The neuromuscular junction is the best understood synapse both structurally and developmentally. Elucidation of the specific molecular mechanisms of its development should provide insights into diseases of muscle and nerve as well as to serve as a model to study central nervous system development. Acetylcholine receptor inducing activity (ARIA) is a soluble factor released from nerve cells prior to nerve-muscle contact which increases the expression of acetylcholine receptors (AChR) in myotubes. Recently, ARIA has been found to be highly homologous to neu differentiation factor (NDF) and heregulin which bind to receptors encoded by the oncogene neu (also known as erbB-2 and HER-2) and lead to changes in cell differentiation and proliferation. This suggests that ARIA operates through similar mechanisms in regulating the development of the neuromuscular junction by interacting with a neu encoded receptor on the muscle cell surface. Experiments proposed here will investigate how ARIA contributes to the development of the neuromuscular junction by examining how ARIA is processed, whether the membrane-bound precursor is functionally active through cell-cell interactions, and how ARIA contributes to synaptic maturation. Methods will utilize both primary cell cultures and transfected cell lines expressing ARIA. Site-directed mutagenesis will be used to produce altered membrane-bound precursors of ARIA in order to study processing and to determine the function of the various structural domains. Long range efforts will attempt to understand ARIA's role in the maturation of the neuromuscular junction by examining the stability of AChR clusters.
