Abstract 5783: β-Arrestin-Dependent Activation of Ca2+/Calmodulin-Dependent Kinase II Following β1-Adrenergic Receptor Activation
Ca2+/calmodulin-dependent kinase II (CaMKII) is a serine/threonine kinase that phosphorylates a wide range of proteins involved in Ca2+ homeostasis and appears to play a central role in the development of heart failure. The mechanism by which β-adrenergic receptor (βAR) stimulation increases the activity of CaMKII remains unclear. In this study, we demonstrate that after isoproterenol (ISO) stimulation, CaMKII activity is significantly increased in hearts of wild-type and β2AR knockout mice, whereas ISO had no effect on CaMKII activity in hearts from β1AR knockout or β1β2AR double knockout mice. Since, recent studies have reported that β-arrestins can bind to calmodulin, we tested whether activation of the CaMKII signaling pathway is regulated by β-arrestin. ISO-mediated CaMKII activation, as well as downstream phospholamban phosphorylation at Threonine-17, was markedly inhibited in β-arrestin knockout mice. Similarly, the depletion of β-arrestin by small interference RNA in HEK-293 cells stably expressing β1ARs strongly inhibited ISO-mediated CaMKII activation indicating the necessity for β-arrestin in CaMKII activation. Using immunoprecipitation and confocal microscopy assays, we show that β1AR stimulation induces the formation of a β-arrestin-CaMKII complex at the plasma membrane. Moreover, after β1AR stimulation, the exchange factor directly activated by cAMP (Epac), which is known to mediate the activation of CaMKII, is also recruited to the membrane in a β-arrestin-dependent manner. Consistent with previous reports, treatment with PKI, an inhibitor of PKA, did not block the ISO-induced activation of CaMKII. In conclusion, we show that β1AR mediated activation of CaMKII requires the scaffolding of CaMKII and Epac by β-arrestin, allowing translocation of this multimeric complex to agonist-occupied β1ARs on the plasma membrane, which brings Epac and CaMKII in close proximity to where activator cAMP is being generated.