Mechanisms of Regulation of Metabotropic Glutamate Receptors

代谢型谷氨酸受体的调节机制

• 批准号: 10660420
• 负责人: Joshua Levitz
• 金额: $ 68.31万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660420
• 关键词: ARRB1 gene; Architecture; Arr2; Binding; Binding Sites; Biological; Biological Assay; Biophysics; C-terminal; Calorimetry; Cell Line; Cells; Complex; Coupling; Cryoelectron Microscopy; Data; Dimensions; Dimerization; Drug Targeting; Electron Microscopy; Endocytosis; Esthesia; Family; Family member; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Proteins; Heterodimerization; Image; In Vitro; Individual; Ligand Binding Domain; Ligands; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Mental disorders; Metabotropic Glutamate Receptors; Molecular; NMR Spectroscopy; Negative Staining; Nerve Degeneration; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Optics; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phosphorylation; Physiological; Process; Property; Proteomics; Receptor Activation; Receptor Down-Regulation; Regulation; Resistance; Resolution; Role; Shapes; Signal Transduction; Signaling Molecule; Signaling Protein; Structure; Synapses; Techniques; Therapeutic; Time; Titrations; Transducers; Work; base; beta-arrestin; biophysical techniques; cell cortex; desensitization; dimer; extracellular; fluorescence imaging; genetic regulatory protein; improved; insight; live cell imaging; metabotropic glutamate receptor 2; metabotropic glutamate receptor 3; metabotropic glutamate receptor 8; molecular imaging; nervous system disorder; neurophysiology; neuroregulation; neurotransmission; pharmacologic; presynaptic; prevent; receptor; receptor downregulation; response; scaffold; single molecule; spatiotemporal; stoichiometry; trafficking

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.

项目摘要 G蛋白偶联受体（GPCR）是对潜水员核心的细调信号分子 神经生理过程，并作为神经和精神疾病的主要药物靶标。专业 GPCR调节的形式是通过多功能β-arrestin（β-arrs）的作用而发生的 受体驱动功能脱敏，控制受体运输并启动G蛋白独立的受体 信号级联。尽管进步，我们对GPCR/β-arr耦合的机械理解是有限的，并且 基于少数原型家族A GPCR。提高我们对GPCR/β-的理解至关重要 通过揭示不同生物物理基础和生物学后果的变异性，而不同 GPCR亚家族和亚型，以及不同药物配体之间。 代谢性谷氨酸受体（MGLURS）形成了一个由八人组成的家族C GPCR，带有A 独特的体系结构由介导一致性的大型细胞外配体结合域组成 二聚化。由于它们在突触神经调节中的作用，mglurs已成为药物靶标的 神经发育，神经退行性和精神病学散布以及癌症。但是，已经 由于缺乏对其基本信号的了解 和监管特性。破译MGLUR管制的机制特别具有挑战性，因为 该亚家族通过异二聚体大大扩展，并由一个正构型面板作为目标 和具有不同特性的变构配体。直到最近，mglur/β-arr耦合的表征还差， 但是我们最近发现，一个mglurs的子集能够具有稳健的β-arr耦合，而其他mglurs具有高度抗性， 为这个GPCR家族提供了分子多样性的另一个维度。 我们将以一系列结构，生物物理和基于细胞的结构，基于细胞的结构，基于我们最近的发现。 测量，以了解mglur/β-arr的潜在机制和物理后果 耦合。在AIM 1中，我们将开发并利用单个分子成像测定法来定义确定词和 mglur/β-arr络合物形成的化学计量法，然后使用电子显微镜（负污渍，冷冻EM）到 解决MGlur/β-Arr复合物的高分辨率结构快照。在AIM 2中，我们将在体外和活细胞中使用 生物物理测定以定义mglur c末端结构域和跨膜核心耦合至β-arrs的基础 跨亚型，并探测不同的配体类型和异二聚化对mglur/β-arr耦合的影响。 在AIM 3中，我们将研究使用高级/β-arr耦合的运输和功能后果 细胞系和皮质神经元中的分辨率光学和接近蛋白质组学技术，重点是 突触前信号传导和贩运​​。该项目将共同提供MGLURS的调节 通过β-arrs，对这个关键受体家族有了更深入的了解，并获得了更广泛的洞察力 GPCR/β-arr耦合。