INVOLVEMENT OF RAP AND RAPGAP IN NMDA RECEPTOR SIGNALING

N-甲基-D-天冬氨酸受体(NMDAR)因其在活性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。定义由NMDAR调控的信号级联对于从机制上理解NMDAR的功能是必不可少的，但NMDAR启动的细胞内信号还没有得到很好的表征。我们的实验室已经对NMDAR胞浆尾部相关的蛋白质进行了系统的分析，其理论基础是这些蛋白质将被理想地定位于通过NMDAR通道向下游信号通路转导钙内流。多域蛋白PSD-95似乎是这些转导元件的组织中心，与NMDAR以及两个GTPase激活蛋白(GAP)等信号分子结合。其中一个是SynGAP，是最近描述的RAS特异性缺口；另一个是可能调节Rap小GTP酶的新缺口。这项建议将集中在span GAP和Rap在NMDAR信号转导中的潜在作用，检查该途径(与RAS途径相比)在NMDAR信号级联中的相对贡献。显性负性突变体和反义策略将被用来剖析培养的原代神经元的特定转导机制。将通过使用各种转录报告以及针对MAP激酶和CREB的磷酸特异性抗体来分析效果。这项研究的结果可能有助于阐明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.