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。

基本信息

批准号：
10704588
负责人：
Dagan Marx
金额：
$ 6.95万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
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/10704588
关键词：
Affect Affinity Agonist Alanine Alzheimer&apos s Disease Anxiety Arrestin Beta 1 Arrestins Award Binding Biochemical Biological Assay Biophysics C-terminal Cell physiology Cells Cellular Assay Clathrin Collaborations Complex Coupling Cryoelectron Microscopy Cytoplasm Data Development Disease Dissociation Environment Event Face Family Fluorescence Foundations G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors GTP-Binding Proteins Glutamates Goals Heterotrimeric GTP-Binding Proteins In Vitro Institution 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 autism spectrum disorder beta-arrestin biophysical techniques career cellular imaging desensitization dimer experience extracellular high throughput screening improved in vivo Model innovation medical schools meetings monomer nervous system disorder novel postsynaptic presynaptic programs protein function receptor receptor function receptor internalization recruit response single molecule stoichiometry

项目摘要

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职业职业生涯的过渡专注于理解膜蛋白功能如何通过与蛋白质相互作用和磷脂膜，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一项企业和雄心勃勃的提议。背景：G蛋白包装的受体（GPCR）是膜蛋白，感知刺激性刺激和通过与胞质G蛋白相互作用，启动细胞内信号通路被认为是通过与β-arrestin的相互作用进行多步灭活和降低过程固有的GPCR C末端结构域（CTD）的高磷酸化也可以除了分子外，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未得到充分理解。两种β-甲蛋白同工型，被认为是启动不同的细胞途径。负责感应主要的兴奋性神经递质谷氨酸，均在突触后。鉴定出一种单一的同工型MGLUR3，可以进行稳健的β介导的内在化和β-arrestin- 确定了CTD的结合区域。阻止蛋白，导致有关复杂化学计量和结构的新问题初步数据表明MGLUR3的β-arrestin结合军团进一步复杂化。 CTD还与磷脂膜相互作用。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您的确定词 MGLUR3-β-arrestin复合物的形成，以及2）冷冻电子显微镜和核磁磁性共振光谱法评估了MGLUR3，Bβ-甲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使用地窖测定和成像进行验证。 K99/R00提案旨在应用该F32研究的方法和发现， GPCR并在体内训练中追求GPCR-β-arrestin耦合的功能构建。和指导：培训目标得到1）联合赞助商DRS的支持。合作者（Meyerson博士）具有生物物理和生化研究专业知识，2）富裕研究所 Weill Cornell医学院的环境以及3）科学会议，研讨会和计划出版物。影响：奖励期间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。