MOLECULAR MECHANISM OF ACETYLCHOLINE RECEPTOR CLUSTERING

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The overall goal of this proposal is to elucidate the molecular mechanism of nerve-induced acetylcholine receptor (AChR) clustering. Nerve-induced AChR clustering is one of the key steps involved in formation of the neuromuscular junction (NMJ). We propose to study the complex process of AChR clustering by greatly simplifying both the nerve and muscle cell. The nerve cell will be replaced by putative clustering agents. These will be derivatives of neural tissue or basal lamina from muscle cells or Torpedo electric organ that have been shown to have AChR clustering activity in cultured muscle cells. The muscle cell will be replaced by cultured mouse fibroblasts that stably express functional cell surface AChRs (AChR-fibroblasts). This system has a number of advantages over standard nerve-muscle coculture systems for studying AChR clustering. There are 1) no nerve or muscle cell maturational changes, 2) no nerve or basal lamina factors other than those introduced and 3) no intrinsic muscle cell proteins other than the AChR and those specifically introduced for the study. The specific aims of this proposal can be divided into three major projects. Project #1 will assay putative AChR clustering agents for their ability to induce AChR clusters on the surface of AChR-fibroblasts. AChR-fibroblasts will be incubated with clustering agents and the clusters formed will be quantitatively evaluated using fluorescent labelling, photomicroscopy and computer analysis. Project #2 will determine if the clustering agents induce clustering by direct interaction with AChR or by interaction with non-AChR components of the target cell membrane or cytoskeleton. Fibroblasts will be exposed to clustering agents and then the agent will be removed before the fibroblasts are induced to express surface AChRs. If clusters do not form, it will be suggestive that the clustering agent directly interacts with AChRs. If clusters do form, it will suggest that the AChR is not the primary target of the clustering agent. Project #3 will determine which AChR subunits and domains of subunits are involved in clustering. We will develop fibroblast cell lines that express specifically mutated AChRs and analyze those which are fully functional, but have lost the ability to cluster; any defects in clustering can be attributed to the mutated subunit or domain. Study of AChR clustering and its role in neuromuscular synaptogenesis has wide ranging medical significance. The ability to understand and manipulate AChR dynamics at the NMJ may be useful in preventing and treating such diseases as amyotrophic lateral sclerosis, inflammatory myopathies and muscular dystrophies and respiratory failure due to neuromuscular transmission failure at NMJs in the diaphragm. In addition, the NMJ and its AChRs are a model for nerve-nerve synapses and the ligand-gated receptor channels involved in nerve-nerve synaptic transmission. Thus, study of the mechanisms by which the motoneuron exerts its influence upon the muscle cell may help elucidate the mechanisms by which central nervous system neurons exert influences upon each other across synapses. Such effects may be important for the development of learning and memory.