MUSCARINIC RECEPTOR COUPLING MECHANISMS

这些研究将调查毒蕈碱受体蛋白的能力， 与效应蛋白相互作用。 在从大鼠脑中制备的膜中， 区域，毒蕈碱受体与效应蛋白的相互作用将 使用双功能蛋白质-蛋白质交联剂进行研究， 随后依次进行溶解、亲和层析或 免疫沉淀、亲和标记和凝胶电泳。 重点 这些膜研究将建立条件， 形成共价连接的生物特异性复合物， 受体-Ni，其已知调节腺苷酸环化酶活性。 毒蕈碱受体与其他效应物的相互作用将被研究。 鉴定，特别是最近鉴定的GTP结合蛋白。 为 在这些研究中，将膜与毒蕈碱激动剂一起孵育， 然后用交联剂。 由此形成的共价复合物将是 溶解并使用亲和色谱法部分纯化，或 免疫沉淀。 含有释放的蛋白质的级分将被 使用凝胶电泳分析。 释放的蛋白质的身份 将建立IAP底物和毒蕈碱配体结合蛋白 随后对共价标记的蛋白质进行SDS-凝胶电泳。 这些 研究应该提供有关毒蕈碱的有用信息 受体-效应器偶联机制，以及建立方法 用于检查毒蕈碱受体介导的应答的方案。

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.