Novel Regulation of Beta Andregenic Receptor Function

项目摘要 β-肾上腺素能受体（bAR）是心脏功能的强有力调节剂之一。bAR下调 (loss和脱敏（受损的G蛋白偶联）是心力衰竭的标志。 脱敏是由bAR激酶1（bARK 1或GRK 2）对bAR的磷酸化启动的， 导致心脏反应降低的儿茶酚胺。b-抑制蛋白结合磷酸化的bAR，促进 下调（downregulation）。磷酸化的bAR通过去磷酸化介导的 通过内体中的蛋白磷酸酶2A（PP 2A）并再循环回到质膜 作为准备激活的幼稚受体。与重新敏感化是一个维持 在体内平衡方面，我们在当前RO 1中的机制研究已经确定PI 3 Kg抑制PP 2A活性， 从而负调节bAR再敏感化。我们已经证明PI 3 Kg内源性磷酸化 丝氨酸9和93（S9，93）上的PP 2A（I2 PP 2A）抑制剂，其与PP 2A牢固结合，抑制PP 2A活性， 阻断bAR再敏化，有效确定PI 3 Kg-I2 PP 2A-PP 2A通路调节bAR 再敏感化虽然我们已经确定了调节bAR再致敏的途径， 这一途径是否被改变并导致心脏肥大和心力衰竭。因此，目标 更新的目的是确定在心力衰竭的情况下， 测试使用基因改变小鼠的靶向脱敏成分是否可以提供有益的 在压力下重塑。我们已经产生了心脏过表达野生型的转基因小鼠， I2 PP 2A（Wt I2 PP 2A Tg）或I2 PP 2A突变体S9，93 A（I2 PP 2A S9，93 A Tg，模拟脱磷酸状态）或 S9，93 D（I2 PP 2A S9，93 D Tg，磷酸态）。我们的初步研究表明，使Wt I2 PP 2A Tg 小鼠或I2 PP 2A S9，93 D Tg小鼠对心脏应激的反应导致加速的心功能障碍， 在I2 PP 2A S9，93 A Tg小鼠中显著改善，表明bAR再致敏在 有害的重塑此外，我们还观察到非经典的不依赖于Gbg的PI 3 Kg募集， bAR对促炎细胞因子TNF α的反应，TNF α可能抑制PP 2A活性，导致bAR丧失 再次致敏表明TNF α可以通过改变PI 3 Kg-I2 PP 2A-PP 2A轴来调节bAR功能。 基于这些初步研究，我们假设bAR再敏感化被PI 3 Kg抑制。 I2 PP 2A-PP 2A通路对心脏应激的影响及靶向解除对bAR再敏感的抑制作用 I2 PP 2A可保护bAR功能，防止有害的心脏重构。我们提议下列 具体目的是解决假设：具体目的1：确定是否调节心脏bAR 再致敏途径预防心脏应激后的有害心脏重塑，具体目标 2.鉴定PI 3 Kg介导的I2 PP 2A-PP 2A相互作用的分子基础及其在bAR中的作用 特异性目的3：研究介导激动剂非依赖性 PI 3 Kg的非典型募集和响应于TNF α的bAR再敏化的抑制。确定 PI 3 Kg-I2 PP 2A-PP 2A轴在调节bAR对心脏应激再敏感中的作用将 提供对bAR功能障碍的全面机制理解，从而鉴定新的 通过靶向bAR再增敏通路治疗心力衰竭的策略。

PROJECT SUMMARY b-adrenergic receptors (bARs) are one of the powerful regulators of cardiac function. bAR downregulation (loss of surface receptors) and desensitization (impaired G-protein coupling) are hallmarks of a failing heart. Desensitization is initiated by phosphorylation of bARs by bAR kinase 1 (bARK1 or GRK2) in response to catecholamines resulting in reduced cardiac responses. b-arrestin binds phosphorylated bARs facilitating their endocytosis (downregulation). Phosphorylated bARs are resensitized by dephosphorylation mediated through protein phosphatase 2A (PP2A) in the endosomes and are recycled back to the plasma membrane as na¿ve receptor ready for activation. Contrary to the belief that resensitization is a process that maintains homeostasis, our mechanistic studies in the current RO1 have identified that PI3Kg inhibits PP2A activity, thereby negatively regulating bAR resensitization. We have shown that PI3Kg phosphorylates endogenous inhibitor of PP2A (I2PP2A) on serine 9 & 93 (S9, 93) that robustly binds to PP2A, inhibiting PP2A activity and blocking bAR resensitization, effectively determining that the PI3Kg-I2PP2A-PP2A pathway regulates bAR resensitization. Although we have identified the pathway that regulates bAR resensitization, it is not known whether this pathway is altered and contributes to cardiac hypertrophy and heart failure. Therefore, the goal of the renewal is to determine whether the resensitization pathway is altered in conditions of heart failure and test whether targeting components of resensitization using genetically altered mice could provide beneficial remodeling upon stress. We have generated transgenic mice with cardiac overexpression of wild type I2PP2A (Wt I2PP2A Tg) or I2PP2A mutants S9, 93A (I2PP2A S9, 93A Tg, mimicking dephospho state) or S9, 93D (I2PP2A S9, 93D Tg, phospho state). Our preliminary studies show that subjecting Wt I2PP2A Tg mice or I2PP2A S9, 93D Tg mice to cardiac stress results in accelerated cardiac dysfunction which is markedly ameliorated in I2PP2A S9, 93A Tg mice suggesting a critical role for bAR resensitization in deleterious remodeling. Additionally, we also observed non-classical Gbg-independent PI3Kg recruitment to bARs in response to pro-inflammatory cytokine TNFa which may inhibit PP2A activity leading to loss in bAR resensitization suggesting that TNFa could regulate bAR function by altering the PI3Kg-I2PP2A-PP2A axis. Based on these preliminary studies, we hypothesize that bAR resensitization is inhibited by the PI3Kg- I2PP2A-PP2A pathway upon cardiac stress and releasing the inhibition on bAR resensitization by targeting I2PP2A could preserve bAR function and prevent deleterious cardiac remodeling. We propose the following specific aims to address the hypothesis: Specific aim 1: To determine whether regulating cardiac bAR resensitization pathway prevents deleterious cardiac remodeling in response to cardiac stress, Specific aim 2: To identify the molecular basis of PI3Kg mediated I2PP2A-PP2A interaction and its role in bAR resensitization, Specific aim 3: To investigate the molecular mechanisms mediating agonist independent non-canonical recruitment of PI3Kg and inhibition of bAR resensitization in response to TNFa. Determining the role of PI3Kg-I2PP2A-PP2A axis in regulating bAR resensitization in response to cardiac stress will provide comprehensive mechanistic understanding of bAR dysfunction leading to identification of novel therapeutic strategies for heart failure by targeting bAR resensitization pathway.