PHOSPHOANTIBODIES AS RESEARCH TOOLS TO STUDY THE NMDAR

DESCRIPTION (provided by applicant): The NMDA receptor appears to play a critical role in memory and it is widely thought to play important roles in development and in pathological conditions such as alcoholism, and the age-related dementias including Alzheimer?s. There is good evidence that phosphorylation of the NMDAR may be important for regulation of NMDAR function. The specific goal of the present application will be to identify the sites on the NMDAR that are phosphorylated and to prepare antibodies specific for those phosphorylation sites. The ultimate goal of these studies will be to use the antibodies to study the role of NMDAR phosphorylation in forms of plasticity that may underlie learning as well as its role in various pathological conditions. Thus, in this application we will test the hypothesis that specific phosphorylation sites on the NR2 subunits of the NMDAR are subject to LIP-related regulation in situ in the hippocampal slice. In Part 1 of the proposal, the specific sites on the NR2 subunits of the NMDAR that are phosphorylated will be identified. The essential milestone of this aim in Phase I will first be mass spectrometry of NR2 fusion proteins to optimize sequence coverage of the protein. The fusion proteins will then be phosphorylated in vitro followed by mass spectrometry. Analysis of immunoisolated, native NR2B and NR2B subunits will take place in Phase II. In that phase, particular emphasis will be placed on determining the sites basally phosphorylated and those influenced by LTP stimulation. In Part 2, phosphosite specific antibodies for Tyr 842 in NR2A and for sties on NR2A and NR2B identified in Part 1 will be prepared. Production of an antibody specific for phosphosite Tyr 842 will be the principal milestone of this Part of Phase I. In Phase II phosphospecific antibodies for sites identified in Part 1 will be prepared.