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 postsynaptic apparatus, which includes aggregates of acetylcholine receptors (AChRs). Several lines of evidence indicate that agrin, a protein purified from the synapse- rich electric organ of the marine ray Torpedo californica, mediates the nerve-induced formation of postsynaptic specializations at embryonic and regenerating neuromuscular junctions. For example, agrin causes the formation of patches on the surface of myotubes in culture at which AChRs and other components of the postsynaptic apparatus are aggregated. Agrin also causes AChRs to be phosphorylated, a modification that could regulate 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 agrin-induced phosphorylation of AChRs may play a role in receptor aggregation.
The specific aims are: 1. To characterize agrin-induced phosphorylation AChR phosphorylation and aggregation. 2. To compare the properties of agrin-induced AChR phosphorylation and aggregation. 3. To measure changes in AChR phosphorylation at developing neuromuscular junctions. 4. To identify the agrin receptor/kinase. 5. To search for agrin-induced phosphorylation of other proteins. The experiments outlined in this proposal involve protein chemistry, immunochemistry, and immunohistochemistry. Studies such as these are essential to understanding the molecular mechanisms of the formation of the neuromuscular junction and so may provide insights into ways to diagnose and treat developmental abnormalities or diseases of the neuromuscular system and to enhance neuromuscular regeneration after trauma.
