We will study the mechanism of trafficking of the GluR2 subunit of the AMPA receptor to synapses in cultured hippocampal neurons. Regulation of trafficking of AMPA receptors is thought to control AMPA receptor synaptic abundance and hence synaptic strength. We have identified three PDZ-containing proteins that associate with GluR2, ABP, GRIP and PICK1. These factors bind to the extreme carboxy terminal region of GluR2 and may serve as adaptors that link the receptor to the trafficking machinery or tether AMPA receptors at the synapse. We have also shown that the chaperone, N-ethylmaleimide sensitive fusion protein (NSF), binds specifically to GluR2. NSF may dissociate SNARE complexes associated with AMPA receptors and thereby """"""""prime"""""""" vesicles containing GluR2 for transit to the synapse. Our work also suggests functional interactions between these two sets of proteins. We will determine the biochemical, molecular and cell biological consequences of mutating the GluR2 C terminus. We will express mutant GluR2 subunits and single pass chimeras bearing the GluR2 C terminus in hippocampal neurons from Sindbis virus vectors and measure a series of phenotypes during synapse formation and modification. This will divulge the contributions of different binding proteins to GluR2 trafficking and function. We will study subcellular structures formed by ABP and GRIP and analyze the contributions of ABP subdomains to ABP function. To assess NSF function in a possible priming of vesicles bearing GluR2, we will test for interaction of the NSF-GluR2 complex with the SNARE core complex. This will entail identifying proteins that bind to NSF while it is in contact with GluR2. We will also study the effects of mutation on activity dependent translocation of GluR2 subunits to synapses in cultured neurons. Together these studies will give a mechanistic view of the contributions of protein binding the GluR2 C terminal domain in AMPA receptor trafficking. These studies are pertinent to normal mechanisms of learning and to learning disabilities that occur during aging or as a result of neural degeneration or genetic mutation.
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