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.

项目摘要/摘要 G蛋白偶联受体（GPCR）是一类大的细胞表面受体，负责 调节人体中几乎每个身体过程。结合与细胞外表面后 GPCR，配体触发会议变化，导致结合和激活信号蛋白在 受体的胞质表面。许多GPCR通过包括不同的途径发出的信号 异三聚体G蛋白亚型以及逮捕蛋白，“偏见”配体对接收器的调节不同 通过这些各种途径发出信号。 GPCR依靠高度的构象灵活性来实现 这种信号复杂性以及基于结构的药物设计领域的主要目标之一是确定 生成特定信号剖面的构象。已经取得了这一目标的重大进展， 然而，关于内源调节剂的影响，包括蛋白质 - 脂质相互作用，后 GPCR结构和动力学上的转化修饰（PTM）和附件蛋白。效果 构象景观上的内源调节剂以及内源调节剂与 需要对潜在药物进行表征以实现理性药物设计的目标，这是一个主要的 我的实验室的长期目标。在这个项目中，我们将重点关注β2-肾上腺素受体（β2AR）的两个PTM- 棕榈酰化和糖基化 - 以确定其结构和动态基础的目的 调节β2AR信号传导。连续波和脉冲EPR技术的完整组合， 这些研究将使用质谱和功能测定法。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械见解 构象景观。首先，我们将在细胞质表面绘制构象景观 受体的参考状态，本文定义为嵌入植物糖基化和棕榈酰化受体 在脂质双层中。然后，我们将对一次删除PTM进行实验，以识别 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和棕榈酰化的配体结合口袋的局部影响 螺旋8和C末端尾巴。结果将为这些PTM的理解但至关重要的作用提供洞察力 作为β2AR信号的调节剂，并且由于β2AR是GPCR超家族的典型成员，所以我们 预计结果适用于其他具有相似PTM的GPCR。详细说明内源性的影响 在构象环境中调节器，我们的结果将提高研究人员合理设计的能力 药物通过允许内源性调节剂与体内相互作用与 候选药物将被更准确地预测。