Abstract 765: Novel Regulation of β1-adrenergic Receptor Function by Phosphoinositide 3-kinase
Agonist activation of beta-adrenergic receptor (βAR) leads to desensitization due to phosphorylation by βAR kinase 1 (βARK1) followed by recruitment of β-arrestin resulting in endocytosis. βAR resensitization is mediated by dephosphorylation of the internalized receptor by protein phosphatase 2A (PP2A) in the endosomes before being recycled back to the plasma membrane. Contrary to this classical paradigm, cardiac specific expression of PI3K mutant (PI3Kγinact) leads to inhibition of receptor internalization and is associated with a novel phenomenon of receptor resensitization that is cardio-protective. Therefore, we hypothesize that PI3K activity negatively regulates receptor resensitization at the plasma membrane. We have generated HEK cells stably expressing FLAG-β1AR (single stable) or FLAG-β1AR and PI3Kγinact (double stable). Cells were pre-treated with ICI-118,551 (β2AR antagonist) prior to challenge and rechallenge with β1AR specific agonist dobutamine (Dob). Plasma membrane adenylyl cyclase (AC) activity and cAMP generation were measured. Single stables showed significant receptor desensitization as measured by reduced cAMP generation (35.6 ± 4.6 pmol/mg protein) or blunted AC activity (38.7 ± 7.9 pmol/mg protein). In contrast, double stables or wortmannin (Wort, PI3K inhibitor) pre-treated single stables showed marked generation of cAMP (87.4 ± 3.2 or 83.1 ± 5.7) and AC activity (60.7 ± 10.5 or 54.7 ± 11.3) respectively, indicating β1AR resensitization. Inhibition of PP2A activity by Fostreiscin resulted in complete loss of β1AR resensitization despite expression of PI3Kγinact. β1AR-associated phosphatase activity was significantly inhibited upon Dob stimulation, in contrast to Wort treatment (Veh, 4131 ± 14; Dob, 3180 ± 111; Dob+ Wort, 17123 ± 680 pmoles/ mg protein). Dob stimulation of transgenic mice with cardiac FLAG-β1AR resulted in significant inhibition of β1AR-associated phosphatase activity (Veh, 87 ± 12; Dob, 61.7 ± 8.3). In contrast, dob stimulation resulted in activation of phosphatase activity in the double transgenic mice expressing FLAG-β1AR and PI3Kγinact (Veh, 93 ± 9; Dob, 118 ± 6). The molecular mechanism underlying this novel regulation of β1AR associated phosphatase activity by PI3K will be discussed.