Mechanisms of Regulation of Metabotropic Glutamate Receptors

项目摘要 G蛋白偶联受体（GPCR）是一种精细调节的信号分子，是多种免疫反应的核心。 神经生理过程，并作为神经和精神疾病的主要药物靶标。一个主要 一种GPCR调节形式通过多功能β-抑制蛋白（β-arrs）的作用发生，β-arrs结合活化的 受体驱动功能性脱敏，控制受体运输，并启动G蛋白非依赖性 信号级联。尽管取得了进展，但我们对GPCR/β-环糊精偶联的机理理解有限， 基于少量的原型家族A GPCR。提高我们对GPCR/β- 通过解开生物物理学基础和生物学后果之间的差异， GPCR亚家族和亚型，以及不同的药理学配体之间。 代谢型谷氨酸受体（mGluRs）形成了一个由八个成员组成的C家族GPCR家族， 独特的结构，由大的胞外配体结合结构域组成， 二聚化。由于它们在突触神经调节中的作用，mGluRs已经成为突触神经调节的药物靶标。 神经发育、神经变性和精神疾病以及癌症。然而已经 由于缺乏对mGluRs基本信号传导的了解， 和监管属性。破译mGluR调节机制特别具有挑战性，因为 该亚家族通过异源二聚化而显著扩增， 和具有不同性质的变构配体。直到最近，mGluR/β-GluR偶联的特征还很差， 但我们最近发现mGluRs的一个子集能够进行稳健的β-GluRs偶联，而其他mGluRs则具有高度抗性， 为GPCR家族提供了另一种分子多样性。 我们将建立在我们最近的研究结果与电池的结构，生物物理，和细胞为基础的 测量，以了解mGluR/β-arr的潜在机制和生理后果 偶合器.在目标1中，我们将开发和利用单分子成像分析来确定决定因素， mGluR/β-GluR复合物形成的化学计量，然后使用电子显微镜（负染色，cryo-EM） 解决mGluR/β-GluR复合物的高分辨率结构快照。在目标2中，我们将使用体外和活细胞 确定mGluR C-末端结构域和跨膜核心偶联至β-arrs的基础的生物物理测定 并探索不同配体类型和异源二聚化对mGluR/β-GluR偶联的影响。 在目标3中，我们将研究mGluR/β-GluR偶联的运输和功能后果，使用高浓度的 分辨率光学和邻近蛋白质组学技术在细胞系和皮层神经元，重点是 突触前信号和运输。总之，该项目将提供mGluRs调控的全貌， 通过β-arrs，提供了对这一关键受体家族的更深入了解，并获得了对 GPCR/β-环糊精偶联。

PROJECT SUMMARY G protein-coupled receptors (GPCRs) are finely tuned signaling molecules that are central to diverse neurophysiological processes and serve as major drug targets for neurological and psychiatric disorders. A major form of GPCR regulation occurs through the action of multifunctional β-arrestins (β-arrs) which bind to activated receptors to drive functional desensitization, control receptor trafficking, and initiate G protein-independent signaling cascades. Despite progress, our mechanistic understanding of GPCR/β-arr coupling is limited and based on a small number of prototypical family A GPCRs. It is critical to improve our understanding of GPCR/β- arr coupling by unraveling the biophysical basis and biological consequences of variability between different GPCR subfamilies and subtypes, as well as between different pharmacological ligands. The metabotropic glutamate receptors (mGluRs) form an eight-member family of family C GPCRs with a unique architecture consisting of large extracellular, ligand binding domains that mediate constitutive dimerization. Due to their roles in synaptic neuromodulation, mGluRs have emerged as drug targets for neurodevelopmental, neurodegenerative, and psychiatric disoders, as well as cancers. However, it has been difficult to harness mGluRs therapeutically because of a lack of understanding of their basic signaling and regulatory properties. Deciphering the mechanisms of mGluR regulation is particularly challenging since this subfamily is dramatically expanded by heterodimerization and is targeted by a broad panel of orthosteric and allosteric ligands with distinct properties. Until recently, mGluR/β-arr coupling has been poorly characterized, but we recently found that a subset of mGluRs is capable of robust β-arr coupling while others are highly resistant, providing another dimension of molecular diversity to this GPCR family. We will build on our recent findings with a battery of structural, biophysical, and cell-based measurements, to understand the underlying mechanisms and physiological consequences of mGluR/β-arr coupling. In aim 1, we will develop and harness a single molecule imaging assay to define the determinants and stoichiometry of mGluR/β-arr complex formation and then use electron microscopy (negative stain, cryo-EM) to solve high resolution structural snapshots of mGluR/β-arr complexes. In aim 2, we will use in vitro and live cell biophysical assays to define the basis of mGluR C-terminal domain and transmembrane core coupling to β-arrs across subtypes and probe the effects of distinct ligand types and heterodimerization on mGluR/β-arr coupling. In aim 3, we will investigate the trafficking and functional consequences of mGluR/β-arr coupling using high- resolution optical and proximity proteomics techniques in both cell lines and cortical neurons, with a focus on presynaptic signaling and trafficking. Together, this project will provide a full picture of the regulation of mGluRs by β-arrs, providing a deeper understanding of this critical receptor family and gaining broader insight into GPCR/β-arr coupling.