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.

项目总结 这项提案包含一个全面的计划，以支持向独立研究事业的过渡 侧重于了解膜蛋白功能是如何通过与其他蛋白质的相互作用和 磷脂膜，以及这种调节如何影响细胞过程和在疾病状态下的变化。 这份申请描述了一个与我的职业和培训目标直接相关的创新和雄心勃勃的建议。 背景：G蛋白偶联受体(G蛋白偶联受体)是一种膜蛋白，它能感觉细胞外刺激和 通过与胞浆G蛋白的相互作用启动细胞内信号通路。传统上，GPCR是 认为通过与β-arrestins的相互作用经历了一个多步骤的失活和脱敏过程 固有无序的GPCRC-末端结构域(CTD)的过度磷酸化。β-Drastins也可以 除了脱敏和内化GPCRs外，还启动了不同的信号级联反应，尽管分子 每条途径的决定因素还没有被很好地理解。这一系统因有了 两种β-arrestin亚型，被认为倾向于不同的GPCRCTD磷酸化模式，并可能 启动不同的细胞通路。代谢性谷氨酸受体(MGluR)是一种二聚体神经性GPCR， 负责感觉突触前和突触后的主要兴奋性神经递质谷氨酸。最近 一个单一的亚型，mGluR3，被鉴定为经历了β-arrestin介导的内化和β-arrestin- 鉴定了CTD的结合区。这是首次报道二聚体gpr与β之间的相互作用。 逮捕，导致了关于复杂化学计量和结构的新问题。此mGluR3-β-arrestin 初步数据表明mGluR3的β-arrestin结合区的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 研究mGluR3与β抑制蛋白和膜之间的相互作用 1)单分子和系综荧光结合方法研究决定因素 MGluR3-β-arrestin络合物的形成，以及2)冷冻电子显微镜和核磁共振 光谱分析以评估mGluR3、β-arrestins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分析和成像得到验证。调查结果将向 K99/R00提案旨在将此F32研究计划的方法和结果广泛应用于其他 并在活体模型中探索GPCRs-β-arrestin偶联的功能后果。培训 和指导：培训目标得到了1)由莱维茨博士和埃利泽博士组成的共同赞助者团队的支持，以及一个收尾 合作者(迈耶森博士)，在生物物理和生化研究方面具有专业知识，2)拥有丰富的机构 韦尔康奈尔医学院的环境，以及3)科学会议、研讨会和计划出版的出版物。 影响：在这个奖项中获得的经验将为我的独立职业生涯奠定基础 蛋白质生物物理学研究人员，并开发一种新的膜蛋白质研究计划。