INVOLVEMENT OF RAP AND RAPGAP IN NMDA RECEPTOR SIGNALING

N-甲基-D-天冬氨酸受体（NMDAR）因其在活动依赖性突触可塑性和兴奋性毒性中的关键作用而被广泛研究。 定义 NMDAR 调节的信号级联对于理解 NMDAR 功能的机制至关重要，但 NMDAR 引发的细胞内信号尚未得到很好的表征。 我们的实验室对与 NMDAR 细胞质尾部相关的蛋白质进行了系统分析，理由是此类蛋白质能够理想地通过 NMDAR 通道将钙流入转导至下游信号通路。 多域蛋白 PSD-95 似乎是这些转导元件的组织中心，与 NMDAR 以及信号分子（例如两个 GTP 酶激活蛋白 (GAP)）结合。 其中之一 SynGAP 是最近描述的 Ras 特异性 GAP；另一个是 SpanGAP，是一种可能调节 Rap 小 GTPase 的新型 GAP。 该提案将重点关注 SpanGAP 和 Rap 在 NMDAR 信号传导中的潜在作用，检查该通路（与 Ras 通路相比）在 NMDAR 信号级联中的相对贡献。 将采用显性失活突变体和反义策略来剖析培养的原代神经元中的特定转导机制。将使用各种转录报告基因以及针对 MAP 激酶和 CREB ​​的磷酸化特异性抗体来分析效果。 这项研究的结果可能揭示 NMDAR 介导的突触可塑性和兴奋性毒性的新机制。

The N-methyl-D-aspartate receptor (NMDAR) has been extensively studied due to its critical role in activity-dependent synaptic plasticity and excitotoxicity. Defining the signaling cascades regulated by the NMDAR is essential for a mechanistic understanding of NMDAR function, but the intracellular signals initiated by NMDARs are not well characterized. Our laboratory has undertaken a systematic analysis of the proteins associated with the cytoplasmic tails of NMDARs with the rationale that such proteins would be ideally positioned to transduce calcium influx through the NMDAR channel to downstream signaling pathways. The multidomain protein PSD-95 appears to be an organizing center for these transducing elements, binding to NMDARs as well as to signaling molecules such as two GTPase activating proteins (GAPs). One of these, SynGAP, is a recently described Ras-specific GAP; the other, SpanGAP, is a novel GAP likely to regulate Rap small GTPases. This proposal will focus on the potential role of SpanGAP and Rap in NMDAR signaling, examining the relative contribution of this pathway (compared with the Ras pathway) in the NMDAR signaling cascade. Dominant negative mutants and antisense strategies will be employed to dissect specific transduction mechanisms in cultured primary neurons. Effects will be analyzed by using a variety of transcriptional reporters as well as phospho-specific antibodies against MAP kinases and CREB. The results of this study may shed light on novel mechanisms contributing to NMDAR-mediated synaptic plasticity and excitotoxicity.