Characterization of the Molecular Mechanisms Regulating the Agrin Signaling Pathway: a Dissertation

Agrin is an extracellular matrix protein that directs neuromuscular junction formation. Early signal transduction events in agrin-mediated postsynaptic differentiation include activation of a receptor tyrosine kinase and phosphorylation of AChRs , but later steps in this pathway are unknown. Here, we have investigated the role of intracellular calcium in agrin-induced AChR clustering on cultured myotubes. Clamping intracellular calcium levels by loading with the fast chelator BAPT A inhibited agrin-induced AChR aggregation. In addition , preexisting AChR aggregates dispersed under these conditions, indicating that the maintenance of AChR clusters is similarly dependent on intracellular calcium fluxes. The decrease in AChR clusters in BAPT A-loaded cells was dosedependent and reversible, and no change in the number or mobilty of AChRs was observed. Clamping intracellular calcium did not block agrin-induced tyrosine phosphorylation ofthe AChR ~-subunit, indicating that intracellular calcium fluxes are likely to act downstream from or parallel to AChR phosphorylation. Finally, the targets of the intracellular calcium are likely to be close to the calcium source, since agrininduced AChR clustering was unaffected in cells loaded with EGT A, a slower-binding calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibilty that the pathways mediating agrinand activity-driven changes in synaptic architecture could intersect at the level of intracellular