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: 10019579
• 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/10019579
• 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; 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.

项目总结/摘要 G蛋白偶联受体（GPCR）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。在结合到细胞外表面后， GPCR，配体触发构象变化，导致结合和激活信号蛋白， 受体的细胞质表面。许多GPCR信号通过几种途径，包括不同的 异三聚体G蛋白亚型以及抑制蛋白和“偏向”配体差异调节受体 通过这些不同的途径传递信号。GPCR依赖于高度的构象灵活性来实现 基于结构的药物设计领域的主要目标之一是确定 产生特定信号传导谱的构象。在实现这一目标方面已经取得了重大进展， 然而，对于内源性调节剂的作用，包括蛋白质-脂质相互作用， 翻译修饰（PTM）和辅助蛋白对GPCR结构和动力学的影响。的影响 内源性调节剂对构象景观的影响以及内源性调节剂与 潜在的药物需要进行表征，以实现合理药物设计的目标，这是一个主要的 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- 棕榈酰化和糖基化的目标是确定其结构和动力学基础， β2AR信号的调节。连续波和脉冲EPR技术的互补组合， 质谱和功能测定将用于这些研究。重要的是，尖端变量- 压力EPR技术将提供独特的机制的见解，通过映射人口稀少的地区， 构象景观首先，我们将绘制在细胞质表面的构象景观， 受体的参考状态，在本文中定义为天然糖基化和棕榈酰化的受体嵌入 在脂质双层中。然后，我们将进行实验与PTM删除，一次一个，以确定 棕榈酰化和糖基化对细胞质表面的变构作用。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作用和棕榈酰化对 螺旋8和C末端尾部。这些结果将为深入了解这些PTM的未充分研究但关键作用提供帮助 作为β2AR信号的调节因子，并且因为β2AR是GPCR超家族的典型成员，我们 预期结果适用于具有类似PTM的其他GPCR。在详细说明内源性 调节剂的构象景观，我们的研究结果将提高研究人员的能力，合理设计 通过允许内源性调节剂与药物的体内相互作用， 更准确地预测候选药物。