Mechanism of Stargazin-Regulated AMPAR Trafficking

描述（由申请人提供）：本申请的长期目标是描述突触分化和调节的分子机制。调节离子型谷氨酸受体靶向突触是突触发生和突触可塑性的关键步骤。最近的研究表明，stargazin及其家族成员是谷氨酸受体靶向所必需的。涉及两种不同的机制：Stargazin和AMPA受体亚基之间的相互作用是受体表面递送所必需的; Stargazin和PSD-95家族蛋白之间的相互作用是将AMPA受体靶向突触所必需的。 关于AMPA受体运输的这种新的调节仍然存在许多问题：1）Stargazin参与AMPA受体靶向过程的哪些步骤？2)Stargazin是否直接或通过其他中间蛋白与AMPA受体相互作用？3)如果它们直接相互作用，那么stargazin中与AMPA受体相互作用的功能结构域在哪里，将它们转运到质膜并将它们靶向突触？4)AMPA受体中与stargazin相互作用的功能域在哪里？我建议通过集中于以下目标来解决这些问题。 本申请的第一个目的是研究stargazin在AMPA受体靶向的不同阶段中的作用。Stargazin可能在其转运途径沿着不同阶段控制AMPA受体靶向，即ER ->高尔基体->膜融合机制->内化机制。我们将有选择地阻止其中的一些步骤，并检查其对AMPA受体贩运的影响。 第二个目标是绘制stargazin和AMPA受体中介导其相互作用并控制其靶向的功能结构域。Stargazin和AMPA受体中可能存在两种功能结构域，一种负责将AMPA受体和Stargazin结合到同一蛋白复合物中（相互作用结构域），另一种负责将整个复合物转运到质膜上（转运结构域）。我们将使用Co-IP和FRET方法来绘制相互作用结构域，并使用表面生物素化和电生理方法来确定运输结构域。 大量的脑功能障碍与大脑发育和可塑性异常有关。拟议的研究将促进我们对突触发生和可塑性的分子机制的理解，从而为这些脑疾病及其补救提供见解。

DESCRIPTION (provided by applicant): The application's long-term objective is to delineate the molecular mechanisms underlying synaptic differentiation and modulation. Regulated targeting of ionotropic glutamate receptors to the synapse is a critical step in synaptogenesis and synaptic plasticity. Recent studies indicate that stargazin and its family members are essential for glutamate receptor targeting. Two distinct mechanisms are involved: interactions between stargazin and AMPA receptor subunits are required for surface delivery of the receptors; and interactions between stargazin and PSD-95 family proteins is required for targeting AMPA receptors to the synapse. Many questions remain regarding this novel regulation of AMPA receptor trafficking: 1) In which steps of the AMPA receptor targeting processes is stargazin involved? 2) Does stargazin interact with AMPA receptors directly or through other intermediate proteins? 3) If they interact directly, where are the functional domains in stargazin that interact with AMPA receptors, transport them to the plasma membrane and target them to the synapse? 4) Where are the functional domains in AMPA receptors that interact with stargazin? I propose to address these questions by focusing on the following aims. The first aim of this application is to investigate the roles of stargazin in different stages of AMPA receptor targeting. Stargazin may potentially control AMPA receptor targeting at different stages along its transport pathway-i.e. ER -> Golgi apparatus -> membrane fusion machinery -> internalization machinery. We will selectively block some of these steps and examine its impact on AMPA receptor trafficking. The second aim is to map the functional domains in stargazin and the AMPA receptor that mediate their interactions and control their targeting. There are potentially two types of functional domains in stargazin and the AMPA receptor, one responsible for bringing AMPA receptor and stargazin into same protein complex (the interaction domains), the other for bring the whole complex to plasma membrane (the trafficking domains). We will use Co-IP and FRET methods to map the interaction domains, and surface biotinylation and electrophysiological approaches to determine the trafficking domains. A large number of brain disorders are related to abnormal brain development and plasticity. The proposed studies will advance our understanding on molecular mechanisms of synaptogenesis and plasticity, thereby providing insights in these brain diseases, and their remediation.