The long-range goal of this work is to understand how signal transduction is established and maintained at CNS cholinergic synapses; its focus is on the biochemistry, development and cellular regulation of muscarinic ACh receptors. These receptors mediate a large proportion of CNS cholinergic signaling, have been implicated in many behaviorally and clinically significant phenomena and show a broad spectrum of transductional and regulatory responses. At present, it appears most appropriate to isolate and begin characterizing receptor system proteins and to generate receptor specific antibodies for studies of muscarinic cell and developmental biology. The following three aims have therefore been chosen:
AIM #1 --To generate a library of muscarinic receptor-specific monoclonal antibodies. Partially purified (250-fold) bovine brain receptors will be used for antibody induction using a combination of methods already tested for their effectiveness. Binding assays and western blots will be used in testing antibody specificity.
AIM #2 --To purify and begin characterization of receptor molecules. Immunoaffinity chromatography will be added to tested separations based on subcellular fractionation, hydrophobicity, charge, molecular weight and specific glycosylation. 2D-Gel, sedimentation equilibrium and N-terminal analyses will be used in testing purity. The ligand specificity, number of subunits, number of binding sites, and overall amino acid composition of the purified molecules will be assessed.
AIM #3 --To localize receptors as a function of synaptic development, post-translational modification, and cholinergic stimulation. The monoclonal antibodies obtained above will be used to localize receptors on avian retina neurons at the subcellular and ultrastructural levels. A novel approach employing isolation of differentiated neurons and HVEM whole mount analysis will be used.
