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 activ...