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(GPCRs)提供了动态之间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，GPCRs作为 神经递质释放的精确模式的探测器，并能够反过来调节神经元的兴奋性和突触 变速箱。尤其重要的是C类代谢性谷氨酸(MGluR)和GABA受体(GABABR)，它们 分别对主要的兴奋性和抑制性神经递质作出反应，并作为神经和神经的药物靶点。 精神障碍。不幸的是，我们对它们潜在的信号分子机制的了解仍然有限。 由于缺乏直接测量和操纵其活性的方法，具有高度的特异性和空间和 时间精确度。此外，C类GPCRs的生物物理激活机制尤其难以破译 因为与A类GPCRs不同，如视紫红质或肾上腺素能受体，它们含有大量的细胞外配体结合 结构域(LBD)，通过一种鲜为人知的机制，多聚化并耦合到跨膜结构域(TMD)。 我们最近的工作建立了直接测量mGluR组装和构象的新的光学方法 在单分子水平上的动力学，还产生了一种光遗传学方法来操纵具有亚型的受体 使用可光切换的系链配体的选择性和高时空精度。这些突破推动了我们的 对mGluRs如何二聚化以及导致合作受体激活的初始分子运动的了解，但有许多 根本问题依然存在。在研究领域1，我们将剖析mGluRs和GABABRs的激活机制 一种使用光学方法的定量、跨学科的方法，包括单分子福斯特共振能量 转移(FRET)，以测量构象动力学，结合功能记者和详细的结构 分析。长期的目标是从生物物理的角度理解变构结构域间和亚单位间的耦合作用 允许正构和变构配体结合以产生G蛋白激活。这项工作将使我们深入了解 一大类膜受体的基本激活过程，应该有助于更深入地了解它们的 分子药理学。在研究领域2，我们将改进和利用光学传感器的激活和 光遗传控制的受体，以探索不同亚型的mGluR在激活，信号， 和脱敏，并剖析它们在海马突触的时空信号特征。从长远来看 我们计划利用这一信息来探索突触前、突触后、突触前、突触后、突触后、突触前、突触后 和神经胶质细胞mGluR群体。这项工作将提供现场缺失的mGluR信号的动态图像 并将加强我们对这些受体在健康和疾病中突触调节中作用的分子理解。

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.