Novel regulation of beta-adrenergic receptor function by phosphoinositide 3-kinase

磷酸肌醇 3-激酶对 β-肾上腺素能受体功能的新调节

• 批准号: 10591688
• 负责人: Sathyamangla V Prasad
• 金额: $ 63.77万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591688
• 关键词: Acceleration; Acute; Address; Adrenergic beta-Antagonists; Adult; Amino Acids; Belief; Binding; Biological Assay; Breeding; C-terminal; Cardiac; Cardiac Myocytes; Cell surface; Cells; Coupling; Cyclic AMP; Data; Diagnosis; Dimerization; Docking; Dominant-Negative Mutation; Down-Regulation; Endocytosis; Endosomes; Epinephrine; Functional disorder; G protein coupled receptor kinase; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Heart failure; Homodimerization; Hormones; Human; Impairment; In Vitro; Isoproterenol; Knockout Mice; Knowledge; Measures; Mediating; Mus; Myocardial dysfunction; Nature; Norepinephrine; Operative Surgical Procedures; Outcome; Pathway interactions; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiology; Process; Protein Dephosphorylation; Protein Phosphatase Inhibitor; Protein Region; Protein phosphatase; Proteomics; Regulation; Role; SET gene; Stress; Testing; Therapeutic; Transgenic Mice; Transgenic Organisms; aorta constriction; beta-adrenergic receptor; desensitization; dimer; flexibility; heart function; heart preservation; in vivo; insight; mimetics; mouse model; novel; phosphoproteomics; preservation; promoter; protein purification; receptor function; tool

Project Summary/Abstract The current proposal is focused on determining protein phosphatase 2A (PP2A) regulation of β-adrenergic receptors (βARs) function as βAR dysfunction is a hallmark of heart failure. Hormones binding to βAR results in phosphorylation by βAR kinases (GRKs) promoting desensitization and endocytosis. βAR undergoes resensitization by PP2A-mediated dephosphorylation in the endosomes. Studies on regulation of βAR function have majorly focused on kinases due to the belief that PP2A regulation is homeostatic in nature. Unexpectedly, we identified that PI3Kγ acutely regulates PP2A by phosphorylating an endogenous inhibitor of PP2A(I2PP2A). Phosphorylated I2PP2A binds to PP2A inhibiting its activity that impairs βAR resensitization. Studies in human heart failure showed accumulation of β1 and β2ARs in the endosomes with reduction in βAR-associated phosphatase activity reflecting inhibition of resensitization. Subjecting mice with cardiomyocyte-specific expression of wild type I2PP2A (WT I2PP2A) to transverse aortic constriction (TAC) resulted in dilation, while expression of phospho-I2PP2A (pI2PP2A) mimetic that persistently inhibits βAR resensitization did not survive past four weeks TAC. In contrast, expression of dephospho-I2PP2A (de-pI2PP2A) mimetic that preserves βAR resensitization showed significant amelioration of cardiac dysfunction post-TAC reflecting a quintessential role for resensitization in cardiac remodeling. Since mechanistic underpinnings of pI2PP2A interaction with PP2A is not known, we used a combination of computational and experimental approaches to show that isoproterenol (ISO) stimulation of βARs leads to PI3Kγ-mediated phosphorylation of I2PPA, priming its homo-dimerization resulting in robust binding to PP2A. Docking studies further showed that I2PP2A binds to the C-terminal region of PP2A (PP2A-CT). Expression of PP2A-CT as a dominant negative strategy in cells preserved βAR resensitization, while cardiomyocyte-specific expression of PP2A-CT in mice resulted in preservation of cardiac function following 2 weeks of ISO administration supporting the premise that targeting resensitization may be beneficial. Based on these findings, we hypothesize that relieving PP2A inhibition from pI2PP2A preserves βAR resensitization and function underlying beneficial cardiac remodeling, counter-intuitive to the current option of blocking G-protein coupling with β-blockers in heart failure. In this regard, we surprisingly observed that βARs in the de-pI2PP2A mice switch their G-protein coupling from stimulatory Gαs (cAMP generating) to the inhibitory Gαi upon TAC. This suggests that sustaining resensitization allows the βARs to flexibly switch between G-proteins maintaining cardiac function despite cardiac stress and sympathetic overdrive indicating a, yet to be understood fundamental mechanism of βAR regulation and therefore, propose the following aims- 1) determine whether targeting I2PP2A dimerization preserves PP2A activity and restores βAR function, 2) test whether relieving of PP2A inhibition preserves β1 and β2AR resensitization and cardiac function post-stress, 3) to investigate how preservation of βAR function in conditions of cardiac stress mediates beneficial effects.

项目总结/摘要 目前的建议集中在确定蛋白磷酸酶2A（PP 2A）对β-肾上腺素能受体的调节。 β受体（β AR）功能障碍是心力衰竭的标志。激素与βAR的结合结果 在βAR激酶（GRKs）促进脱敏和内吞作用的磷酸化中。βAR经历 通过内体中PP 2A介导的去磷酸化再敏化。β-AR功能调节的研究 由于相信PP 2A调节在本质上是稳态的，因此主要集中在激酶上。没想到， 我们发现，PI 3 K γ通过磷酸化PP 2A的内源性抑制剂（I2 PP 2A）来急性调节PP 2A。 磷酸化I2 PP 2A与PP 2A结合，抑制其损害βAR再敏感的活性。人体研究 心力衰竭显示β1和β 2AR在内体中积聚，β AR相关的 磷酸酶活性反映了再敏化的抑制。对小鼠进行心肌细胞特异性 表达野生型I2 PP 2A（WT I2 PP 2A）导致主动脉横缩窄（TAC）扩张，而 持续抑制βAR再敏感的磷酸化I2 PP 2A（pI 2 PP 2A）模拟物的表达没有存活 已经过去四周了与此相反，表达脱磷酸-I2 PP 2A（de-pI 2 PP 2A）模拟物，保留βAR， 再致敏显示TAC后心功能不全的显著改善，反映了其典型作用 用于心脏重塑的再敏感化。由于pI 2 PP 2A与PP 2A相互作用的机制基础是 未知，我们使用计算和实验方法相结合，以表明异丙肾上腺素， (ISO)刺激β AR导致PI 3 K γ介导的I2 PPA磷酸化，引发其同源二聚化 导致与PP 2A的强结合。对接研究进一步表明，I2 PP 2A结合到C-末端区域 PP2A（PP2A-CT）。PP 2A-CT作为显性负性策略在保留βAR的细胞中的表达 而小鼠心肌细胞特异性表达PP 2A-CT导致心肌细胞的保护， ISO给药2周后的功能，支持靶向脱敏可能 有利于基于这些发现，我们假设从pI 2 PP 2A中解除PP 2A抑制作用， βAR再敏化和功能是有益的心脏重塑的基础，与目前的研究相反 β受体阻滞剂阻断G蛋白偶联治疗心力衰竭的一种选择。在这方面，我们惊讶地观察到， 在de-pI 2 PP 2A小鼠中，β AR将其G蛋白偶联从刺激性Gα（产生cAMP）转换为 TAC对Gαi的抑制作用。这表明持续的再敏感化允许β AR灵活地转换 在尽管心脏应激但仍维持心脏功能的G蛋白和交感神经兴奋之间， βAR调节的基本机制尚不清楚，因此，提出以下目标：1） 确定靶向I2 PP 2A二聚化是否保留PP 2A活性并恢复βAR功能，2）测试 解除PP 2A抑制是否能保护应激后β1和β2AR再敏感和心功能，3） 研究在心脏应激条件下βAR功能的保留如何介导有益作用。