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

项目概要： G蛋白偶联受体（GPCR）运输和信号传导的错误调节涉及引起几种 疾病和药物耐受性的发展，对人类健康产生重大影响。GPCR发展到 是高等生物细胞和组织之间最重要的通讯手段。他们是 对包括光、气味剂、肽、神经递质和激素在内的广泛刺激作出反应， 使GPCR在调节人体生理学方面发挥重要作用。由于他们的重要性，他们是目标 三分之一的FDA批准的药物。对于暂时调节的信号传导，在激动剂刺激后，GPCR 是不敏感的。这种脱敏作用分两步进行：首先通过磷酸化，然后通过与 这种蛋白质叫做β-抑制蛋白。β-arrestin结合通过阻断G蛋白通路促进急性脱敏 到受体。此外，β-抑制蛋白作为与网格蛋白介导的内吞作用有关的蛋白质的接头， 促进全球化学还原的国际化。一旦内化，GPCR的命运就会大不相同， 迅速再循环回到质膜被降解。虽然经典的GPCR信号被认为是 为了限制于质膜，现在认识到GPCR也可以从各种细胞中发出信号。 细胞内区室虽然我们对G蛋白介导的信号传导的理解已经成熟多年 然而，我们对GPCR如何被识别为内吞货物的理解仍然有限。一个重要 用于该过程的蛋白质复合物是retromer，其在核内体处对货物进行分类以进行再循环。的关键组成部分 retromer，VPS 26，在结构上类似于β-抑制蛋白，并且对于货物识别是重要的。我假设 抑制蛋白结构域蛋白是用于识别和运输膜蛋白的特殊支架。作为 结果，了解决定GPCR-β-arrestins如何组装的分子机制， 以及它们如何在细胞中被运输，将对我们对信号的理解产生深远的影响。 GPCR和药物的作用。使用V2 R和NTSR 1作为模型受体， (1)描述GPCR-β-arrestin复合物如何组装和拆卸，以及如何受到 膜脂质、GPCR磷酸化和其他结合伴侣的存在。（2）识别 GPCR-抑制蛋白复合物的蛋白质相互作用伙伴在细胞中，以了解哪些因素调节快速 或者这些受体的缓慢循环行为。最后，（3）我将通过以下方式描述全球保护化学品还原的参与： retromer。这些目标将使用单分子荧光光谱，最先进的质量 光谱法和电池内光交联。这些目标将回答长期存在的问题， 抑制蛋白的功能，并开辟了新的研究路线，在核内体的GPCR的调节。我的导师，博士。 Kobilka，共同导师冯Zastrow博士和专家顾问在蛋白质组学和蛋白质-蛋白质相互作用（博士。 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，开始我的独立研究生涯。

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.