The goals of this research are to elucidate the molecular mechanism of acetylcholine receptor (AChR) clustering on cell surfaces, study the process of subunit assembly and define the acetylcholine (ACh) binding site using the techniques of gene cloning, in vitro mutagenesis and gene transfer. The long-term objectives are to further define the roles the AChR plays in synaptic transmission, in synaptogenesis and in the autoimmune disease, myasthenia gravis. Project I: Clustering Mammalian tissue culture cells will be transformed with the 4 Torpedo AChR subunit cDNA clones engineered into appropriate expression vectors, and cell lines which stably express functional AChR complexes on their surface will be established. Extensive biochemical, kinetic, immunological and electrophysiological analyses will be performed on these cell lines before they will be used to study the process of AChR clustering. The transformed cell lines will be co-cultured with nerve cells or treated with various factors in order to induce cluster formation. New cell lines will next be created that contain mutant AChR subunit genes. Receptors which possess all normal properties except the ability to cluster will allow identification of those regions of the protein required for clustering. Project II: Assembly Cells will be transformed with all possible combinations of 1, 2 and 3 genes. Biochemical and immunological analyses of these lines will reveal whether subunits can stably self-assemble and whether one subunit can substitute for any other subunit in terms of assembly or function. If stable receptor complexes can be formed with fewer than 4 different subunits, then it may be possible to assign certain biological properties to specific subunits. Project III: Ligand Binding Site Biochemical studies have identified specific regions of the AChR as the putative ACh binding sites. In vitro site-specific mutations creating single base-pair changes that lead to single amino acid changes will further define the binding site region, identify amino acids in the site critical for agonist and antagonist binding and ultimately further our understanding of how agonist binding and channel opening are coupled.
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