Nerve-induced clustering of nicotinic acetylcholine receptors (AChRs) is among the earliest detectable changes in the postsynaptic membrane during neuromuscular synaptogenesis. In the past decade, the mechanisms that regulate AChR clustering have been the subject of intense investigations. One approach has focussed on identification of the signaling molecules released by the nerve and their mode of action. Considerable evidence now supports the hypothesis that agrin is a key nerve-derived signal in regulating AChR clustering. The other approach, the focus of our studies, has sought to identify postsynaptic cytoskeletal and peripheral membrane proteins that anchor AChRs at the synaptic site. The 43K protein has received the most attention because of its close association with the AChR and its inherent clustering activity. Coexpression of the 43K protein with AChRs in oocytes causes coclustering of the two proteins. The clusters are small, however, reminiscent of those that appear in the earliest stages of synapse formation. Thus, other synaptic proteins may be needed for full clustering activity. Utrophin, a member of the dystrophin family, is perhaps the strongest candidate for this activity. Of all the proteins concentrated at the neuromuscular junction, utrophin is the only known protein that is confined to precisely the same postsynaptic sites as 43K and AChR. Furthermore, utrophin is believed to be associated via the transmembrane beta-dystroglycan with alpha-dystroglycan, an extracellular protein recently shown to be a major binding site, and perhaps the functional receptor, for agrin. In this proposal, we will test the hypothesis that agrin and the 43K protein are structurally and functionally linked via the dystroglycan/utrophin complex. Targeting of the proteins of this complex to clusters of AChR and 43K protein will be studied by expression of recombinant proteins in oocytes. The effects of agrin on recombinant dystroglycan/utrophin complex assembly and on the clustering of AChRs will be investigated in this system. Biochemical studies will test for direct interactions of the 43K protein with utrophin and other members of the utrophin complex (syntrophin, adhalin, beta- dystroglycan and 35K). Domains of the 43K protein involved in these interactions will be identified. Finally, we will search for other postsynaptic proteins, including tyrosine phosphorylated proteins, that interact with the 43K protein, and may be important for its intrinsic clustering activity and interaction with the utrophin complex. Since a related, dystrophin-based complex is present at postsynaptic sites in the central nervous system, these studies should bear on the mechanisms of synaptogenesis in the brain, and lead to a clearer understanding of the nervous system dysfunction and mental retardation characteristic of patients with Duchenne muscular dystrophy.
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