Cell-cell communication is fundamental to the function of the nervous system. Operations ranging from the simplest reflex arc to the most complex processes of learning and memory rely on the accurate and efficient exchange of information between cells. The preeminent locus of this exchange is the synapse. The overall goal of the work proposed here is to understand the molecular mechanisms that underlie synapse formation. The experiments will focus on agrin, a protein associated with the synaptic basal lamina that is likely to direct the spatial organization of acetylcholine receptors (AChRs) and other postsynaptic components during the formation and regeneration of the neuromuscular junction. An essential step towards understanding agrin's mechanism of action is to identify and characterize the agrin cell surface receptor. In preliminary studies, methods have been developed to visualize and to quantify the binding of exogenously added agrin to both cultured chick myotubes and to AChR-rich membrane fractions from Torpedo electric organ. The putative agrin receptor defined by these methods-co- aggregates with the AChR during agrin-induced AChR clustering on cultured chick myotubes. Putative agrin receptors form Torpedo electoplax, which have agrin binding characteristics similar to those on chick cells, have been solubilized and enriched over 200-fold as compared to crude starting membranes. In the work proposed here, both cell biological and biochemical approaches will be used to further characterize and to identify the putative agrin receptor. The divalent cation, ionic strength, and PH requirements and optima for agrin binding will be determined. The cell surface binding domain of agrin will be determined using fragments of native and recombinant agrin. The mechanism of the redistribution of the putative agrin receptor, and its role in AChR clustering, will be investigated using both pharmacological tools and treatments that selectively inhibit AChR expression. Biochemical, affinity chromatography, and monoclonal antibody approaches will be used to identify and to biochemically characterize the putative agrin receptor. Reconstitution and immunological approaches will be used to functionally characterize the putative agrin receptor on muscle cells. The results of these experiments will provide fundamental insights into synapse formation, modification, loss, and recovery. Knowledge of these basic mechanisms is crucial to the development of rational diagnostic and therapeutic approaches for promoting the regeneration and restoration of function in neural tissue damaged by trauma or disease.
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