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: 10242822
• 负责人: Anthony Nguyen
• 金额: $ 5.1万
• 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/10242822
• 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：7种跨膜受体，又称G蛋白偶联受体 (GPCRs)，参与许多生理过程的信号和调节。目前，超过三分之一的人 在所有临床批准的药物中，靶向GPCRs的药物包括β肾上腺素受体(β-AR)和精氨酸 加压素2型受体(V2R)是具有重要心血管意义的GPCRs。因此，一个更好的 对不同gpr信号模式的理解将极大地促进这方面的药物发现过程 重要的一类受体靶标。经典地说，激动剂结合的活性GPCRs结合并激活 异三聚体G蛋白(Gαβγ)，导致Gα亚基和其余Gβγ亚基解离。 这种解离启动了第二信使分子的产生，如环磷酸腺苷(CAMP)，它 传播一波信号，最终导致生理反应。为了减弱G蛋白信号， GPCRK(GRKs)使GPCRs的C端尾部磷酸化，允许β-1结合和激活。 阻滞素(βARRs)，介导受体脱敏和内化。然而，最近的作品已经 研究表明，一些GPCRs在内化的细胞间隔内参与持续的G蛋白信号传递 在受体内化之后(例如内小体)，而不是保持脱敏。数据直接牵涉到 β在调节这一现象的过程中，我们和其他人进一步表明，上述持续G 蛋白质信号转导可能由两个不同的新的GPCR-转导复合体介导：一个GPCR-G蛋白- β是一个巨型复合体(‘megaplex’)，其中单个gpr直接结合并激活G蛋白和 βARR和G蛋白解离后形成的GPCRGβγ-βARR复合体。这一观察结果是 与上述经典的gpr信号模型不同，该模型认为βARR和G蛋白 与gpr的结合是相互排斥的。虽然这些复合体的发现已经很久了， 为持续的G蛋白信号提供了一个潜在的解释，G蛋白信号是控制相互作用的结构基础 每个复杂成分的相互作用，以及持续的信号是如何被调节的，仍有待阐明。 因此，本提案旨在确定巨型复合体和GPCRGβγ-βARR的结构 复合体，目标如下：(1)优化冷冻-EM的巨型复合体纯化和复合体形成 结构研究，(2)研究GPCRGβγ-βARR复合体对持续G蛋白信号的影响， 并用冷冻-EM法获得其结构。对这些络合物的结构解释直接转化为更好的 对这种新的持续信令模式的理解，并将作为 通过GPCR对信号进行时空控制的疗法的设计，例如那些 对心血管的重要性。特别是，我们的结构方法将生成络合物的原子模型 这将成为基于结构的药物设计的起点。