Differential effects of TARPs on AMPA receptor subunits

描述（由申请人提供）：兴奋性突触传递是所有认知的基础，这种传递的变化被认为在许多神经系统疾病中起作用。兴奋性传递的快速组分主要由ampa型谷氨酸受体携带。这些受体是由四种亚基（GluR1-4）在神经元特异性化学计量中组装而成的四聚体。最近发现，神经元中的AMPA受体也与称为TARPs（跨膜AMPA- r调节蛋白）的辅助亚基相关，TARPs是一个由四种蛋白组成的蛋白家族，其中stargazin （STG）被广泛表征。STG对AMPA受体有两种不同的作用。一种是促进向质膜的运输，并通过PSD-95将AMPA受体锚定在突触上。另一种是通过降低谷氨酸的EC50和减缓受体的失活和脱敏来调节AMPA受体的动力学，这些作用对突触传递的波形有直接的影响。然而，大多数研究都是通过STG与GluR1和2共表达进行的，对于STG对其他受体亚基的影响以及其他三种在皮层和海马中更为普遍的tarp的作用知之甚少。我们最近发现，与GluR1和GluR2不同，受体亚基GluR3几乎不受STG的动力学调节。另一方面，TARPs γ -4和γ -8导致GluR3失活和脱敏的减慢，其幅度大于GluR1和2。有趣的是，STG强烈促进GluR3的表面表达，这表明(i) STG确实与GluR3相关，（ii）受体动力学和运输的调节是独立调节的。在这个项目中，我们将确定四种TARPs对受体亚基的影响如何以及为什么不同。在Specific Aim 1中，我们将完成对它们对GluR3的影响的分析，并将我们的分析扩展到GluR2和GluR4。此外，我们将研究四种TARPs对大脑中普遍存在的异源亚基组合的影响。这些数据对于理解不同脑区突触传递的波形具有直接的相关性。在特定目标2中，我们将确定GluR3中的哪些氨基酸负责TARPs的差异效应。为此，我们将系统地交换GluR2和GluR3之间的片段，从被认为是中心的S1-S2结构域开始，然后使用点突变来识别关键氨基酸。这些实验应该使我们能够确定AMPA受体亚基和TARPs之间的接触点，这对动力学调节很重要。最近的证据表明，GluR和TARP的表达在精神分裂症等疾病中都发生了改变。因此，这项研究的结果也将与理解被认为是许多神经和精神疾病的基础的兴奋性传递的畸变有关。许多证据表明，兴奋性突触传递的紊乱与各种神经和精神疾病的发病机制有关。这种突触传递的紊乱可能是由AMPA型谷氨酸受体的改变引起的，但最近的研究发现，与AMPA受体相关的蛋白质可能同样有助于病理。例如，在精神分裂症患者的大脑中，调节AMPA受体功能的蛋白质TARPs已经被证明发生了改变。在本项目中我们将研究TARPs对不同类型AMPA受体的影响。鉴于AMPA受体在不同的神经元中存在差异，这些研究结果有望为突触传递在大脑不同部位的差异提供新的解释，并可能为各种大脑疾病中兴奋性传递的畸变提供新的见解。

DESCRIPTION (provided by applicant): Excitatory synaptic transmission underlies all cognition, and changes in this transmission are thought to play a role in many neurological disorders. The fast component of excitatory transmission is mainly carried by AMPA-type glutamate receptors. These receptors are tetramers assembled in neuron- specific stoichiometries from four types of subunits (GluR1-4). It has recently been discovered that AMPA receptors in neurons are also associated with auxiliary subunits called TARPs (transmembrane AMPA-R regulatory proteins), a family of four proteins of which stargazin (STG) has been most widely characterized. STG has two distinct effects on AMPA receptors. One is to promote trafficking to the plasma membrane and to anchor AMPA receptors to the synapse via PSD-95. The other one is to modulate the kinetics of AMPA receptors by lowering the EC50 for glutamate and by slowing receptor deactivation and desensitization, effects which have immediate impact on the waveform of synaptic transmission. However, most studies have been carried out by coexpressing STG with GluR1 and 2, and much less is known about STG's effects on other receptor subunits and about the actions of the other three TARPs which are far more prevalent in cortex and hippocampus. We have recently discovered that the receptor subunit GluR3, unlike GluR1 and GluR2, exhibits almost no kinetic modulation by STG. The TARPs gamma-4 and gamma-8, on the other hand, caused a slowing of deactivation and desensitization in GluR3 that was larger than in GluR1 and 2. Interestingly, surface expression of GluR3 was strongly promoted by STG, which indicates (i) that STG did associate with GluR3 and (ii) that modulation of receptor kinetics and trafficking are independently regulated. In this project we will determine how and why the four TARPs differ in their effect on receptor subunits. In Specific Aim 1, we will complete our analysis of their impact on GluR3 and extend our analysis to include GluR2 and GluR4. Moreover, we will examine the effects of the four TARPs on heteromeric subunit combinations that are prevalent in brain. These data will have immediate relevance for understanding the waveform of synaptic transmission in different brain areas. In Specific Aim 2 we will then determine which amino acids in GluR3 are responsible for the differential effects of the TARPs. For this we will systemically exchange segments between GluR2 and GluR3, starting with the S1-S2 domain thought to be central, and then use point mutations to identify the critical amino acids. These experiments should allow us to identify the contact points between AMPA receptor subunits and TARPs that are important for kinetic modulation. Recent evidence suggests that both GluR and TARP expression is altered in disorders such as schizophrenia. Thus, the results of this study will be relevant also to understand aberrations in excitatory transmission that are thought to underlie many neurological and psychiatric disorders. Much evidence suggests that disturbances in excitatory synaptic transmission contribute to the pathogenesis of various neurological and psychiatric disorders. Such disturbances in synaptic transmission may arise from changes in AMPA-type glutamate receptors, but recent studies have found that proteins associated with AMPA receptors may similarly contribute to pathology. For instance, proteins called TARPs which modulate AMPA receptor function have been shown to be altered in the brains of schizophrenic patients. In this project we will study the impact of TARPs on different types of AMPA receptors. Given that AMPA receptors vary across neurons, the results from these studies are expected to offer new explanations as to why synaptic transmission varies in different parts of the brain, and they may also provide new insights about aberrations in excitatory transmission in various brain disorders.