MUSCARINIC RECEPTOR COUPLING MECHANISMS

这个项目的长期目标是研究信号的机制。 大脑中的转导。理解这一过程的第一步是 开发生物化学方法，允许分子表征 受体和效应器实体。为此，针对神经递质的抗体 受体和鸟嘌呤核苷酸结合蛋白(G蛋白)已被 利用最近对它们的主要氨基酸酯的阐明而开发的 D 序列。这些抗体用于免疫亲和纯化 M受体与G蛋白的生物活性复合体。 具体地说，M受体在细胞膜上用 激动剂或拮抗剂，然后从膜上溶解。感受器 通过凝集素亲和树脂纯化，然后免疫沉淀 抗受体抗体。在激动剂存在的情况下，这允许 联合纯化相关G蛋白。G蛋白以复杂的形式存在 使用针对其受体的单特异性抗体进行鉴定 亚单位。这一实验设计将被用来鉴定毒鼠强 受体-G蛋白复合体在不同的大脑区域，并引起人们的兴趣 由于最近克隆了至少5种受体亚型，大多数 它们可以在几个大脑区域中的任何一个中找到。这些实验可以 解决每个受体亚型与哪种G蛋白相互作用的问题 一个给定的大脑区域。不同受体亚型的组合，以及 每种亚型与几种G蛋白相互作用的潜力，例如 D 是细胞反应表观特异性的分子基础 受体激动剂。细胞信号转导功能障碍可OCC R 由于受体或G蛋白亚型表达不当， 从而使其脱离正常的信号通路。这可能会在 形成阿尔茨海默氏症等疾病状态的基础，以及学习障碍的基础 G 以及记忆缺陷，大脑中的胆碱能通路被认为是 被改变。

The long-term objectives of this project are to study mechanisms of signal transduction in the brain. A first step to understanding this process is t develop biochemical methodologies allowing molecular characterization of receptor and effector entities. To this end, antibodies to neurotransmitte receptors and guanine nucleotide binding proteins (G proteins) have been developed, taking advantage of recent elucidation of their primary amino ac d sequences. The antibodies,are used in an immunoaffinity purification of biologically active complexes of muscarinic receptor and G protein. Specifically, muscarinic receptors are labelled in cellular membranes with agonists or antagonists, then solubilized from the membranes. The receptor are purified over a lectin affinity resin, then immunoprecipitated with anti-receptor antibodies. In the presence of agonist, this allows co-purification of associated G proteins. The G proteins present in comple with the receptor are identified using monospecific antibodies to their subunits. This experimental design will be used to identify muscarinic receptor-G protein complexes in various brain regions, and is of interest because of the recent cloning of at least 5 subtypes of the receptor, most of which can be found in any of several brain regions. The experiments can resolve the issue of which G protein each receptor subtype interacts with i a given brain region. The combination of different receptor subtypes, and a potential of each subtype to interact with any of several G proteins, cou d be the molecular basis for the apparent specificity of cellular responses t receptor agonists. A dysfunction of cellular signal transduction could occ r as a result of inappropriate expression of a receptor or G protein subtype, with a consequent subordination off normal signal pathways. This could in turn form the basis for disease states such as Altzheimer's, and for learni g and memory deficits where the cholinergic pathways in brain are believed to be altered.