Differential effects of TARPs on AMPA receptor subunits

描述(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疾病中发挥作用。兴奋性传递的快速成分主要由AMPA型谷氨酸受体携带。这些受体是四种亚基(GluR1-4)在神经元特异性化学计量学中组装的四聚体。最近发现，神经元上的AMPA受体还与被称为跨膜AMPA-R调节蛋白(跨膜AMPA-R调节蛋白)的辅助亚单位相关，这是一个由四个蛋白组成的家族，其中最广泛的是Stargazin(STG)。STG对AMPA受体有两种截然不同的作用。其一是通过PSD-95促进AMPA受体向细胞膜的运输，并将AMPA受体锚定到突触上。另一种是通过降低谷氨酸的EC50和减缓受体失活和脱敏来调节AMPA受体的动力学，这些效应对突触传递的波形有直接影响。然而，大多数研究都是通过与GluR1和2共表达STG来进行的，而对于STG对其他受体亚单位的影响以及其他三种在大脑皮质和海马区普遍存在的Tarp的作用，人们知之甚少。我们最近发现，与GluR1和GluR2不同，受体亚单位GluR3几乎没有受到STG的动力学调节。另一方面，Tarps的Gamma-4和Gamma-8导致了GluR3的失活和脱敏的减缓，慢于GluR1和GluR2。有趣的是，STG强烈地促进了GluR3的表面表达，这表明：(1)STG确实与GluR3有关；(2)受体动力学和转运的调节是独立调节的。在这个项目中，我们将确定四种Tarp如何以及为什么在它们对受体亚单位的影响上存在差异。在具体目标1中，我们将完成对它们对GluR3影响的分析，并将我们的分析扩展到包括GluR2和GluR4。此外，我们将研究四种Tarp对大脑中普遍存在的异构亚单位组合的影响。这些数据将对理解不同脑区突触传递的波形具有直接的相关性。在特定的目标2中，我们将确定GluR3中的哪些氨基酸负责Tarp的不同效果。为此，我们将在GluR2和GluR3之间系统地交换片段，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Tarp之间的接触点，这对动力学调节是重要的。最近的证据表明，在精神分裂症等疾病中，Glur和TARP的表达都发生了变化。因此，这项研究的结果对于理解兴奋性传递中的异常也是相关的，这些异常被认为是许多神经和精神障碍的基础。许多证据表明，兴奋性突触传递的障碍参与了各种神经和精神疾病的发病。突触传递的这种障碍可能是由于AMPA型谷氨酸受体的变化引起的，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，精神分裂症患者大脑中调节AMPA受体功能的被称为Tarp的蛋白质已被证明是改变的。在这个项目中，我们将研究Tarp对不同类型AMPA受体的影响。鉴于AMPA受体在不同神经元之间的差异，这些研究的结果有望为为什么突触传递在大脑的不同部位有所不同提供新的解释，它们也可能为各种大脑疾病中兴奋性传递的异常提供新的见解。

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.