Regulation of beta2-adrenergic receptor signaling by post-translational modifications

通过翻译后修饰调节β2-肾上腺素受体信号传导

• 批准号: 10678654
• 负责人: MICHAEL TAYLOR LERCH
• 金额: $ 33.88万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10678654
• 关键词: Acylation; Adrenergic Receptor; Affinity; Agonist; Arrestins; Asthma; Attenuated; Binding; Biological Assay; C-terminal; Cardiovascular Physiology; Cell Surface Receptors; Cell membrane; Cytoplasm; Cytoplasmic Receptors; Disease; Drug Design; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Excision; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Goals; Heart failure; Heterogeneity; Heterotrimeric GTP-Binding Proteins; Human body; Kinetics; Knowledge; Laboratories; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Maps; Mass Spectrum Analysis; Measures; Mediating; Membrane; Modeling; Molecular; Molecular Conformation; N-terminal; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Physiologic pulse; Physiological Processes; Play; Polysaccharides; Population; Post-Translational Protein Processing; Proteins; Receptor Signaling; Regulation; Research Personnel; Role; Signal Transduction; Signaling Protein; Site; Structure; Surface; Tail; Techniques; Technology; Therapeutic Effect; Time; beta-2 Adrenergic Receptors; drug candidate; drug development; drug discovery; experimental study; extracellular; flexibility; glycosylation; in vivo; insight; member; non-Native; palmitoylation; pressure; radioligand; rational design; receptor; receptor internalization; recruit

PROJECT SUMMARY/ABSTRACT G-protein-coupled receptors (GPCRs) are a large and diverse class of cell surface receptors responsible for regulating nearly every physiological process in the human body. Upon binding to the extracellular surface of GPCRs, ligands trigger conformational changes that lead to binding and activating signaling proteins at the cytoplasmic surface of the receptor. Many GPCRs signal through several pathways that include different heterotrimeric G-protein subtypes as well as arrestins, and “biased” ligands differentially modulate receptor signaling through these various pathways. GPCRs rely on a high degree of conformational flexibility to achieve this signaling complexity, and one of the major goals in the field of structure-based drug design is to identify the conformations that generate a particular signaling profile. Significant progress toward this goal has been made, yet relatively little is known about the effects of endogenous modulators, including protein-lipid interactions, post- translational modifications (PTMs), and accessory proteins, on GPCR structure and dynamics. The effect of endogenous modulators on the conformational landscape and the interplay of endogenous modulators with potential drugs need to be characterized to achieve the objectives of rational drug design, and this is a major long-term goal of my laboratory. In this project, we will focus on two PTMs of the β2-adrenergic receptor (β2AR)— palmitoylation and glycosylation—with the goal of identifying the structural and dynamical basis for their regulation of β2AR signaling. A complementary combination of continuous-wave and pulsed EPR techniques, mass spectrometry, and functional assays will be used in these studies. Importantly, cutting-edge variable- pressure EPR technology will provide unique mechanistic insights by mapping sparsely populated regions of the conformational landscape. First, we will map the conformational landscape at the cytoplasmic surface in the reference state of the receptor, defined herein as the natively glycosylated and palmitoylated receptor embedded in a lipid bilayer. Then, we will perform experiments with the PTMs removed, one at a time, to identify the allosteric effects of palmitoylation and glycosylation on the cytoplasmic surface. Finally, we will determine the local effects of glycosylation on the ligand-binding pocket at the extracellular surface and of palmitoylation on helix 8 and the C-terminal tail. The results will provide insight into the understudied yet critical role of these PTMs as regulators of β2AR signaling, and because the β2AR is a prototypical member of the GPCR superfamily, we anticipate the results to be applicable to other GPCRs with similar PTMs. In detailing the effects of endogenous modulators on the conformational landscape, our results will increase researchers' ability to rationally design drugs to achieve the desired therapeutic effect by allowing the in vivo interplay of endogenous modulators with drug candidates to be predicted more accurately.

项目摘要/摘要 G蛋白偶联受体(GPCRs)是一大类不同的细胞表面受体，负责 调节人体内几乎所有的生理过程。在与胞外表面结合时 GPCRs，配体触发构象变化，导致结合和激活信号蛋白在 受体的细胞质表面。许多GPCR通过几条途径发出信号，包括不同的 异源三聚体G蛋白亚型和拦阻蛋白，以及“有偏向”的配体对受体进行差异调节 通过这些不同的途径发出信号。GPCRs依赖于高度的构象灵活性来实现 这种信号的复杂性，以及基于结构的药物设计领域的主要目标之一是识别 生成特定信令配置文件的构象。在实现这一目标方面取得了重大进展， 然而，人们对内源性调节剂的影响知之甚少，包括蛋白质-脂质相互作用，后 翻译修饰(PTM)和辅助蛋白对GPCR结构和动力学的影响。的影响 构象格局上的内源调节剂以及内源调节剂与 为了实现合理的药物设计目标，需要对潜在的药物进行表征，这是一个主要的 我实验室的长期目标。在这个项目中，我们将重点研究β2肾上腺素能受体(β2AR)的两个PTM- 棕榈酰化和糖基化-目的是确定它们的结构和动力学基础 β-2AR信号的调控。连续波和脉冲EPR技术的互补组合， 在这些研究中，将使用质谱仪和功能分析。重要的是，尖端变量- 压力EPR技术将通过绘制人口稀少的地区地图来提供独特的机械洞察 构造地貌。首先，我们将绘制在细胞质表面的构象地貌 受体的参考状态，这里定义为天然糖基化和棕榈酰化嵌入的受体 在脂质双层中。然后，我们将对移除的PTM进行实验，一次一个，以确定 棕榈酰化和糖基化在细胞质表面的变构效应。最后，我们将确定 糖基化对细胞外表面配体结合口袋的局部影响和棕榈酰化对细胞外表面配体结合口袋的影响 螺旋8和C末端的尾巴。这些结果将使人们深入了解这些PTM尚未被研究但却至关重要的作用 作为β2AR信号的调节者，并且由于β2AR是gPCR超家族的典型成员，我们 预期研究结果将适用于其他具有类似技术指标的GPCR。在详细描述内源性的影响 对于构象景观上的调节器，我们的结果将提高研究人员合理设计的能力 药物通过允许体内内源性调节剂与 对候选药物进行更准确的预测。