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.

项目总结/摘要 心力衰竭是西方国家发病率和死亡率的主要原因，并且代表了最后的常见疾病。 许多心血管疾病的临床事件。心脏β2肾上腺素能信号传导决定心输出量 并已被广泛研究，因为针对β2肾上腺素能信号传导的治疗已被证明有效 改善心脏功能和心力衰竭的结果。β2肾上腺素能受体（β2AR）是一种G蛋白， 偶联受体（GPCR），代表最大和最通用的一组膜蛋白家族， 细胞表面受体它们是普遍存在的膜蛋白，促进在细胞膜的转导中的初始作用。 通过广泛的细胞外信号-受体相互作用将信息输入细胞。虽然取得了很大进展， 已经在阐明配体介导的GPCR信号传导的构象见解方面取得了进展，但还没有 已经对GPCR信号传导的内源性调节剂进行了相应的研究。内源性调节剂 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 交互.β 2 AR有一个S-棕榈酰化位点，棕榈酸酯共价连接到半胱氨酸上， C-末端螺旋8（H8）。有趣的是，β2AR的H8参与β-抑制蛋白的募集，导致非- 经典的信号通路，但很少有人知道的影响，棕榈酰化的H8细胞内信号 和受体的结构动力学。我推测β2AR的S-棕榈酰化具有变构作用， 对其中构象可塑性的影响在上下文中调制功能性换能器耦合 配体介导的蛋白质激活。该项目的目标是确定S-棕榈酰化的效果 在以下两个具体目标中对β2AR信号转导进行研究。目标1。为了确定s-棕榈酰化对 在β 2 AR的胞质表面的构象动力学及其对与 使用电子顺磁共振（EPR）技术促进细胞内信号传导的传感器 和信号传导测定。在初步实验中，我们进行了定点自旋标记，以生成一组 氮氧自由基自旋标记的受体突变体进行EPR光谱实验。目标二。确定 使用EPR技术研究S-棕榈酰化对H8和β2AR尾部动力学的影响。这些实验将 帮助我们更好地理解β2AR信号传导，并为GPCR信号传导的新方式提供新的见解 通过内源性调节剂调节。此外，这些实验将为S- 棕榈酰化对构象可塑性的影响，并最终影响β2AR的信号传导。这些结果将 在生理学相关的细胞环境中， 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。