At synapses throughout the nervous system there are structural specializations that play a direct role in synaptic transmission. The formation and maintenance of such structural specializations relies on communication between the axon terminal and its target. For example, at the vertebrate skeletal neuromuscular junction the axon terminal releases signals that direct the muscle fiber to organize a postsynaptic apparatus, which includes aggregates of nicotinic acetylcholine receptors (AChRs) and other extracellular, membrane, and cytoplasmic components. Several lines of evidence indicate that agrin, a protein originally identified in extracts of the synapse-rich electric organ of the marine ray Torpedo, is such a signal, mediating the nerve-induced formation of postsynaptic specialization at embryonic and regenerating neuromuscular junctions. When added to myotubes in culture, agrin causes the formation of patches at which AChRs and other components of the postsynaptic apparatus are aggregated. Components of such aggregates appear to be held in place by attachment to the underlying cytoskeleton. Agrin also causes AChRs to be phosphorylated, a modification that appears to be involved in regulating receptor distribution. The goal of this project is to understand the mechanism of action of agrin at the molecular level and, in particular, to test the hypothesis that tyrosine phosphorylation of AChRs, and perhaps other proteins as well, plays a role in receptor aggregation.
The specific aims are: i) To determine if tyrosine phosphorylation of the AChR beta-subunit is necessary for agrin-induced AChR aggregation, ii) To determine the role of tyrosine phosphorylation and AChR aggregation in the accumulation of other postsynaptic components in agrin-induced specializations, iii) To determine how AchR microaggregates are assembled and incorporated into large agrin-induced specializations, iv) To examine changes in the association of AChRs with other proteins during agrin-induced receptor aggregation, and v) To analyze changes in phosphorylation of components of the postsynaptic apparatus during synapse formation and maturation of rat neuromuscular junctions in vivo. The experiments outlined in this proposal involve protein chemistry, immunochemistry, immunohistochemistry, and site-directed mutagenesis. Studies such as these are essential to understanding the molecular mechanisms of the formation of the neuromuscular junction. Such information may provide insights into ways to diagnose and treat developmental abnormalities or diseases of the neuromuscular system and to enhance neuromuscular regeneration after trauma.
