Mechanisms of Regulation of Metabotropic Glutamate Receptors

项目总结 G蛋白偶联受体(Gpcr)是一种微调的信号分子，是多种信号分子的中心。 并作为治疗神经和精神疾病的主要药物靶点。一位少校 Gpr的调节形式是通过与激活的多功能β-arr结合的作用来实现的。 受体驱动功能性脱敏，控制受体转运，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gpr/β-arr偶联的机械理解是有限的。 基于少数原型家系A的GPCR。提高对gpr/β的认识是至关重要的。 通过ARR耦合来揭开不同生物之间变异性的生物物理基础和生物学后果 GPCR亚家族和亚型之间，以及不同的药理配体之间。 代谢性谷氨酸受体(MGluRs)是C家族GPCRs的一个八人家族，具有一个 独特的结构，由大的胞外配体结合域组成，介导构成成分 二聚化。由于它们在突触神经调节中的作用，mGluR已成为治疗的药物靶点 神经发育障碍、神经退行性疾病、精神疾病以及癌症。然而，它一直是 由于缺乏对mGluRs基本信号的了解，难以从治疗上利用mGluRs 和监管性质。破译mGluR调节机制尤其具有挑战性，因为 这个亚家族通过异源二聚作用得到了极大的扩展，并成为一大批正构体药物的靶标。 和具有不同性质的变构配体。直到最近，mGluR/β-ARR偶联还没有得到很好的描述， 但我们最近发现，mGluR的一个子集能够实现强大的β-ARR偶联，而其他的则高度抵抗， 为这个GPCR家族提供了分子多样性的另一个维度。 我们将以我们最近的发现为基础，用一组结构、生物物理和细胞为基础的电池 测量，以了解mGluR/β-ARR的潜在机制和生理后果 耦合。在目标1中，我们将开发和利用单分子成像分析来定义决定因素和 MGluR/β-ARR复合体形成的化学计量，然后使用电子显微镜(负染，冷冻-EM)来 解决mGluR/β-ARR复合体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 生物物理方法确定β-ARRs的mGluR C-末端结构域和跨膜核心偶联的基础 跨亚型，并探讨不同的配体类型和异源二聚化对mGluR/β-ARR偶联的影响。 在目标3中，我们将调查mGluR/β-ARR偶联的贩运和功能后果。 细胞系和皮质神经元中的分辨光学和邻近蛋白质组学技术，重点是 突触前信号和交易。综上所述，该项目将全面了解mGluRs的监管情况 通过β-ARRS，提供了对这个关键受体家族的更深层次的了解，并获得了更广泛的洞察力 GPCR/β-ARR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.