Signaling Mechanisms Behind nAChR Clustering at the NMJ

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The neuromuscular junction (NMJ) has a structure that is optimized to relay signals from nerve to muscle. As part of this organizational scheme, certain muscular proteins, like the nicotinic acetylcholine receptor (nAChR), are clustered preferentially at the NMJ. Clustering of the nAChR at the NMJ is essential for efficient nerve and muscle transmission. A major factor that strengthens and sustains the NMJ localization of the nAChR is the neural-derived protein agrin. Agrin acts via a receptor complex that includes muscle-specific kinase (MuSK). Although MuSK has been well characterized, the signaling pathway by which it leads to agrin-induced clustering of the nAChR remains poorly understood. Elucidation of the components of this cascade may provide insights into future therapies for the treatment of a variety of neurological and muscle weakness disorders, such as myasthenia gravis and muscular dystrophy. For instance, treatments can be designed to circumvent, enhance, or inhibit specific steps of the pathway in diseases where the cascade is disrupted. In addition, this pathway may reveal mechanisms important for the formation and/or maintenance of synapses. In an effort to uncover the initial steps of the agrin -" MuSK -> nAChR signaling pathway, a bacterial two hybrid assay was used to identify a protein, a heat shock protein 40 homologue (hsp40h), that interacts strongly with the cytoplasmic region of MuSK. In the present proposal, it is hypothesized that interaction between MuSK and hsp40h is a key component of the signaling pathway responsible for agrin-mediated clustering of the nAChR. The proposed research aims to confirm the interaction between MuSK and hsp40h, to determine how interaction between MuSK and hsp40h affects agrin-mediated clustering of the nAChR, and to examine hsp40h's role in the development and maintenance of the NMJ. These goals will be accomplished by performing immunoblotting, coimmunoprecipitation, immunofluorescent staining, mutational analyses, RNAi-mediated protein knock-down, and denervation studies with cultured myotubes and rodent muscles. The experiments outlined in this proposal are likely to provide insights into the mechanisms responsible for the organization of the neuromuscular junction. Many diseases result from disruptions of the normal structures of nerves and muscles. Thus, studying the fundamental process of neuromuscular development is critical to understanding the causes of and to designing more effective treatments for these debilitating conditions.