Proper development and functioning of the brain relies on the precise and rapid flow of signals between neurons. Synapses play the foremost role in this information exchange. While the physiology and structure of mature synapses are understood in considerable depth, much less is known about the molecular cues mediating synaptic differentiation, maintenance, and turnover. A cardinal event in synapse formation is the marshalling of neurotransmitter receptors to the postsynaptic membrane. Mounting evidence suggests that agrin, a protein that directs postsynaptic differentiation at developing neuromuscular junctions, may also play a role in synaptogenesis in the brain. A full understanding of agrin's function in the brain requires the characterization of its cell surface receptor. In preliminary experiments we have found that agrin binds to cultured neurons and to synaptosomes. In some cells, agrin elicits the redistribution of these binding sites, suggesting that they constitute functional agrin receptors. Further, we have identified two agrin binding polypeptides in brain membranes. In the proposed studies we will characterize neuronal agrin receptor(s) at the cellular, biochemical, and functional levels, with the overall goal of elucidating the molecular events underlying synapse formation among neurons. Using well characterized primary hippocampal cultures, we will determine the time course of expression, the cellular localization, and the functional properties of agrin receptors on differentiating neurons. We will compare the distribution of agrin receptors to that of neurotransmitter receptors and other synaptic specializations. The subcellular localization of agrin receptors will be analyzed at the ultrastructural level. We will also determine whether agrin induces changes in the distribution of its own receptor or of related markers of synaptic differentiation. In parallel with this cell biological approach, we will characterize neural agrin receptors biochemically. Ligand overlay techniques and affinity chromatography methods will be used to identify and to determine the subunit composition of agrin receptors on neurons, and partial amino acid sequence of the purified receptors will be obtained. In addition, we will determine how alternative splicing of agrin may regulate binding to agrin receptors, and investigate the ensuing functional consequences. The results of these experiments will provide fundamental insights into synapse formation, modification, loss, and recovery. Knowledge of these basic mechanisms is crucial to our understanding of normal synaptic function, and forms a bedrock for the development of diagnostic and therapeutic approaches aimed at ameliorating or curing mental illnesses.
