The mammalian neuromuscular junction undergoes a number of developmental changes between the time of initial nerve-muscle contact and the establishment of a mature, fully-developed synapse. Little is known about the molecular mechanisms responsible for these alterations but it seems likely that synaptic proteins other than the acetylcholine receptor are involved, particularly in the process of anchoring receptors at the synapse. For anchoring, the submembrane postsynaptic proteins (the postsynaptic cytoskeleton) are probably of special importance. This proposal focuses on these proteins, taking advantage of the Torpedo electrocyte synapse as a model for the neuromuscular junction. Several experimental approaches, including the use of monoclonal antibodies in our laboratory, have shown that a major Mr 43,000 peripheral membrane protein (43K protein) that is distinct from actin resides in situ on the cytoplasmic side of the postsynaptic membrane of Torpedo electrocytes in a distribution coextensive with the receptors. A component related to this protein is present in rat muscle endplates and is associated with clusters of receptors found on cultured Xenopus and chick myotubes. We will continue our work on this protein and other components of the postsynaptic cytoskeleton with particular emphasis on the cellular mechanisms involved in the formation and maintenance of receptor accumulations and other neuromuscular junction specializations. Specifically, the temporal appearance of 43K protein will be related to functional and structural changes that occur at developing embryonic rat synapses, at ectopic synapses, and at receptor clusters on myotubes in vitro. Monoclonal antibodies to the 43K protein will be microinjected into myotubes to examine their effects on the formation of receptor patches. The interaction of purified 43K protein with other components of the postsynaptic membrane, including the receptor, will be examined. A similar approach will be used to study a recently-discovered Mr 58,000 protein that may exist in a complex with the 43K protein. Ultimately, we intend to determine the function of these and other proteins of the cytoplasmic specialization. Abnormalities in the structure or expression of the components of the postsynaptic cytoskeleton undoubtedly have serious effects on the formation, function, and stability of the neuromuscular junction.
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