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

• 批准号: 10014630
• 负责人: Anthony Nguyen
• 金额: $ 3.76万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10014630
• 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/antagonist; insight; interest; men; novel; preservation; protein complex; receptor; receptor binding; receptor internalization; receptor-mediated signaling; response; scaffold; screening; spatiotemporal

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：七种跨膜受体（7 TMR），也称为G蛋白偶联受体 （GPCR）参与许多生理过程的信号传导和调节。目前，超过三分之一 所有临床批准的药物靶向GPCR，包括β-肾上腺素受体（β-AR）和精氨酸 加压素2型受体（V2 R），其是具有重要心血管意义的GPCR。一个更好的 对GPCR信号传导的不同模式的理解将极大地为药物发现过程提供信息。 一类重要的受体靶点。经典地，激动剂结合的活性GPCR结合并激活GPCR。 异源三聚体G蛋白（Gαβγ），导致Gα亚基和其余Gβγ亚基之间的解离。 这种解离引发第二信使分子如环AMP（cAMP）的产生， 传播信号波最终导致生理反应。为了减弱G蛋白信号， GPCR激酶（GRKs）磷酸化GPCR的C末端尾，允许β-GPCR的结合和激活。 抑制蛋白（βarrs）介导受体脱敏和内化。然而，最近的作品 表明一些GPCR参与来自内在化细胞区室的持续G蛋白信号传导， (e.g.内体），而不是保持脱敏。数据直接表明 βarrs在介导这一现象，我们和其他人进一步表明，上述模式的持续G 蛋白质信号传导可能由两种不同的新型GPCR-转导复合物介导：一种GPCR-G蛋白- β-GPCR大复合物（“megaplex”），其中单个GPCR直接接合并激活G蛋白和 GPCR-Gβγ-β γ复合物，随后在G蛋白解离后形成。该观察结果 这与上述GPCR信号传导的经典模型不一致，该模型指出β 1和G蛋白 与GPCR的结合是互斥的。虽然这些复合物的发现是公认的， 为持续的G蛋白信号传导提供了一个潜在的解释，即控制相互作用的结构基础 每一个复杂的组成部分，并通过扩展如何持续的信号是介导的，仍然有待阐明。 因此，该建议旨在确定megaplex和GPCR-Gβγ-β β的结构， （1）优化冷冻电镜的megaplex纯化和复合物形成 结构研究，（2）研究GPCR-Gβγ-β-D复合物对G蛋白信号传导的影响， 用冷冻电镜观察其结构。这些复合物的结构阐明直接转化为更好的 理解这种新的持续信号模式，并将作为基础， 设计通过GPCR赋予信号传导的时空控制的治疗剂，例如 心血管的重要性特别是，我们的结构方法将生成复合物的原子模型 这将作为基于结构的药物设计的起点。