Extracellular Modulation of Metabotropic GluRs

描述（由申请人提供）：谷氨酸是中枢神经系统（CNS）中的主要兴奋性氨基酸神经递质，并激活两个不同的谷氨酸受体（GluR）家族。离子型谷氨酸受体（iGluR）家族由介导快速突触传递的NMDA（N-甲基-D-天冬氨酸）、AMPA（α-氨基-3-羟基-5-甲基-异恶唑-4-丙酸）和红藻氨酸受体组成，而细胞兴奋性和突触传递的调节由谷氨酸激活的G蛋白偶联受体（GPCR）家族、代谢型谷氨酸受体（mGluR）介导。mGluRs的分子克隆揭示了八种不同的受体亚型：I组mGluRs（mGluRs 1和5）与Gq信号传导途径偶联，而II组（mGluRs 2和3）和III组（mGluRs 4、6、7和8）mGluRs在异源表达系统中与Gi/Go信号传导途径偶联。最近对蛋白质-蛋白质相互作用的研究将联合收割机共免疫沉淀（colP）与质谱（MS）相结合，以产生一个推定的相互作用蛋白质的目录，该目录被测试以验证或否定它们与靶蛋白的相互作用。因此，本研究的目的是确定蛋白质相互作用的第一组受体mGluR 5a使用蛋白质组学的方法。I组mGluR激动剂变化很大，它们可以放大或减弱兴奋性毒性细胞死亡。三个变量影响I组mGluR对激动剂的反应：1）细胞中NMDA受体的异聚体组成，2）暴露于药物的时间，和3）星形胶质细胞的存在（参见Nicoletti等人，1999年）。我们认为，细胞特异性的mGluR-蛋白质相互作用也可能占，在一定程度上，为双重反应的第一组mGluR激动剂。mGluR的胞外氨基末端结构域（ATD）约为500个氨基酸，或单个mGluR的蛋白质质量的一半。在谷氨酸结合位点之外，尚未描述大的细胞外ATD的功能作用，然而ATD可用于与突触间隙或细胞外基质中的蛋白质相互作用。如果ATD结合蛋白被鉴定，它们可能为CNS中mGluR信号的调节提供替代机制。我们的初步研究已经确定了已知的，以及新的，mGluR 5a相互作用的蛋白质，包括调节mGluR 5a信号的蛋白质。这些数据提供了概念证明，colP/MS可以识别新的mGluR 5a相互作用蛋白，这些蛋白质可能揭示了mGluR 5a在CNS中的不同作用的分子机制，并且这些相互作用可能被忽略使用更传统的蛋白质-蛋白质相互作用研究。

DESCRIPTION (provided by applicant): Glutamate is the principal excitatory amino acid neurotransmitter in the central nervous system (CNS) and activates two distinct families of glutamate receptors (GluRs). The ionotropic glutamate receptor (iGluR) family is composed of NMDA (N-methyI-D-aspartate), AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid), and kainate receptors that mediate fast synaptic transmission, while modulation of cell excitability and synaptic transmission is mediated by the family of glutamate activated G protein-coupled receptors (GPCRs), the metabotropic glutamate receptors (mGluRs). Molecular cloning of mGluRs revealed eight distinct receptor subtypes: Group I mGluRs (mGluRs 1 and 5) couple to the Gq signaling pathway, whereas group II (mGluRs 2 and 3) and group III (mGluRs 4, 6, 7 and 8) mGluRs couple to Gi/Go signaling pathways in heterologous expression systems. Recent studies on protein-protein interactions combine coimmunoprecipitation (colP) with mass spectrometry (MS) to generate a catalog of putative interacting proteins that are tested to validate or negate their interaction with the target protein. Thus the goal of this study is to identify proteins that interact with the group I receptor mGluR5a using a proteomics approach. Group I mGluR agonists vary widely and they can either amplify or attenuate excitotoxic cell death. Three variables affect group I mGluR responses to agonists: 1) the heteromeric composition of NMDA receptors in the cell, 2) the time of exposure to drugs, and 3) the presence of astrocytes (see Nicoletti et al., 1999). We submit that cell-specific mGluR-protein interactions may also account, in part, for the dual response of group I mGluRs to agonists. The extracellular amino terminal domain (ATD) of mGluRs is approximately 500 amino acids, or one half of the protein mass of an individual mGluR. Outside of the glutamate-binding site, a functional role for the large extracellular ATD has not been described however the ATD may serve to interact with proteins in the synaptic cleft or the extracellular matrix. If ATD-binding proteins are identified, they may provide alternative mechanisms for the regulation of mGluR signaling in the CNS. Our preliminary studies have identified known, as well as novel, mGluR5a-interacting proteins, including a protein that regulates mGluR5a signaling. These data provide proof of concept that colP/MS can identify novel mGluR5a interacting proteins, that these proteins may shed light on the molecular mechanisms that contribute to the diverse roles of mGluR5a in the CNS, and that these interactions may be overlooked using more traditional protein-protein interaction studies.