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，以支持向独立的研究生涯过渡 专注于了解膜蛋白功能是如何通过与其他蛋白质的相互作用来调节的， 磷脂膜，以及这种调节如何影响细胞过程，并在疾病状态下改变。 此申请描述了一个创新和雄心勃勃的建议，直接关系到我的职业和培训目标。 背景：G蛋白偶联受体（GPCR）是一种感受细胞外刺激的膜蛋白， 通过与胞质G蛋白相互作用启动胞内信号传导途径。传统上，GPCR是 被认为通过与β-抑制蛋白的相互作用经历多步失活和脱敏过程， 固有无序的GPCR C末端结构域（CTD）的过度磷酸化。β-抑制蛋白还可以 除了脱敏和内化GPCR外，还启动不同的信号级联反应，尽管分子 每种途径的决定因素还不清楚。该系统由于以下因素的存在而进一步复杂化： 两种β-抑制蛋白同种型被认为偏好不同的GPCR CTD磷酸化模式， 启动不同的细胞通路。代谢型谷氨酸受体（mGluR）是二聚的神经元GPCR， 负责在突触前和突触后感知主要兴奋性神经递质谷氨酸。最近 一种单一的同种型mGluR 3被鉴定为经历强有力的β-抑制蛋白介导的内化，β-抑制蛋白- 鉴定了CTD的结合区。这是第一次报道二聚体GPCR和β- 抑制蛋白，导致复杂的化学计量和结构的新问题。这种mGluR 3-β-抑制蛋白 初步数据表明mGluR 3的β-抑制蛋白结合区 CTD还与磷脂膜相互作用。具体目标和研究设计：拟议研究 研究了mGluR 3与β-抑制蛋白和膜之间的相互作用， 1)单分子和合奏荧光结合方法，以调查的决定因素， mGluR 3-β-抑制蛋白复合物的形成，以及2）冷冻电子显微镜和核磁共振 通过质谱分析来评估mGluR 3、β-抑制蛋白和膜之间相互作用的结构特征。 我的体外和结构研究结果将通过细胞分析和成像来验证。调查结果将告知 K99/R 00提案旨在将F32研究计划的方法和发现广泛应用于其他领域。 GPCR和追求在体内模型中GPCR-β-抑制蛋白偶联的功能后果。培训 和指导：培训目标得到以下方面的支持：1）一个由Levitz和Eliezer博士组成的共同赞助团队， 合作者（Meyerson博士）在生物物理和生物化学研究方面具有专业知识，2）丰富的机构 环境在威尔康奈尔医学院，和3）科学会议，研讨会，并计划出版物。 影响：在这个奖项期间获得的经验将为我作为一个独立的职业生涯奠定基础。 蛋白质生物物理学研究员，并为膜蛋白研究的新计划的发展。