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.

项目摘要/摘要 心力衰竭是西方国家发病和死亡率的主要原因，代表了最终的共同点 许多心血管疾病的临床事件。心脏β2肾上腺素信号决定心脏输出 并且由于针对β2肾上腺肾上腺素信号的处理已被证明有效，并且已被广泛研究 改善心脏表现和心力衰竭结果。 β2肾上腺素受体（β2AR）是G蛋白 耦合受体（GPCR），这是一个代表最大，最通用群的膜蛋白家族 细胞表面受体。它们是无处不在的膜蛋白，可促进在转移中的初始作用 通过广泛的细胞外信号受体相互作用向细胞中的信息。虽然取得了很多进展 在阐明了对GPCR配体介导的信号的会议见解中，没有 对GPCR信号的内源调节剂进行了相称的研究。内源调节剂 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 互动。 β2AR具有用于S-顶酰化的位点 C末端螺旋8（H8）。有趣的是，β2AR的H8在β-arrestin募集中暗示，导致非 - 规范信号通路，但对H8的棕榈酰化对细胞内信号的影响鲜为人知 和接收器的结构动力学。我假设β2AR的s-膜酰化具有变构 在上下文中调节功能传感器耦合的构象可塑性的影响 配体介导的蛋白质激活。该项目的目的是确定s-膜烯二酰化的影响 在以下两个特定目标中，在β2AR信号传导上。目的1。定义s-甲米二酰化的影响 β2AR的细胞质表面的构象动力学及其对与与 使用两种电子顺磁共振（EPR）技术促进细胞内信号传导的传感器 和信号测定。在初步实验中，我们进行了定向的自旋标记，以生成一组 硝基自旋标记的受体突变体进行EPR光谱实验。目标2。确定 使用EPR技术的S-膜酰化对β2AR的H8和尾部动力学的影响。这些实验会 帮助我们更好地理解整个β2AR信号传导，并为GPCR信号的新型方式提供新的见解 通过内源调节剂进行调节。此外，实验将为S-的影响提供新的见解 对构象可塑性的棕榈酰化，最终是β2AR的信号传导。这些结果将会 在物理相关的细胞背景下，对β2AR信号传导和调节提供了更大的清晰度 环境将有助于设计针对心脏β2肾上腺素信号传导的有效治疗方法。