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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要目前的提案重点是确定蛋白磷酸酶 2A (PP2A) 对 β-肾上腺素能的调节 βAR 受体（βAR）功能障碍是激素与 βAR 结合导致心力衰竭的标志。 βAR 激酶 (GRK) 磷酸化，促进脱敏和内吞作用。内体中 PP2A 介导的去磷酸化再敏化 βAR 功能调节的研究。由于相信 PP2A 调节本质上是稳态的，所以主要关注激酶。我们发现 PI3Kγ 通过磷酸化 PP2A 的内源性抑制剂（I2PP2A）来敏锐地调节 PP2A。磷酸化 I2PP2A 与 PP2A 结合，抑制其活性，从而损害 βAR 重新敏化作用。心力衰竭显示 β1 和 β2AR 在内体中积累，同时 βAR 相关的减少磷酸酶活性反映了对心肌细胞特异性抑制的小鼠。野生型 I2PP2A (WT I2PP2A) 的表达导致横主动脉缩窄 (TAC) 导致扩张，而持续抑制 βAR 再敏化的磷酸化 I2PP2A (pI2PP2A) 模拟物的表达未能存活相比之下，过去 4 周的 TAC 中，去磷酸-I2PP2A (de-pI2PP2A) 模拟物的表达保留了 βAR。再敏化显示 TAC 后心功能障碍的显着改善，反映了典型的作用由于 pI2PP2A 与 PP2A 相互作用的机制基础是心脏重塑的再敏化。未知，我们结合使用计算和实验方法来证明异丙肾上腺素 (ISO) 刺激 βAR 导致 PI3Kγ 介导的 I2PPA 磷酸化，引发其同源二聚化对接研究进一步表明 I2PP2A 与 C 末端区域结合。 PP2A (PP2A-CT) 的表达作为保存的 βAR 细胞中的显性失活策略。再敏化，而小鼠心肌细胞特异性表达 PP2A-CT 导致心脏保存 ISO 管理 2 周后的功能支持目标重新敏化可能是的前提基于这些发现，我们勇敢地解除 pI2PP2A 的 PP2A 抑制可以保留。 βAR再敏化和有益心脏重塑的功能，与当前的直觉相反在心力衰竭中用β受体阻滞剂阻断G蛋白偶联的选择在这方面，我们令人惊讶地观察到。 de-pI2PP2A 小鼠中的 βAR 将其 G 蛋白偶联从刺激性 Gα（产生 cAMP）转换为 TAC 上的抑制性 Gαi 这表明持续的再敏化允许 βAR 灵活地切换。尽管心脏压力和交感神经过度兴奋，G 蛋白仍能维持心脏功能，这表明， βAR调节的基本机制尚待了解，因此，提出以下目标- 1) 确定靶向 I2PP2A 二聚化是否保留 PP2A 活性并恢复 βAR 功能，2) 测试解除 PP2A 抑制是否可以保留 β1 和 β2AR 再敏化和应激后心脏功能，3) 研究心脏应激条件下βAR功能的保存如何介导有益作用。