Structural basis for regulation of beta2 adrenergic receptor signaling by the dynamic post-translational modification S-palmitoylation

动态翻译后修饰S-棕榈酰化调节β2肾上腺素受体信号传导的结构基础

• 批准号: 10603466
• 负责人: Patrick C Brennan
• 金额: $ 4.77万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10603466
• 关键词: Acylation; Address; Adrenergic Agents; Adrenergic Receptor; Affinity; Agonist; Biological Assay; C-terminal; Cardiac; Cardiac Output; Cardiovascular Diseases; Cell Surface Receptors; Cell membrane; Cells; Clinical; Country; Coupling; Cysteine; Cytoplasm; Cytoplasmic Receptors; Dimensions; Drug Design; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Environment; Equilibrium; Event; Family; Fellowship; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Goals; Heart failure; Heterogeneity; Human; Human body; Investigation; Kinetics; Ligands; Link; Lipid Bilayers; Lipids; Maps; Measures; Mediating; Membrane Proteins; Modeling; Molecular; Molecular Conformation; Morbidity - disease rate; Outcome; Palmitates; Palmitic Acylation Site; Pathway interactions; Performance; Pharmacology; Phosphorylation; Phosphorylation Site; Physiologic pulse; Physiological; Physiological Processes; Physiology; Post-Translational Protein Processing; Process; Protein Dynamics; Proteins; Receptor Signaling; Regulation; Research Personnel; Role; Side; Signal Pathway; Signal Transduction; Site; Solvents; Spin Labels; Structure; Surface; Tail; Techniques; Transducers; Transferase; beta-2 Adrenergic Receptors; beta-arrestin; design; drug development; effective therapy; experience; experimental study; extracellular; improved; insight; member; mortality; mutant; novel; palmitoylation; pharmacologic; protein activation; protein function; protein structure; receptor; recruit

PROJECT SUMMARY/ABSTRACT Heart failure is a leading cause of morbidity and mortality in western countries and represents the final common clinical event of numerous cardiovascular diseases. Cardiac β2 adrenergic signaling determines cardiac output and has been extensively studied as treatments directed toward β2 adrenergic signaling have proven effective at improving cardiac performance and heart failure outcomes. The β2 adrenergic receptor (β2AR) is a G-protein- coupled receptor (GPCR), a family of membrane proteins representing the largest and most versatile group of cell surface receptors. They are ubiquitous membrane proteins that facilitate the initial role in the transduction of information into the cell via a wide array of extracellular signal-receptor interactions. Although much progress has been made in elucidating conformational insights into ligand-mediated signaling of GPCRs, there has not been commensurate investigation into endogenous modulators of GPCR signaling. Endogenous modulators include but are not limited to protein-lipid interactions, post-translational modifications, and accessory protein interactions. The β2AR has a site for S-palmitoylation, the covalent attachment of palmitate to a cysteine, in the C-terminal helix 8 (H8). Interestingly, H8 of the β2AR is implicated in β-arrestin recruitment, resulting in a non- canonical signaling pathway, yet little is known of the effect that palmitoylation of H8 has on intracellular signaling and structural dynamics of the receptor. I hypothesize that S-palmitoylation of the β2AR has an allosteric effect on the conformational plasticity therein modulating functional transducer coupling in the context of ligand mediated protein activation. The goal of this project is to determine the effect S-palmitoylation has on β2AR signaling in the following two specific aims. Aim 1. To define the impact of s-palmitoylation on the conformational dynamics at the cytoplasmic surface of the β2AR and the effect it has on the interaction with transducers to facilitate intracellular signaling using both electron paramagnetic resonance (EPR) techniques and signaling assays. In preliminary experiments, we performed site-directed spin-labeling to generate a set of nitroxide spin labeled receptor mutants to perform EPR spectroscopy experiments. Aim 2. To determine the impact of S-palmitoylation on H8 and tail dynamics of the β2AR using EPR techniques. These experiments will help us better understand β2AR signaling as a whole and provide new insight into novel ways of GPCR signaling regulation via endogenous modulators. Further, the experiments will provide new insight into the effects of S- palmitoylation on the conformational plasticity and, ultimately, the signaling of the β2AR. These results will provide a greater clarity on β2AR signaling and regulation within the context of a physiologically relevant cellular environment, which will help with the design of effective treatments targeting cardiac β2 adrenergic signaling.

项目概要/摘要 心力衰竭是西方国家发病和死亡的主要原因，也是最常见的疾病 心脏 β2 肾上腺素信号传导决定了心输出量。 并且已被广泛研究，针对 β2 肾上腺素信号传导的治疗已被证明是有效的 β2 肾上腺素受体 (β2AR) 是一种 G 蛋白。 偶联受体（GPCR），一个膜蛋白家族，代表最大和最通用的群体 它们是普遍存在的膜蛋白，有助于在转导中发挥初始作用。 尽管取得了很大进展，但信息通过广泛的细胞外信号-受体相互作用进入细胞。 已在阐明配体介导的 GPCR 信号传导的构象方面取得了进展，但尚未有 对 GPCR 信号传导的内源性调节剂进行了相应的研究。 包括但不限于蛋白质-脂质相互作用、翻译后修饰和辅助蛋白 β2AR 具有 S-棕榈酰化位点，即棕榈酸酯与半胱氨酸的共价连接。 C 末端螺旋 8 (H8) 表明，β2AR 的 H8 参与了 β-arrestin 募集，导致非-抑制蛋白。 经典信号传导途径，但 H8 棕榈酰化对细胞内信号传导的影响知之甚少 和受体的结构动力学，我发现 β2AR 的 S-棕榈酰化具有变构。 对构象可塑性的影响，其中调节功能换能器耦合 该项目的目标是确定 S-棕榈酰化的作用。 β2AR 信号传导有以下两个具体目标 1. 定义 s-棕榈酰化对 β2AR 信号传导的影响。 β2AR细胞质表面的构象动力学及其对与相互作用的影响 使用电子顺磁共振 (EPR) 技术促进细胞内信号传导的传感器 在初步实验中，我们进行了定点自旋标记以生成一组 硝基氧自旋标记受体突变体进行 EPR 光谱实验 目的 2. 确定 这些实验将使用 EPR 技术研究 S-棕榈酰化对 H8 和 β2AR 尾部动力学的影响。 帮助我们更好地理解整个 β2AR 信号传导，并为 GPCR 信号传导的新方法提供新的见解 此外，这些实验将为 S- 的作用提供新的见解。 棕榈酰化对构象可塑性的影响以及最终对 β2AR 信号传导的影响。 在生理相关细胞的背景下提供更清晰的 β2AR 信号传导和调节 环境，这将有助于设计针对心脏 β2 肾上腺素能信号传导的有效治疗方法。