Proteomic mining of intra- and extracellular proteins that modulate AMPA receptor function

项目摘要 AMPA型谷氨酸受体（AMPAR）是脑内主要的兴奋性神经递质受体， 中枢神经系统AMPAR的密度、亚基组成和运输的动态调节 和兴奋性突触外是促进突触强度变化的关键机制， 与学习和记忆相关的各种形式的突触可塑性。在过去的20年里，从 Huganir实验室和其他人为揭示分子机制做出了重大贡献 增强（长时程增强，LTP）或减弱（长时程增强，LTP）的AMPAR的潜在突触靶向 抑郁症，有限公司）突触传递。这些研究为LTP的流行模型做出了贡献， 由此亚基特异性蛋白质相互作用和胞质内的翻译后修饰， AMPAR的（C）-末端促进AMPAR向突触的运输，从而增强突触连接。 传输最近的工作挑战了这一模型和对AMPAR的C-末端的要求，以及 它们的相互作用的伴侣，来介导LTP。研究发现，不仅缺乏C- 在所有AMPAR（GluA1-A3）缺陷的小鼠中，末端结构域拯救了LTP，但GluK1也可以，GluK1属于 离子型谷氨酸受体的不同红藻氨酸亚类通常在这些突触中不存在。而 这些数据仍然是一个有争议的话题，AMPA和红藻氨酸受体共享保守的结构体系， 他们的细胞外结构域，提高了有趣的和较少探索的可能性，细胞外结构域的 特异性AMPAR亚基也可能在调节受体的突触靶向中起关键作用。 在本研究提案中，我们将调查AMPAR的突触靶向需要的假设 诱导突触强度的长期变化是由协调的蛋白质-蛋白质双向调节的 与N-和C-末端的相互作用。为了验证这一假设，我们开发了一种检测方法， 用APEX2标记AMPAR亚基的N-或C-末端，以进行无偏倚的蛋白质组学分析。 筛选以鉴定与细胞外或细胞内结构域内源性相互作用的蛋白质， GluA1和GluA2的AMPAR在突触可塑性。选择的候选蛋白质， 证明验证，动态和具体的相互作用与AMPAR亚基将进一步表征 使用过表达和敲低方法来评估它们在调节突触靶向中的作用， AMPAR在突触可塑性中的功能。通过识别和表征新的功能相关的 突触蛋白和AMPAR之间的相互作用，拟议的研究将提供重要的见解， 可塑性与学习、记忆和高级脑功能相关的分子机制。 此外，这些研究对涉及突触功能障碍的大脑疾病具有广泛的影响，因为它们可能 揭示了治疗神经和精神疾病的新的治疗靶点。

PROJECT SUMMARY AMPA-type glutamate receptors (AMPARs) are the major excitatory neurotransmitter receptor in the central nervous system. Dynamic regulation of the density, subunit composition and trafficking of AMPARs into and out of excitatory synapses is a key mechanism to facilitate changes in synaptic strength important for various forms of synaptic plasticity relevant to learning and memory. Over the last twenty years, work from Huganir Laboratory and others has made significant contributions to uncovering the molecular mechanisms underlying synaptic targeting of AMPARs that strengthens (long-term potentiation, LTP) or weakens (long-term depression, LTD) synaptic transmission. These studies have contributed to the prevailing model of LTP, whereby subunit-specific protein interactions and post-translational modifications within the cytosolic, carboxy (C)-terminus of AMPARs facilitate the trafficking of AMPARs to synapses and thereby enhance synaptic transmission. Recent work challenged this model and the requirement for the C-termini of AMPARs, as well as their interacting partners, to mediate LTP. It was found that not only could AMPAR mutants that lack the C- terminal domain rescue LTP in mice deficient all AMPARs (GluA1-A3), but so could GluK1, which belongs to the distinct, Kainate subclass of ionotropic glutamate receptors not normally found at these synapses. While these data remain a topic of debate, AMPA and Kainate receptors shared conserved structural architecture in their extracellular domains, raising the intriguing and less explored possibility that the extracellular domains of specific AMPAR subunits might also play key roles in regulating the synaptic targeting of the receptors. In this research proposal we will investigate the hypothesis that synaptic targeting of AMPARs required to induce long-term changes in synaptic strength is bi-directionally regulated by coordinated protein-protein interactions with the N- and C-termini. To test this hypothesis we have developed an assay that uses proximity- labeling with APEX2-tagged to the N- or C-termini of AMPAR subunits to perform an unbiased, proteomic screen to identify proteins that endogenously interact with either the extracellular or intracellular domains of GluA1- and GluA2-containing AMPARs during synaptic plasticity. Selected candidate proteins that demonstrate validated, dynamic and specific interactions with AMPAR subunits will be further characterized using overexpression and knockdown approaches to evaluate their role in regulating the synaptic targeting and function of AMPARs during synaptic plasticity. By identifying and characterizing new, functionally-relevant interactions between synaptic proteins and AMPARs, the proposed research will provide significant insight into the molecular mechanisms underlying plasticity relevant to learning, memory and higher brain function. Further, these studies have broad implications for brain disorders involving synaptic dysfunction as they might reveal novel therapeutic targets for the treatment of neurological and psychiatric diseases.