MUSCARINIC RECEPTOR COUPLING MECHANISMS

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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.