TRANSPORT AND SORTING OF NEURONAL MEMBRANE PROTEINS

神经元膜蛋白的运输和分选

• 批准号: 3411005
• 负责人: Richard Lee Rotundo
• 金额: $ 9.01万
• 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: 1988-02-01 至 1992-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3411005
• 关键词: Hirudinea; acetylcholinesterase; biological transport; cell membrane; gene expression; membrane activity; membrane permeability; membrane proteins; messenger RNA; molecular cloning; nerve /myelin protein; neurons; nucleic acid probes; protein structure; protein transport; 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 acetylcholinesterase (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 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，分类和运输许多膜蛋白 将其本地化到高度专业的功能域上 质膜。 他们完成的分子机制 这个目标是未知的。 该项目的远距离目标 是确定负责靶向的分子信号 神经元膜蛋白到其适当的目的地 细胞表面及其保留在内的机制 质膜的选定区域。 作为第一步 了解这些机制，我们选择了 乙酰胆碱酯酶的加工，运输和定位 （ACHE）在组织培养的中枢神经系统神经元中。 疼痛是一种酶 水解胆碱能神经释放的乙酰胆碱。 这 酶作为一个复杂的寡聚形式家族，一个子集 迅速向下运输的轴突。 另外，一个 ACHE的整体膜形式针对表面等离子体 组织培养的神经元中的膜，该神经元位于该神经元中 沿着神经突。 机制很可能 神经元用来分类和运输ACHE分子将是 大多数（即使不是全部迅速运输的神经元膜）共享 蛋白质，而最终靶向将取决于特定 膜蛋白子集的特征。 这些是基本的 将导致了解所有神经细胞的研究 将膜蛋白分类并运输到其特定区域 质膜。 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 CNS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以研究命运 鸡肉ACHE多肽和4）注入合成 ACHE，VSV G-糖蛋白和IL2受体mRNA识别为 水ech神经元（Hirudo Medicinalis）研究运输 以及确定的快速运输蛋白质和 体内外膜蛋白。