Detailed characterizations of neuronal signal transduction and protein phosphorylation/dephosphorylation are critical for understanding many brain functions. For example, activation of NMDA-type glutamate receptors stimulates both protein kinases and protein phosphatases, which feedback to modulate AMPA- and NMDA-type glutamate receptors. Ca-+/calmodulin-dependent protein kinase II (CaMKII) is a major dendritic kinase activated by NMDA receptor stimulation, resulting in Thr286-autophosphorylation and phosphorylation of AMPA- and NMDA-receptors and several other proteins including densin-180, an O-sialoglycoprotein with a PDZ domain. We showed that CaMKII autophosphorylation promotes its translocation to postsynaptic densities (PSDs), submembranous cytoskeletal specializations, and identified the NR2B subunit of NMDA receptors and densin- 180 as two proteins that likely contribute to translocation.
Five Specific Aims address our hypothesis that binding to NR2B and densin-180 modulates CaMKII, resulting in synapse-specific regulation of glutamate receptors. 1. Neuronal interaction of CaMKII and dens in- 180 will be verified by colocalization using immunofluorescent confocal microscopy and by coimmunoprecipitation assays. Relative contributions of NR2B and densin- 180 to CaMKII binding activities in PSDs will be determined. 2. Interaction domains in NR2B, densin-180 and CaMKII will be identified in vitro by truncation/deletion and site-directed mutagenesis, and their importance will be confirmed in HEK293 cells and neurons. This information will be used to develop reagents that specifically manipulate CaMKII localization in cells. 3. Dynamics of CaMKII.densin-180 and CaMKII.NR2B interactions, and regulatory roles of phosphorylation/dephosphorylation of densin-180, NR2B and CaMKII, as well as NMDA receptor activation, will be examined in vitro and in intact cells. 4. Effects of interaction with NR2B or densin-180 on CaMKJI autophosphorylation will be investigated in vitro and in intact cells. AMPA receptor phosphorylation and potentiation in HEK293 cells and neurons will be compared under conditions where NR2B and densin-180 are used to differentially target CaMKJI. 5. Roles of CaMKII-binding and CaMKII-mediated NR2B phosphorylation in regulation of NMDA receptors will be determined. More long-range goals are to establish the roles of CaMKII targeting by NR2B and densin-180 in regulation of synaptic transmission and synapse-specific synaptic plasticity. These studies will provide fundamental insights into signal transduction mechanisms underlying normal brain functions such as learning and memory. Reagents that block these protein.protein interactions also have potential for development as therapeutic compounds to treat mental disorders, such as schizophrenia or depression, and possibly brain injuries.
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