Structural Determination of Novel GPCR Signaling Complexes that Mediate Sustained G Protein Signaling

介导持续 G 蛋白信号传导的新型 GPCR 信号复合物的结构测定

基本信息

批准号：
10471272
负责人：
Anthony Nguyen
金额：
$ 4.29万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10471272
关键词：
Accounting Acute Adrenergic Receptor Agonist American Architecture Argipressin Arrestins Attenuated Binding Binding Proteins Biochemical Biological Assay Bioluminescence C-terminal Cardiovascular Diseases Cardiovascular system Cause of Death Cell membrane Cells Cessation of life Characteristics Clinical Complex Cryoelectron Microscopy Cyclic AMP Data Dissociation Drug Design Drug Targeting Drug usage Electron Microscopy Endosomes Energy Transfer Exhibits Fingers Foundations G Protein-Coupled Receptor Signaling G protein coupled receptor kinase G-Protein-Coupled Receptors GTP-Binding Protein alpha Subunits, Gs GTP-Binding Proteins Generations Goals Health Heterotrimeric GTP-Binding Proteins Individual Investigation Knowledge Mediating Membrane Proteins Modality Modeling Molecular Molecular Conformation Negative Staining Nucleotides Output Patient-Focused Outcomes Pharmacotherapy Physiological Physiological Processes Plasma Preparation Process Proteins Receptor Signaling Regulation Reporting Resolution Role Sampling Second Messenger Systems Signal Transduction Signaling Protein Structure Tail Testing Therapeutic Therapeutic Uses Time Transducers Translating United States Vasopressins Woman Work base beta-adrenergic receptor desensitization design drug discovery improved inhibitor insight interest men novel preservation protein complex receptor receptor binding receptor internalization receptor-mediated signaling response scaffold screening spatiotemporal

项目摘要

ABSTRACT: Seven transmembrane receptors (7TMRs), alternatively known as G protein-coupled receptors (GPCRs), are involved in the signaling and regulation of many physiological processes. Currently, over one third of all clinically approved drugs target GPCRs, including the β-adrenoceptors (β-ARs) and the arginine vasopressin type 2 receptor (V2R), which are GPCRs of great cardiovascular significance. Thus, a better understanding of different modes of GPCR signaling stands to greatly inform the drug discovery process for this important class of receptor targets. Classically, agonist-bound active GPCRs binds to and activate the heterotrimeric G protein (Gαβγ), leading to dissociation between the Gα subunit and the remaining Gβγ subunits. This dissociation initiates the generation of second messenger molecules such as cyclic AMP (cAMP), which propagate a wave of signaling that eventually leads to a physiological response. To attenuate G protein signaling, GPCR kinases (GRKs) phosphorylate the C-terminal tail of GPCRs, allowing for the binding and activation of β- arrestins (βarrs), which mediates receptor desensitization and internalization. However, recent works have shown that some GPCRs engage in sustained G protein signaling from within internalized cellular compartments (e.g. endosomes) after receptor internalization rather than staying desensitized. With data directly implicating βarrs in mediating this phenomenon, we and others further show that the aforementioned mode of sustained G protein signaling is potentially mediated by two distinct novel GPCR-transducer complexes: a GPCR–G protein– βarr mega-complex (‘megaplex’), whereby a single GPCR directly engages and activates both G protein and βarr, and a GPCR–Gβγ–βarr complex that subsequently forms after G protein dissociation. This observation is inconsistent with the classical model of GPCR signaling described above, which states that βarr and G protein binding to a GPCR is mutually exclusive. While the discovery of these complexes is well established, and provides a potential explanation for sustained G protein signaling, the structural basis governing the interactions of each complex component, and by extension how sustained signaling is mediated, remains to be elucidated. Accordingly, this proposal aims to determine the structure of both the megaplex and the GPCR–Gβγ–βarr complex, with the following aims: (1) To optimize megaplex purification and complex formation for cryo-EM structural studies, (2) to investigate the effects of the GPCR–Gβγ–βarr complex on sustained G protein signaling, and to obtain its structure by cryo-EM. Structural elucidation of these complexes translates directly into a better understanding of this newly appreciated mode of sustained signaling, and will serve as the foundation for the design of therapeutics that confers spatiotemporal control of signaling by GPCRs, such as those of cardiovascular importance. In particular, our structural approach will generate atomic models of the complexes of interest, which will serve as starting points for structure-based drug design.

摘要：七个跨膜受体（7TMR），或者称为G蛋白偶联受体（GPCR）参与了许多物理过程的信号传导和调节。目前，超过三分之一在所有临床批准的药物中，包括β-肾上腺素受体（β-ARS）和精氨酸加压素2型受体（V2R），它是心血管意义极高的GPCR。那，更好理解不同模式的GPCR信号传导可以很好地告知药物发现过程重要的受体靶标。从经典上讲，激动剂结合的活性GPCR结合并激活异三聚体G蛋白（Gαβγ），导致Gα亚基与剩余的Gβγ亚基之间的解离。这种解离启动了第二信使分子的产生，例如环状AMP（CAMP），传播一波信号传导，有时会导致物理反应。为了减弱G蛋白信号传导， GPCR激酶（GRKS）磷酸化GPCR的C末端尾巴，从而允许β-的结合和激活抑制蛋白（βarrs），介导受体脱敏和内在化。但是，最近的作品有表明某些GPCR从内在的细胞室内参与持续的G蛋白信号传导（例如内体）受体内在化而不是保持脱敏。数据直接隐含在介导这种现象的βarrs中，我们和其他人进一步表明了持续G的近似模式蛋白质信号传导可能是由两个不同新型的GPCR转换剂复合物介导的：GPCR – G蛋白 - βarrmega-complex（'Megaplex'），单个GPCR直接参与并激活G蛋白和 βARR和GPCR -Gβγ -βarr复合物随后在G蛋白解离后形成。这个观察是与上述GPCR信号传导的经典模型不一致，该模型指出βARR和G蛋白与GPCR的结合是互斥的。虽然发现这些综合体的发现已经很好，并且为持续G蛋白信号传导提供了潜在的解释，该结构基础管理相互作用每个复杂的成分以及延伸的持续信号传导的介导，仍有待阐明。根据该提议，旨在确定Megaplex和GPCR – GPCR -Gβγ -βarr的结构复杂的，以下目的：（1）优化Cero-EM的巨型纯化和复杂形成结构研究，（2）研究GPCR -Gβγ -βarr复合物对持续G蛋白信号传导的影响，并通过冷冻EM获得其结构。这些复合物的结构阐明直接转化为更好的了解这种新欣赏的持续信号模式，并将作为供认GPCR对信号传导的时空控制的治疗设计，例如心血管重要性。特别是，我们的结构方法将产生复合物的原子模型有趣的是，它将用作基于结构的药物设计的起点。