Structural and Allosteric Mechanisms of mGluR Activation

摘要 在神经系统中，G蛋白偶联受体(GPCRs)用于检测精确的空间和时间 调节突触传递的神经递质释放模式。代谢性谷氨酸受体 (MGluRs)是C家族GPCRs，感受兴奋性神经递质谷氨酸。MGluR是结构性的 具有连接到七螺旋跨膜的大的胞外配体结合域的二聚体 结构域通过一个富含半胱氨酸的中间连接子。有八个mGluR子类型，它们以 大脑的重叠区域，在突触上扮演着不同的角色。此外，mGluR很容易形成 异二聚体，增加了该体系的分子多样性和功能复杂性。最近的突破 在低温电子显微镜(Cryo-EM)中，已经导致了mGluRs的全长结构，提供了改进的 了解这个受体的整体架构。然而，这些结构引发了进一步的问题 关于在激活时发生的动态重排。而细胞外结构域已知为 经历谷氨酸诱导的重排，这些构象变化是如何耦合到产物激活的 G蛋白在TMD的募集仍有待确定。此外，这种耦合是如何调整的 导致观察到的活化性质差异的mGluR同源和异源二聚体亚型是 未知。第二组mGluR是解决这些问题的理想候选者，也是本报告的重点 求婚。第二组mGluRs由mGluR2和mGluR3组成，它们在所有序列中具有最高的同源性 MGluRs，但仍显示出明显的谷氨酸亲和力、动力学和基础活性。此外，mGluR2和mGluR3 很容易在大脑中形成异二聚体。在本提案中，我将结合使用低温电磁结构分析和 除亚型特定药理化合物外的功能分析。使用这些工具，我将探索 第二组mGluRs的构象动力学，重点是偶联配体的变构机制 结合到跨膜结构域的激活。总而言之，这项工作将提供一幅高分辨率的 激活mGluR同源和异源二聚体。

ABSTRACT In the nervous system, G protein coupled receptors (GPCRs) serve to detect the precise spatial and temporal pattern of neurotransmitter release to modulate synaptic transmission. The metabotropic glutamate receptors (mGluRs) are family C GPCRs that sense the excitatory neurotransmitter glutamate. mGluRs are constitutive dimers with a large extracellular ligand binding domain which is connected to a seven-helix transmembrane domain via an intermediate cysteine-rich linker. There are eight mGluR subtypes, which are expressed in overlapping regions of the brain and play distinct roles at the synapse. Additionally, mGluRs readily form heterodimers, increasing the molecular diversity and functional complexity of this system. Recent breakthroughs in cryogenic electron microscopy (cryo-EM) have led to full-length structures of mGluRs, providing an improved understanding of the overall architecture of this receptor. However, these structures raise further questions regarding the dynamic rearrangements that occur upon activation. While the extracellular domain is known to undergo glutamate-induced rearrangements, how these conformational changes are coupled to yield activation and G protein recruitment at the TMD remains to be defined. Additionally, how this coupling is tuned across mGluR homo- and heterodimer subtypes to produce the observed differences in activation properties is unknown. The group II mGluRs are ideal candidates for addressing these questions and are the focus of this proposal. Group II mGluRs consist of mGluR2 and mGluR3, which have the highest sequence homology of all mGluRs, yet still show distinct glutamate affinity, kinetics, and basal activity. Furthermore, mGluR2 and mGluR3 readily heterodimerize in the brain. In this proposal, I will use a combination of cryo-EM structural analysis and functional assays in addition to subtype specific pharmacological compounds. Using these tools, I will probe the conformational dynamics of group II mGluRs, with a focus on the allosteric mechanisms that couple ligand binding to transmembrane domain activation. Together, this work will provide a high-resolution picture of activation for mGluR homo- and heterodimers.