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.

该项目的总体目标是识别几种细胞 因素 有助于组装和细胞表面表达 烟碱型乙酰胆碱受体通过转染表达 哺乳动物的 细胞。第一个目的是确定是否遵守 伴侣蛋白钙连接蛋白对未组装的受体亚基起作用 在内质网中保留受体亚基，直到 集会 发生。 使用缺乏 ER 的截短的钙联蛋白 cDNA 保留 信号并被转运至细胞表面，截短的钙连接蛋白 cDNA 和受体亚基cDNA将被共转染并且细胞 将表征受体亚基的定位。 这 第二个目标 是为了确定假定的内质网保留 代码 位于受体δ亚基的羧基末端 有助于其保留。 定点诱变将改变 这 代码，并将使用监控到高尔基体的运输 双重的- 免疫荧光染色。 第三个目标是确定是否 这 钙联蛋白与未组装亚基的关联提供了 保护 免于退化。 钙连接蛋白通常通过以下方式识别糖蛋白： 成型 与 N-连接寡糖链的合同，这可以是 删除者 改变 Asn-X-Ser/Thr 信号。 全烟碱乙酰胆碱 受体 亚基在 Asn-141 处具有保守的糖基化位点。 这 Asn-141 糖基化位点将被改变并且钙连接蛋白关联， 受体 将评估积累和运输到内体的情况。