Abstract 229: Novel Plasma Membrane Beta Adrenergic Receptor Resensitization By Protein Phosphatase 2a
Resensitization of β-adrenergic receptor (βAR) occurs by dephosphorylation of the internalized βAR by protein phosphatase 2A (PP2A) before being recycled back to plasma membrane (PM). Contrary to this classical paradigm, cardiac specific expression of inactive PI3Kγ (PI3Kγinact) leads to receptor resensitization at the plasma membrane as measured by adenylyl cyclase activity in mice chronically treated with the agonist. We hypothesized that PI3K activity inhibits PM receptor resensitization. Using cells stably expressing FLAG-β1AR alone (single) or along with PI3Kγinact (double), we show that inhibition of PI3K activity results in novel PM receptor resensitization. Agonist activation of single stables showed significant receptor desensitization as measured by reduced cAMP generation (35.6 ± 4.6 pmol/mg protein). In contrast, double-or wortmannin (Wort, PI3K inhibitor) pre-treated single- stables showed marked generation of cAMP (87.4 ± 3.2 or 83.1 ± 5.7) showing β1AR resensitization. Agonist stimulation of metabolically labeled β1ARs pre-treated with inhibitors of internalization, sucrose and β-cyclodextrin resulted in accumulation of phosphorylated receptors at the PM, which was abolished with PI3Kγinact suggesting dephosphorylation of β1ARs. Inhibition of PP2A by okadaic acid or Fostriecin resulted in complete loss of β1AR resensitization despite the presence of PI3Kγinact showing that PI3K regulates PP2A activity at the β1AR complex. Pre-treatment of single stable cells with Wort resulted in significant increase in β1AR-associated phosphatase activity following dobutamine (Dob) treatment (Veh, 4131 ± 14; Dob, 3180 ± 111; Dob + Wort, 17123 ± 680 pmoles/mg protein). Consistently, similar results were obtained in vivo using single transgenic (FLAG-β1AR, Veh, 87 ± 12; Dob, 61.7 ± 8.3) and double transgenic (FLAG-β1AR and PI3Kγinact, Veh, 93 ± 9; Dob, 118 ± 6) mice. In vivo metabolic labeling, co-immunoprecipitation and in vitro kinase assays showed that inhibitor of PP2A (I2PP2A) protein as a target of PI3K in regulating PP2A activity at the β1AR complex. Indeed, siRNA knock down of I2PP2A results in preservation of β1AR function by PM receptor resensitization demonstrating a novel role for PI3K in receptor resensitization.
This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).