This application seeks funds to continue the on-going project of producing and characterizing site-directed anti-peptide antibodies to muscarinic receptor (MR) subtypes. The goal of the proposal is to study the distinct structure, binding properties, function and location of MR subtypes in mammalian brain. MR subtypes postulated on the basis of pharmacological binding data have been verified by the recent molecular biological evidence for five distinct MR genes coding for unique primary sequences. Nevertheless, correlation between the pharmacology, biochemistry and function of each subtype remain unclear, primarily because there are no available methodologies which distinguish the five receptor proteins. Currently available muscarinic ligands distinguish only three MR subtypes, and nucleotide probes do not recognize gene product, or receptor, but only DNA or RNA receptor message. An immunological approach, therefore, has the advantage of 1) distinguishing between the highly homologous receptor proteins and 2) directly recognizing receptor proteins. Specifically, mono- and polyclonal antibodies to unique sequences of m1-m5 MR subtypes will be produced. Thus far, eight monoclonal antibodies from hybridomas and ascites-producing mice have been generated to peptide uniquely corresponding to m1. Polyclonal antibodies have also been generated to peptides m2 and m3. A combination of immunodot, ELISA, immunoblotting, immunoprecipitation and immunohistochemistry will be used to screen and characterize antibodies with respect to specificity for the immunogen, MR, MR subtype, and molecular location. Ultrastructural location of MR subtypes in the rodent and human brain will be assessed at the light and electron microscopic levels. Associated G proteins will be identified electrophoretically by specific immunoprecipitation of soluble MR-G protein complexes. Distinction in primary structure between the MR subtypes will be made using two-dimensional electrophoresis after selective immunoprecipitation or immunoaffinity chromatography. Results of these studies will provide a clearer understanding of central MR which remain important targets for therapeutic intervention in treating memory disorders associated with advanced age and Alzheimer's Disease.
