MUSCARINIC RECEPTOR COUPLING MECHANISMS

这些研究将探讨M受体蛋白的能力。 与效应器蛋白相互作用。在大鼠脑内制备的膜中 毒蛾碱受体与效应蛋白的相互作用 使用双功能蛋白质-蛋白质交联剂进行研究， 随后依次进行增溶、亲和层析或 免疫沉淀、亲和标记和凝胶电泳法。焦点 这些在膜上的研究将为 形成以下物种的紧密连接的生物特异性复合体 受体-Ni，这是已知的调节腺苷酸环化酶的活性。 M受体与其他效应器的相互作用将是 特别是最近发现的GTP结合蛋白。为 在这些研究中，膜将与毒碱激动剂孵育，以及 然后用交联剂。这样形成的共价络合物将是 用亲和层析或亲和层析进行溶解和部分纯化 免疫沉淀。含有释放的蛋白质的级分将是 用凝胶电泳法进行分析。释放的蛋白质的同一性为 将建立IAP底物和毒鼠碱配体结合蛋白 共价标记蛋白质的十二烷基硫酸钠凝胶电泳法。这些 研究应提供有关毒鼠强的有用信息 受体-效应器耦合机制，以及建立方法学 检测M受体介导的反应的方案。

These studies will investigate the ability of muscarinic receptor proteins to interact with effector proteins. In membranes prepared from rat brain regions, interactions of muscarinic receptors with effector proteins will be studied using bifunctional protein-protein cross-linking agents, followed sequentially by solubilization, affinity chromatogrphy or immunoprecipitation, affinity labeling, and gel electrophoresis. The focus of these studies in membranes will be to establish conditions for the formation of covantly linked biospecific complexes of such species as receptor-Ni, which are known to modulate adenylate cyclase activity. Interaction of the muscarinic receptor with other effectors will be identified, in particular, recently identifiedd GTP-binding proteins. For these studies, membranes will be incubated with muscarinic agonists, and then with cross-linking agents. The covalent complexes thus formed will be solubilized and partially purified using affinity chromatograhy or immunoprecipitation. Fractions containing released proteins will be analyzed using gel electrophoresis. The identity of released proteins as IAP substrates and muscarinic ligand-binding proteins will be established following SDS-gel electrophoresis of covalently labelled proteins. These studies should provide useful information about muscarinic receptor-effector coupling mechanisms, as well as establish methodological protocols for examining muscarinic receptor-mediated responses.