Phosphoantibodies as Research Tools to Study the NMDAR

磷酸化抗体作为 NMDAR 研究工具

• 批准号: 6484433
• 负责人: ANDREW J CZERNIK
• 金额: $ 9.88万
• 依托单位: PHOSPHOSOLUTIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6484433
• 关键词: NMDA receptors; immunologic substance development /preparation; long term potentiation; mass spectrometry; method development; monoclonal antibody; phosphorus compounds; phosphorylation; posttranslational modifications; recombinant proteins

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.

描述(申请人提供)：NMDA受体似乎扮演着一种 在记忆中起着关键作用，人们普遍认为它在 发展和病理条件，如酒精中毒，以及 与年龄相关的痴呆，包括阿尔茨海默病？S。有很好的证据表明，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 磷酸化的NMDAR，并制备针对这些蛋白的抗体 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的抗体 这可能是学习及其在各种病理变化中的作用的基础 条件。因此，在此应用程序中，我们将测试特定的假设 NMDAR的NR2亚基上的磷酸化位点受 大鼠海马片中与嘴唇相关的原位调节。在第一部分中 提案中，NMDAR的NR2亚基上的特定位点是 磷酸化将被识别出来。这一目标在阶段中的重要里程碑 我将首先用质谱仪对NR2融合蛋白进行序列优化 蛋白质的覆盖率。然后，融合蛋白将被磷酸化 体外实验，然后进行质谱分析。免疫分离、天然NR2B的分析 和NR2B亚基将发生在第二阶段。在那个阶段，特别是 重点将放在确定碱基磷酸化和 受LTP刺激影响的患者。在第二部分中，亚磷酸盐特异性抗体 对于NR2A中的轮胎842以及在第1部分中确定的NR2A和NR2B上的STIES，将 准备好了。生产针对亚磷酸盐Tyr 842的抗体将是 这部分第一阶段的主要里程碑是第二阶段 将针对第1部分中确定的位置制备抗体。