AMPA Receptor Trafficking and Signaling Complexes

描述(由申请人提供)： 我们将研究AMPA的GluR2亚单位的运输机制 培养的海马神经元中突触的受体。对贩运活动的监管 被认为控制AMPA受体突触丰度和 因此突触的力量。我们已经鉴定出三种含有PDZ的蛋白质 与GluR2、ABP、GRIP和PICK1关联。这些因素束缚到了极致。 GluR2的羧基末端区域，可作为连接 运输机械的受体或将AMPA受体系在突触。 我们还证明了伴侣、N-乙基马来酰亚胺的敏感融合 蛋白质(NSF)，与GluR2特异性结合。NSF可能解离SNARE复合体 与AMPA受体相关，从而使含有GluR2的囊泡“启动” 以转移到突触。我们的工作还表明，功能相互作用 在这两组蛋白质之间。我们将测定生化指标， GluR2 C末端突变的分子和细胞生物学后果。我们 将表达突变的GluR2亚基和携带GluR2 C的单代嵌合体 Sindbis病毒载体在海马神经元中的末端和一系列测量 突触形成和修饰过程中的表型。这将泄露 不同结合蛋白在GluR2转运和功能中的作用 我们将研究ABP和GRIP形成的亚细胞结构，并分析 ABP亚区对ABP功能的贡献。要评估NSF在 可能启动含GluR2的囊泡，我们将测试 NSF-GluR2复合体与SNARE核心复合体。这将需要确定 当NSF与GluR2接触时与其结合的蛋白质。我们还将研究 突变对GluR2亚基活性依赖易位的影响 培养的神经元中的突触。总而言之，这些研究将给出一种机械论 结合GluR2 C末端结构域的蛋白质在体内的作用 AMPA受体交易。这些研究与正常的心力衰竭机制有关。 学习和学习障碍发生在衰老过程中或由于以下原因 神经变性或基因突变。

DESCRIPTION (provided by applicant): We will study the mechanism of trafficking of the GluR2 subunit of the AMPA receptor to synapses in cultured hippocampal neurons. Regulation of trafficking of AMPA receptors is thought to control AMPA receptor synaptic abundance and hence synaptic strength. We have identified three PDZ-containing proteins that associate with GluR2, ABP, GRIP and PICK1. These factors bind to the extreme carboxy terminal region of GluR2 and may serve as adaptors that link the receptor to the trafficking machinery or tether AMPA receptors at the synapse. We have also shown that the chaperone, N-ethylmaleimide sensitive fusion protein (NSF), binds specifically to GluR2. NSF may dissociate SNARE complexes associated with AMPA receptors and thereby "prime" vesicles containing GluR2 for transit to the synapse. Our work also suggests functional interactions between these two sets of proteins. We will determine the biochemical, molecular and cell biological consequences of mutating the GluR2 C terminus. We will express mutant GluR2 subunits and single pass chimeras bearing the GluR2 C terminus in hippocampal neurons from Sindbis virus vectors and measure a series of phenotypes during synapse formation and modification. This will divulge the contributions of different binding proteins to GluR2 trafficking and function. We will study subcellular structures formed by ABP and GRIP and analyze the contributions of ABP subdomains to ABP function. To assess NSF function in a possible priming of vesicles bearing GluR2, we will test for interaction of the NSF-GluR2 complex with the SNARE core complex. This will entail identifying proteins that bind to NSF while it is in contact with GluR2. We will also study the effects of mutation on activity dependent translocation of GluR2 subunits to synapses in cultured neurons. Together these studies will give a mechanistic view of the contributions of protein binding the GluR2 C terminal domain in AMPA receptor trafficking. These studies are pertinent to normal mechanisms of learning and to learning disabilities that occur during aging or as a result of neural degeneration or genetic mutation.