Structure and function of mGluR3 interactions with beta-arrestins and the membrane.

mGluR3 与 β-arrestins 和膜相互作用的结构和功能。

• 批准号: 10536255
• 负责人: Dagan Marx
• 金额: $ 6.72万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536255
• 关键词: Affect; Affinity; Agonist; Alanine; Alzheimer' s Disease; Anxiety; Arrestins; Award; Binding; Biochemical; Biological Assay; Biophysics; C-terminal; Cell physiology; Cells; Cellular Assay; Clathrin; Collaborations; Complex; Coupling; Cryoelectron Microscopy; Data; Development; Disease; Environment; Event; Face; Family; Fluorescence; Foundations; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Glutamates; Goals; Heterotrimeric GTP-Binding Proteins; In Vitro; Lead; Length; Libraries; Measurement; Measures; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Mental Depression; Mental disorders; Mentors; Metabotropic Glutamate Receptors; Methods; Molecular; Molecular Conformation; Monomeric GTP-Binding Proteins; Mutation; NMR Spectroscopy; Neurons; Neurotransmitters; Nucleotides; Outcome; Pathway interactions; Pattern; Phospholipids; Phosphorylation; Photobleaching; Population; Process; Protein Isoforms; Proteins; Protomer; Publications; Regulation; Reporting; Research; Research Design; Research Personnel; Resolution; Schizophrenia; Serine; Serine/Threonine Phosphorylation; Signal Pathway; Signal Transduction; Site; Stimulus; Structure; Synapses; System; Techniques; Tertiary Protein Structure; Thermodynamics; Threonine; Training; Transmembrane Domain; Variant; Work; arrestin 1; autism spectrum disorder; base; beta-arrestin; biophysical techniques; career; cellular imaging; desensitization; dimer; experience; extracellular; high throughput screening; improved; in vivo Model; innovation; medical schools; meetings; nervous system disorder; novel; programs; protein function; receptor; receptor function; receptor internalization; recruit; response; single molecule; stoichiometry

PROJECT SUMMARY This proposal contains a comprehensive program to support a transition toward an independent research career focused on understanding how membrane protein function is modulated by interactions with other proteins and phospholipid membranes, and how this modulation impacts cellular processes and is altered in disease-states. This application describes an innovative and ambitious proposal directly tied to my career and training goals. Background: G protein-coupled receptors (GPCR) are membrane proteins that sense extracelluar stimuli and initiate intracellular signaling pathways through interactions with cytosolic G proteins. Classically, GPCRs are thought to undergo a multi-step inactivation and desensitization process via interactions with β-arrestins following hyper-phosphorylation of the intrinsically-disordered GPCR C-terminal domain (CTD). β-arrestins can also initiate distinct signaling cascades in addition to desensitizing and internalizing GPCRs, though the molecular determinants for each pathway are not well understood. This system is further complicated by the presence of two β-arrestin isoforms which are thought to prefer different GPCR CTD phosphorylation patterns and may initiate different cellular pathways. Metabotropic glutamate receptors (mGluR) are dimeric, neuronal GPCRs that are responsible for sensing main excitatory neurotransmitter glutamate both pre- and post-synaptically. Recently a single isoform, mGluR3, was identified to undergo robust β-arrestin-mediated internalization and the β-arrestin- binding region of the CTD was identified. This was the first reported interaction between a dimeric GPCR and β- arrestins, leading to novel questions about complex stoichiometry and structure. This mGluR3-β-arrestin interaction is further complicated by preliminary data indicating that the β-arrestin binding region of the mGluR3 CTD also interacts with the phospholipid membrane. Specific Aims and Research Design: The proposed study investigates the interactions between mGluR3 and both β-arrestins and the membrane using a combination of 1) single-molecule and ensemble fluorescence-based binding methods to investigate the determinants of the formation of mGluR3-β-arrestin complexes, and 2) cryo-electron microscopy and nuclear magnetic resonance spectroscopy to assess the structural features of interactions between mGluR3, β-arrestins, and the membrane. My in vitro and structural findings will be validated using cellular assays and imaging. Findings will inform a K99/R00 proposal aimed to apply the approaches and findings from this F32 research program broadly to other GPCRs and to pursue the functional consequences of GPCR-β-arrestin coupling in in vivo models. Training and Mentoring: Training goals are supported by 1) a team of co-sponsors Drs. Levitz and Eliezer and a close collaborator (Dr. Meyerson) with expertise in biophysical and biochemical research, 2) a rich institutional environment at Weill Cornell Medical College, and 3) scientific meetings, seminars, and planned publications. Impact: The experience gained during this award will serve at the foundation for my independent career as a protein biophysics researcher and for the development of a novel program of membrane protein research.

项目概要 该提案包含一个全面的计划，以支持向独立研究职业的过渡 专注于了解膜蛋白功能如何通过与其他蛋白质的相互作用进行调节，以及 磷脂膜，以及这种调节如何影响细胞过程并在疾病状态下发生改变。 该申请描述了一个与我的职业和培训目标直接相关的创新且雄心勃勃的提案。 背景：G蛋白偶联受体（GPCR）是感知细胞外刺激和 通过与胞质 G 蛋白相互作用启动细胞内信号传导途径。传统上，GPCR 是 被认为通过与 β-arrestins 相互作用经历多步失活和脱敏过程 本质上无序的 GPCR C 端结构域 (CTD) 的过度磷酸化。 β-抑制素还可以 除了使 GPCR 脱敏和内化之外，还启动不同的信号级联反应，尽管分子机制 每个途径的决定因素尚不清楚。该系统因存在而变得更加复杂 两种 β-arrestin 亚型被认为更喜欢不同的 GPCR CTD 磷酸化模式，并且可能 启动不同的细胞途径。代谢型谷氨酸受体 (mGluR) 是二聚体神经元 GPCR， 负责感知突触前和突触后的主要兴奋性神经递质谷氨酸。最近 单一异构体 mGluR3 被鉴定为经历强大的 β-arrestin 介导的内化，并且 β-arrestin- CTD 的结合区域已被鉴定。这是二聚体 GPCR 和 β- 之间首次报道的相互作用 视紫红质抑制蛋白，引发了有关复杂化学计量和结构的新问题。这种 mGluR3-β-arrestin 初步数据表明 mGluR3 的 β-arrestin 结合区域使相互作用进一步复杂化 CTD 还与磷脂膜相互作用。具体目标和研究设计：拟议的研究 使用以下组合研究 mGluR3 与 β-arrestins 和膜之间的相互作用 1）基于单分子和整体荧光的结合方法来研究结合的决定因素 mGluR3-β-arrestin 复合物的形成，以及 2) 冷冻电子显微镜和核磁共振 光谱法评估 mGluR3、β-抑制蛋白和膜之间相互作用的结构特征。 我的体外和结构发现将通过细胞测定和成像进行验证。调查结果将告知 K99/R00 提案旨在将该 F32 研究计划的方法和结果广泛应用于其他领域 GPCR 并在体内模型中探究 GPCR-β-arrestin 偶联的功能后果。训练 和指导：培训目标由 1) 共同发起人 Drs. 团队支持。莱维茨和埃利泽以及结束 合作者（Meyerson 博士）在生物物理和生化研究方面拥有专业知识，2）丰富的机构 威尔康奈尔医学院的环境，以及 3) 科学会议、研讨会和计划的出版物。 影响：在这个奖项中获得的经验将为我作为独立职业者奠定基础 蛋白质生物物理学研究员，并开发了膜蛋白研究的新计划。