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.

项目摘要 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和 磷脂膜，以及该调节如何影响细胞过程并在疾病范围内改变。 该应用程序描述了与我的职业和培训目标直接相关的创新和雄心勃勃的提议。 背景：G蛋白偶联受体（GPCR）是膜蛋白，感知外部刺激和 通过与胞质G蛋白相互作用来引发细胞内信号通路。经典，GPCR是 被认为是通过与β-arrestin的相互作用的多步灭活和脱敏过程 内部排序的GPCR C末端结构域（CTD）的高磷酸化。 β-arrestin也可以 除了分子外，还可以启动不同的信号级联反应和内化GPCR 每种途径的决定因素尚不清楚。由于存在，该系统更加复杂 两种β-甲蛋白同工型被认为偏爱不同的GPCR CTD磷酸化模式，并且可能 启动不同的细胞途径。代谢性谷氨酸受体（MGLUR）是二聚体的神经元GPCR， 负责感测前后的主要兴奋性神经递质谷氨酸。最近 鉴定出一种单一的同工型MGLUR3可以进行鲁棒的β-arrestin介导的内在化和β-arrestin- 确定了CTD的结合区域。这是二聚体GPCR和β-的第一次相互作用 逮捕蛋白，导致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-甲素结合区域进一步复杂化。 CTD还与磷脂膜相互作用。具体目的和研究设计：拟议的研究 通过组合使用 1）基于单分子和集合荧光的结合方法研究了 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-arrestin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 拟议的K99/R00旨在将此F32研究计划的方法和发现广泛应用于其他 GPCR并追求体内模型中GPCR-β-arrestin耦合的功能后果。训练 和指导：培训目标得到1）一组共同赞助商DRS的支持。莱维兹和埃利泽以及关闭 合作者（Meyerson博士）具有生物物理和生化研究专业知识，2）丰富的机构 威尔·康奈尔医学院（Weill Cornell Medical College）的环境，以及3）科学会议，半人选和计划出版物。 影响：该奖项中获得的经验将在我的独立职业基金会上作为一个 蛋白质生物物理学研究人员以及为膜蛋白研究的新计划的发展。