Investigating the molecular details of assembly, disassembly and trafficking of GPCR-arrestin complexes

研究 GPCR-arrestin 复合物组装、拆卸和运输的分子细节

• 批准号: 10507234
• 负责人: John Janetzko
• 金额: $ 10万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10507234
• 关键词: Acute; Address; Adrenergic Receptor; Affect; Agonist; Arrestins; Back; Behavior; Binding; Biological; Biophysics; Cell membrane; Cells; Clathrin; Communication; Complex; Coupled; Data; Dependence; Development; Disease; Dissociation; Down-Regulation; Drug Tolerance; Endocytosis; Endosomes; Environment; FDA approved; Fluorescence Spectroscopy; Foundations; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Health; Hormone Receptor; Hormones; Human; Investigation; Label; Light; Lysosomes; Mass Spectrum Analysis; Mediating; Membrane; Membrane Lipids; Membrane Proteins; Mentors; Microscopy; Modeling; Molecular; Monitor; Nature; Neurotransmitters; Organism; Peptides; Pharmaceutical Preparations; Phase; Phosphatidylinositol 4,5-Diphosphate; Phosphorylation; Physiology; Play; Process; Protein Family; Proteins; Proteomics; Recycling; Regulation; Research; Resolution; Role; Signal Transduction; Stimulus; Structure; Tertiary Protein Structure; Therapeutic; Tissues; Training; V2 Receptors; Vasopressins; Vesicle; Work; career; crosslink; desensitization; drug action; drug development; experimental study; improved; insight; mimetics; molecular modeling; novel; prevent; protein complex; protein protein interaction; protein transport; receptor; receptor recycling; reconstitution; recruit; response; scaffold; single molecule; trafficking; trans-Golgi Network

Project Summary: Mis-regulation of G protein-coupled receptor (GPCR) trafficking and signaling is implicated in causing several diseases and the development of drug tolerance, having a major impact on human health. GPCRs evolved to be the most important means for communication between cells and tissues in higher organisms. They are responsive to a wide range of stimuli including light, odorants, peptides, neurotransmitters, and hormones, making GPCRs critical players in regulating human physiology. Owing to their importance, they are the targets for a third of all FDA-approved drugs. For signaling to be temporally regulated, after agonist stimulation, GPCRs are desensitized. This desensitization occurs as a two-step process: first by phosphorylation, then by binding to proteins called -arrestins. -arrestin binding promotes acute desensitization by blocking access of G proteins to receptors. In addition, -arrestins act as adapters to proteins involved in clathrin-mediated endocytosis, facilitating internalization of the GPCR. Once internalized, the fate of a GPCR can differ dramatically, from being rapidly recycled back to the plasma membrane to being degraded. While classically GPCR signaling was thought to be confined to the plasma membrane, it is now appreciated that GPCRs can also signal from various intracellular compartments. Though our understanding of G protein-mediated signaling has matured over years of study, our understanding of how GPCRs are recognized as endocytic cargo remains limited. An important protein complex for this process is retromer, which sorts cargo at endosomes for recycling. A key component of retromer, vps26, is structurally similar to -arrestins, and is important for cargo recognition. I hypothesize that arrestin domain proteins are a privileged scaffold for recognition and trafficking of membrane proteins. As a result, understanding the molecular mechanisms that determine how GPCR--arrestins assemble and disassemble, and how they are trafficked in a cell, will have a profound impact on our understanding of signaling from GPCRs and the action of drugs. Using the 2AR together with V2R and NTSR1 as model receptors, I will (1) characterize how GPCR--arrestin complexes assemble and disassemble, and how this is affected by membrane lipids, GPCR phosphorylation, and the presence of other binding partners. I will also (2) identify protein interaction partners of GPCR--arrestin complexes in cells to understand which factors regulate the rapid or slow recycling behavior of these receptors. Finally, (3) I will characterize the engagement of a GPCR by retromer. These aims will be addressed using single-molecule fluorescence spectroscopy, state-of-the-art mass spectrometry, and in-cell photo-crosslinking. These aims will answer long-standing questions pertaining to arrestin function, and open new lines of investigation into regulation of GPCRs at endosomes. My Mentor, Dr. Kobilka, co-mentor Dr. von Zastrow and expert advisors in proteomics and protein-protein interactions (Drs. Hüttenhain, Krogan, Ting) and arrestin proteins (Dr. Benovic), will provide me with the training necessary to complete these aims and launch my independent research career.

项目摘要： G蛋白偶联受体（GPCR）运输和信号传导的错误调节暗示引起了几种 疾病和药物耐受性的发展，对人类健康产生重大影响。 GPCR进化为 成为较高生物体中细胞和组织之间通信的最重要手段。他们是 对广泛的刺激有反应，包括光，气味，辣椒，神经递质和激素， 使GPCR在控制人类生理学方面成为关键参与者。由于它们的重要性，它们是目标 为了暂时调节信号，在激动剂刺激后，GPCR 脱敏。这种脱敏是作为两个步骤的过程：首先是通过磷酸化，然后结合到 蛋白质称为-arrestins。 -arrestin结合通过阻止G蛋白的访问来促进急性脱敏 到受体。此外，rarstin是参与网格蛋白介导的内吞作用的蛋白质的衔接子， 促进GPCR的内在化。一旦内部化，GPCR的命运就可以大大不同，而不是 迅速回收回到质膜以降解。虽然考虑了古典的GPCR信号 要局限于质膜，现在众所周知，GPCR也可以发出各种信号 细胞内室。尽管我们对G蛋白介导的信号传导的理解已经成熟 在研究中，我们对GPCR如何被认为是内吞货物的理解仍然有限。一个重要的 该过程的蛋白质复合物是逆转录器，它与内体的货物进行了对回收的货物。一个关键组成部分 Demer，VPS26在结构上类似于-rarestins，对于货物识别很重要。我假设这一点 逮捕蛋白蛋白是识别和贩运膜蛋白的特权脚手架。作为 结果，了解确定GPCR--rarestins如何组装和 拆卸，以及它们如何被贩运在细胞中，将对我们对信号的理解产生深远的影响 来自GPCR和药物的作用。将2AR与V2R和NTSR1一起用作模型接收器，我将 （1）表征GPCR--arrestin络合物如何组装和拆卸，以及如何影响这 膜脂质，GPCR磷酸化以及其他结合伴侣的存在。我还将（2）确定 细胞中GPCR--arrestin复合物的蛋白质相互作用伙伴，以了解哪些因素调节快速 或这些受体的回收行为缓慢。最后，（3）我将表征GPCR的参与 Demomer。这些目标将使用单分子荧光光谱，最先进的质量来解决 光谱法和电池内照相链接。这些目标将回答有关的长期问题 逮捕蛋白功能，并为内体的GPCR规定开放新的投资线。我的导师博士 Kobilka，von Zastrow博士和蛋白质组学和蛋白质 - 蛋白质相互作用的专家顾问（Drs。 Hüttenhain，Krogan，Ting）和逮捕蛋白（Benovic博士）将为我提供必要的培训 完成这些目标并启动我的独立研究生涯。