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.

项目概要： G 蛋白偶联受体 (GPCR) 运输和信号传导的错误调节可能导致多种疾病 疾病和耐药性的发展，对人类健康产生重大影响。 是高等生物细胞和组织之间最重要的通讯手段。 对各种刺激做出反应，包括光、气味、肽、神经递质和激素， 使 GPCR 在调节人类生理学中发挥关键作用，由于其重要性，它们成为了目标。 对于三分之一的 FDA 批准药物来说，在激动剂刺激后，GPCR 会暂时调节信号。 这种脱敏过程分为两步：首先通过磷酸化，然后通过结合。 称为 -arrestin 的蛋白质结合通过阻止 G 蛋白的进入来促进急性脱敏。 此外，-arrestins 充当参与网格蛋白介导的内吞作用的蛋白质的接头， 促进 GPCR 的内化 一旦内化，GPCR 的命运可能会发生巨大的变化。 快速循环回到质膜并被降解，而经典的 GPCR 信号被认为是。 由于仅限于质膜，现在人们认识到 GPCR 还可以从各种不同的信号发出信号 尽管我们对 G 蛋白介导的信号传导的理解已经成熟多年。 根据研究，我们对 GPCR 如何被识别为内吞货物的理解仍然有限。 该过程的蛋白质复合物是逆转录酶，它对内体中的货物进行分类以进行回收。 逆转录酶 vps26 在结构上与 -arrestins 相似，并且对于货物识别很重要。 抑制蛋白结构域蛋白是识别和运输膜蛋白的特殊支架。 结果，了解决定 GPCR--arrestins 如何组装和的分子机制 分解以及它们如何在细胞中运输，将对我们对信号传导的理解产生深远的影响 通过使用 2AR 以及 V2R 和 NTSR1 作为模型受体，我将了解 GPCR 和药物的作用。 (1) 描述 GPCR--arrestin 复合物如何组装和分解，以及其如何受到以下因素的影响 我还将 (2) 识别膜脂、GPCR 磷酸化和其他结合伙伴的存在。 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 最后，(3) 我将通过以下方式来描述 GPCR 的参与。 这些目标将通过单分子荧光光谱、最先进的质量来实现。 光谱测定法和细胞内光交联技术将回答长期存在的问题。 我的导师，Dr. Kobilka、共同导师 von Zastrow 博士以及蛋白质组学和蛋白质-蛋白质相互作用方面的专家顾问（Drs. Hüttenhain、Krogan、Ting）和抑制蛋白（Benovic 博士）将为我提供必要的培训 完成这些目标并开始我的独立研究生涯。