Molecular Mechanisms of Synaptic G Protein-Coupled Receptors

项目摘要 在许多生物系统中，G蛋白偶联受体（GPCR）在动力学之间提供了至关重要的分子联系。 细胞外环境和相关的细胞内信号反应。在神经系统中，GPCR充当 神经递质释放的精确模式的检测器，并能够反过来调节神经元的兴奋性和突触 传输特别重要的是C类代谢型谷氨酸（mGluR）和GABA受体（GABABR）， 分别对主要的兴奋性和抑制性神经递质作出反应，并作为神经和 精神疾病不幸的是，我们对它们潜在的信号分子机制的理解仍然有限 由于缺乏直接测量和操作其活性的方法， 时间精度此外，C类GPCR的生物物理激活机制特别具有挑战性 因为不像A类GPCR，如视紫红质或β-肾上腺素能受体，它们含有大量的细胞外配体结合， 多聚化结构域（LBD），其通过知之甚少的机制多聚化并偶联至跨膜结构域（TMD）。 我们最近的工作建立了直接测量mGluR组装和构象的新光学方法 在单分子水平的动力学，也产生了光遗传学方法来操纵受体亚型 选择性和高时空精度。这些突破推动了我们的 了解mGluRs如何二聚化和导致合作受体激活的初始分子运动，但许多 根本问题依然存在。在研究领域1中，我们将剖析mGluRs和GABABR的激活机制， 一个定量的，跨学科的方式使用光学方法，包括单分子福斯特共振能量 转移（FRET）来测量构象动力学，结合功能性报告基因和详细的结构 分析.长期的目标是从生物药理学的角度了解变构域间和亚基间的偶联相互作用 允许正构和变构配体结合以产生G蛋白活化。这项工作将使主要洞察到 一个大类膜受体的基本激活过程，并应提供更深入的了解， 分子药理学在研究领域2中，我们将改进和利用光学传感器的激活能力， 受体的光遗传学控制以探测不同mGluR亚型在活化，信号传导， 和脱敏，并剖析他们的时空信号在海马突触。从长远 我们计划利用这些信息来探索突触前，突触后， 和神经胶质mGluR群体。这项工作将提供mGluR信号传导的动态图像，这是该领域所缺少的。 并将加强我们对这些受体在健康和疾病的突触调节中的作用的分子理解。

Project Summary In many biological systems G protein-coupled receptors (GPCRs) provide a crucial molecular link between the dynamics of the extracellular environment and the associated intracellular signaling response. In the nervous system, GPCRs serve as detectors of precise patterns of neurotransmitter release and are able to, in turn, modulate neuronal excitability and synaptic transmission. Of particular importance are the class C metabotropic glutamate (mGluR) and GABA receptors (GABABR), which respond to the major excitatory and inhibitory neurotransmitters, respectively, and serve as drug targets for neurological and psychiatric disorders. Unfortunately, our understanding of their underlying molecular mechanisms of signaling remain limited due to a lack of methods for the direct measurement and manipulation of their activity with high specificity and spatial and temporal precision. Furthermore, the biophysical activation mechanism of class C GPCRs is particularly challenging to decipher because unlike class A GPCRs, such as rhodopsin or ß-adrenergic receptors, they contain large, extracellular ligand binding domains (LBDs) that multimerize and couple, via a poorly understood mechanism, to a transmembrane domain (TMD). Our recent work has established new optical methods for directly measuring mGluR assembly and conformational dynamics at the single molecule level and has also produced an optogenetic method to manipulate receptors with subtype selectivity and high spatiotemporal precision using photoswitchable tethered ligands. These breakthroughs have advanced our understanding of how mGluRs dimerize and the initial molecular motions that lead to cooperative receptor activation, but many fundamental questions remain. In research area 1 we will dissect the activation mechanism of mGluRs and GABABRs in a quantitative, interdisciplinary way using optical approaches, including single molecule Forster resonance energy transfer (FRET) to measure conformational dynamics, in conjunction with functional reporters and detailed structural analysis. The long-term goal is to understand, biophysically, how allosteric inter-domain and inter-subunit coupling interactions permit orthosteric and allosteric ligand binding to produce G protein activation. This work will give major insight into the fundamental activation processes of a large class of membrane receptors and should provide a deeper understanding of their molecular pharmacology. In research area 2 we will improve and harness the power of optical sensors of activation and optogenetic control of receptors to probe the kinetics of different mGluR subtypes at the level of activation, signaling, and desensitization and to dissect their spatiotemporal signaling profiles at hippocampal synapses. In the long term we plan to use this information to probe the mechanism of induction of long-term depression by pre-synaptic, post-synaptic, and glial mGluR populations. This work will provide a dynamic picture of mGluR signaling that has been missing from the field and will strengthen our molecular understanding of the role of these receptors in synaptic modulation in health and disease.