Extracellular Modulation of Metabotropic GluRs

描述（由申请人提供）：谷氨酸是中枢神经系统（CNS）中主要的兴奋性氨基酸神经递质，激活两个不同的谷氨酸受体（GluRs）家族。嗜离子性谷氨酸受体（iGluR）家族由NMDA （n-甲基- d -天冬氨酸）、AMPA （α -氨基-3-羟基-5-甲基-异唑-4-丙酸）和kainate受体组成，介导快速突触传递，而调节细胞兴奋性和突触传递的是谷氨酸激活的G蛋白偶联受体（gpcr）家族，即代谢性谷氨酸受体（mGluRs）。mGluRs的分子克隆揭示了8种不同的受体亚型：在异源表达系统中，I组mGluRs （mGluRs 1和5）与Gq信号通路偶联，而II组（mGluRs 2和3）和III组（mGluRs 4、6、7和8）mGluRs与Gi/Go信号通路偶联。最近关于蛋白质-蛋白质相互作用的研究将共免疫沉淀（colP）与质谱（MS）结合起来，生成了一个假定相互作用的蛋白质目录，这些蛋白质被测试以验证或否定它们与靶蛋白的相互作用。因此，本研究的目的是利用蛋白质组学方法鉴定与I组受体mGluR5a相互作用的蛋白质。I组mGluR激动剂变化很大，它们可以放大或减弱兴奋毒性细胞死亡。三个变量影响I组mGluR对激动剂的反应：1)细胞中NMDA受体的异质组成，2)暴露于药物的时间，以及3)星形胶质细胞的存在（见Nicoletti等人，1999）。我们认为，细胞特异性mglur -蛋白相互作用也可能部分解释了I组mglur对激动剂的双重反应。mGluR的胞外氨基末端结构域（ATD）约为500个氨基酸，或单个mGluR蛋白质质量的一半。在谷氨酸结合位点之外，大细胞外ATD的功能作用尚未被描述，但ATD可能与突触间隙或细胞外基质中的蛋白质相互作用。如果atd结合蛋白被确定，它们可能为CNS中mGluR信号的调控提供替代机制。我们的初步研究已经确定了已知的和新的mGluR5a相互作用蛋白，包括一种调节mGluR5a信号传导的蛋白。这些数据证明了colP/MS可以鉴定出新的mGluR5a相互作用蛋白，这些蛋白可能揭示了mGluR5a在中枢神经系统中发挥不同作用的分子机制，并且这些相互作用可能被传统的蛋白-蛋白相互作用研究所忽视。

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.