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.

项目摘要/摘要 当前的建议集中于确定β-肾上腺素的蛋白质磷酸酶2a（PP2A）调节 接收器（βAR）充当βAR功能障碍是心力衰竭的标志。激素与βAR结果结合 在βAR激酶（GRK）磷酸化中，促进脱敏和内吞作用。 βAR经历 pP2A介导的内体脱磷酸化的脱发。 βAR功能调节的研究 由于认为PP2A调节本质上是稳态的，因此主要关注激酶。不料， 我们确定PI3Kγ通过磷酸化PP2A（I2PP2A）的内源性抑制剂来急性调节PP2A。 磷酸化的I2PP2A与PP2A结合，抑制其活性损害βAR脱激的化。人类研究 心力衰竭显示β1和β2AR在内体中的积累，βAR相关的降低 磷酸酶的活性反映了抑制汇总化。用心肌细胞特异性对小鼠进行小鼠 野生型I2pp2a（WT I2PP2A）表达横向主动脉收缩（TAC）的表达导致扩张，而 持续抑制βAR脱敏的磷酸化磷酸化 - i2pp2a（pi2pp2a）模拟物的表达无法生存 过去四个星期的TAC。相反，dephospho-i2pp2a（de-pi2pp2a）的表达模拟于βAR 发射敏化显示了反映典型作用的TAC后心功能障碍的明显改善 用于心脏重塑中的敏化。由于PI2PP2A与PP2A相互作用的机械基础是 尚不清楚，我们使用了计算和实验方法的组合来表明异丙肾上腺素 （ISO）刺激βAR会导致I2PPA的PI3Kγ介导的磷酸化，从而启动其同二剂量 导致与pp2a的牢固结合。对接研究进一步表明，I2PP2A与C末端区域结合 pp2a（pp2a-ct）。 pp2a-ct的表达是保留βAR的细胞中的主要负策略 敏化，而PP2A-CT在小鼠中的心肌细胞特异性表达导致心脏保存 在ISO给药2周后，功能支持靶向敏感的前提可能是 有利。基于这些发现，我们假设从PI2PP2A保留中缓解PP2A抑制作用 βAR的敏化和功能是有益心脏重塑的基础，与电流相反 在心力衰竭中阻断与β受体阻滞剂的G蛋白耦合的选择。在这方面，我们出奇地观察到 DE-PI2PP2A小鼠中的β会从刺激Gα（CAMP生成）转换为G蛋白耦合到 TAC上的抑制性GαI。这表明持续性敏化允许βAR灵活切换 在维持心脏功能目的地心脏应力的G蛋白和交感神经的超速驾驶之间，表明a 尚未理解βAR调节的基本机制，因此，提议以下目的-1） 确定靶向I2PP2A二聚化是否保留PP2A活性并恢复βAR功能，2）测试 缓解PP2A抑制是否可以保留β1和β2AR脱敏和心脏功能后压力，3） 为了研究βAR在心脏应激条件下的保存如何介导有益作用。