TRANSPORT AND SORTNG OF NEURONAL MEMBRANE PROTEINS

神经元膜蛋白的运输和分类

• 批准号: 3075066
• 负责人: Richard Lee Rotundo
• 金额: $ 6.99万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1993-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3075066
• 关键词: Hirudinea; Torpedo; acetylcholinesterase; cell membrane; central nervous system; chickens; complementary DNA; enzyme mechanism; gene expression; membrane activity; membrane proteins; messenger RNA; molecular cloning; neuronal transport; neurons; nucleic acid probes; nucleic acid sequence; quail; tissue /cell culture

Neurons synthesize, sort, and transport numerous membrane proteins for localization to highly specialized functional domains on their plasma membranes. The molecular mechanisms whereby they accomplish this targeting are unknown. The long range goal of this project is to determine the molecular signal(s) responsible for targeting neuronal membrane proteins to their appropriate destinations on the cell surface and the mechanism whereby they are retained within selected regions of the plasma membrane. As a first step in understanding these mechanisms we have chosen to study the processing, transport, and localization of acetylocholinesterase (AChE) in tissue-cultured CNS neurons. AChE is the enzyme that hydrolyses the acetylcholine released by cholinergic nerves. This enzyme exists as a complex family of oligomeric forms, a subset of which are rapidly transported down the axon. In addition, a single integral membrane form of AChE is targeted to the surface plasma membrane in tissue cultured neurons where it is localized in clusters along the neurites. It is likely that the mechanism(s) employed by neurons to sort and transport AChE molecules will be shared by most if not all rapidly transported neuronal membrane proteins, whereas the final targeting will depend upon specific characteristics of subsets of membrane proteins. These are basic studies that will lead to an understanding of how all nerve cells sort and transport membrane proteins to specific regions of their plasma membranes. Our specific aims during the tenure of this proposal are: 1) to determine where, when, and how the signal specifying membrane localization of AChE in CNS neurons occurs; 2) to clone and identify full length cDNAs encoding the AChE polypeptides from chicken CNS neurons using a full length Torpedo AChE cDNA as a probe, 3) to express these cDNAs in mouse L cells to study the fate of the chicken AChE polypeptides, and 4) to inject in vitro synthesized AChE, VSV G-glycoprotein, and IL2 receptor mRNA into identified neurons of the leech (Hirudo medicinalis) to study the transport and localization of an identified rapidly transported protein and foreign membrane proteins in vivo.

神经元合成，分类和运输许多膜 用于定位到高度专业功能域的蛋白质 在它们的质膜上。 分子机制 他们完成了这个目标是未知的。 远程目标 该项目的是确定负责的分子信号 将神经元膜蛋白靶向其适当 细胞表面的目的地和它们的机制 保留在质膜的选定区域内。 作为 理解这些机制的第一步 研究的处理，运输和本地化 组织培养的中枢神经系统神经元中的乙酰氯乙酸酯酶（ACHE）。 ACHE是水解乙酰胆碱释放的酶 胆碱能神经。 这种酶是一个复杂的家庭 寡聚形式，其子集迅速向下运输 轴突。 另外，酸痛的单一整体膜形式 针对组织培养的组织中的质膜 神经元位于神经突的簇中。 这是 神经元采用的机制可能分类和 大多数（即使不是全部）将共享运输ACHE分子 运输的神经元膜蛋白，而最终 靶向将取决于子集的特定特征 膜蛋白。 这些是将导致的基础研究 了解所有神经细胞如何分类和运输膜 蛋白质到其质膜的特定区域。 我们在本提案任期期间的具体目标是：1） 确定信号在哪里，何时以及如何指定膜 中枢神经系统神经元中的ACHE定位发生； 2）克隆和 确定从中编码ACHE多肽的全长cDNA 鸡CNS神经元使用全长鱼雷ache cDNA作为 探针，3）在小鼠L细胞中表达这些cDNA以研究 鸡肉酸多肽的命运，4）注射体外 合成的ACHE，VSV G-糖蛋白和IL2受体mRNA 进入鉴定的水ech神经元（Hirudo Medicinalis）进行研究 迅速运输的已确定运输的运输和定位 蛋白质和外膜蛋白在体内。