Phosphoantibodies as Research Tools to Study the NMDAR

描述（由申请人提供）：NMDA受体似乎在 在记忆中起着关键作用，人们普遍认为它在记忆中起着重要作用。 发展和病理条件，如酗酒，和 老年痴呆症包括老年痴呆症S.有充分的证据表明，NMDAR的磷酸化可能对NMDAR功能的调节很重要。 本申请的具体目标将是识别在所述区域上的位点。 磷酸化的NMDAR，并制备对这些NMDAR特异的抗体 磷酸化位点。这些研究的最终目标将是使用 研究NMDAR磷酸化在可塑性形式中的作用 这可能是学习的基础，也可能是它在各种病理学中的作用。 条件因此，在本申请中，我们将测试以下假设： NMDAR的NR2亚基上的磷酸化位点受到 海马脑片中的原位脂蛋白相关调节。第1部所 建议，NMDAR的NR2亚基上的特定位点， 将被识别。这一目标的重要里程碑是 我将首先对NR2融合蛋白进行质谱测序优化 蛋白质的覆盖率。然后融合蛋白将被磷酸化， 体外，随后进行质谱分析。免疫分离的天然NR2B的分析 和NR2B亚基将在第二阶段发生。在这个阶段，特别是 重点将放在确定基础磷酸化的位点上， 受LTP刺激影响的那些。在第2部分中，磷酸盐特异性抗体 对于NR2A中的Tyr 842以及第1部分中确定的NR2A和NR2B上的位点， 制备磷酸位点Tyr 842特异性抗体的产生将是 第一阶段本部分的主要里程碑。在II期磷酸特异性 将制备第1部分中确定的位点的抗体。

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.