by transfection in mammalian cells. The first aim is to determine whether the adherence of the chaperone protein calnexin to unassembled receptor subunits acts to retain receptor subunits in the endoplasmic reticulum until assembly occurs. Using a truncated calnexin cDNA that lacks its ER retention signal and is transported to the cell surface, truncated calnexin cDNA and receptor subunit cDNA will be co-transfected and the cellular localization of receptor subunits will be characterized. The second aim is to determine whether a putative endoplasmic reticulum retention code located in the carboxyl-terminus of the receptor's delta-subunit contributes to its retention. Site-directed mutagenesis will alter this code, and transport to the golgi apparatus will be monitored using dual- staining immunofluorescence. The third aim is to determine whether the association of calnexin with unassembled subunits provides protection from degradation. Calnexin usually recognizes glycoproteins by forming contracts with N-linked oligosaccharide chains, which can be removed by altering the signal Asn-X-Ser/Thr. All nicotinic acetylcholine receptor subunits possess a conserved glycosylation site at Asn-141. The Asn-141 glycosylation site will be altered and calnexin association, receptor accumulation, and transport to the endosomes will be assessed.

该项目的总体目的是识别几个蜂窝 因素 这有助于组装和细胞表面表达 通过转染表达的烟碱乙酰胆碱受体 哺乳动物 细胞。第一个目的是确定是否遵守 伴侣蛋白钙钙蛋白钙钙蛋白钙蛋白钙蛋白钙蛋白辣汁油酰之末粉的受体亚基作用至 将受体亚基保留在内质网中直至 集会 发生。 使用缺乏ER的截短的钙网蛋白cDNA 保留 信号并转运到细胞表面，截短的钙钙蛋白 cDNA 和受体亚基cDNA将被共转染，细胞 将表征受体亚基的定位。 这 第二个目标 是确定假定的内质网保留率是否保留 代码 位于受体的三角洲 - 亚基的羧基末端 有助于其保留。 定点诱变将改变 这 代码，将使用运输到高尔基设备 双重的- 染色免疫荧光。 第三目的是确定是否 这 钙钙蛋白与未组装的亚基的关联提供 保护 从退化。 钙钙蛋白通常通过 成型 与N连接的寡糖链合同，可以是 删除 更改信号ASN-X-SER/THR。 所有烟碱乙酰胆碱 受体 亚基在ASN-141处具有保守的糖基化位点。 这 ASN-141 糖基化位点将改变并钙钙蛋白酶结合， 受体 积累，将评估到内体的运输。