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: 10468126
• 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/10468126
• 关键词: Acylation; Adrenergic Receptor; Affinity; Agonist; Arrestins; Asthma; Attenuated; Binding; Biological Assay; C-terminal; Cardiovascular Physiology; Cell Surface Receptors; Cell membrane; Disease; Drug Design; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Excision; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Goals; Heart; Heterogeneity; Heterotrimeric GTP-Binding Proteins; Human body; Kinetics; Knowledge; Laboratories; Lead; Ligand Binding; Ligands; Lipid Bilayers; Lipids; Maps; Mass Spectrum Analysis; Measures; Mediating; 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; base; 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.

项目概要/摘要 G 蛋白偶联受体 (GPCR) 是一大类多样化的细胞表面受体，负责 调节人体几乎所有的生理过程。与细胞外表面结合后 GPCR、配体触发构象变化，导致结合并激活信号蛋白 受体的细胞质表面。许多 GPCR 通过多种途径发出信号，其中包括不同的途径 异三聚体 G 蛋白亚型以及视紫红质抑制蛋白和“偏向”配体差异调节受体 通过这些不同的途径发出信号。 GPCR 依靠高度的构象灵活性来实现 这种信号的复杂性，基于结构的药物设计领域的主要目标之一是确定 生成特定信号传导谱的构象。朝着这一目标已经取得了重大进展， 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 GPCR 结构和动力学的翻译修饰 (PTM) 和辅助蛋白。的效果 内源性调节剂对构象景观的影响以及内源性调节剂与 需要对潜在药物进行表征以实现合理药物设计的目标，这是一个重要的 我实验室的长期目标。在这个项目中，我们将重点关注 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 β2AR 信号传导的调节。连续波和脉冲 EPR 技术的互补组合， 这些研究将使用质谱分析和功能分析。重要的是，尖端变量- 压力 EPR 技术将通过绘制人口稀少的区域来提供独特的机制见解 构象景观。首先，我们将绘制细胞质表面的构象景观 受体的参考状态，本文定义为嵌入的天然糖基化和棕榈酰化受体 在脂质双层中。然后，我们将在移除 PTM 的情况下进行实验，一次一个，以识别 细胞质表面棕榈酰化和糖基化的变构效应。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局部影响 螺旋 8 和 C 末端尾部。结果将深入了解这些 PTM 的尚未充分研究但至关重要的作用 作为 β2AR 信号传导的调节因子，并且由于 β2AR 是 GPCR 超家族的典型成员，我们 预计结果适用于具有类似 PTM 的其他 GPCR。详细介绍内源性的影响 构象景观调节剂，我们的结果将提高研究人员合理设计的能力 药物通过允许内源性调节剂与体内相互作用来达到预期的治疗效果 更准确地预测候选药物。