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.
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