ABSTRACT
BACKGROUND: Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces ß-adrenergic receptor (ßAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. METHODS AND RESULTS: Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate ßAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling ßAR desensitization independent of sympathetic overdrive. TNFα-mediated ßAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in ß2AR-overexpressing human embryonic kidney 293 cells showed significant ßAR desensitization, GRK2 upregulation, and recruitment to the ßAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated ßAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated ßAR phosphorylation was not blocked with ßAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gßγ-sequestering peptide ßARK-ct could not prevent ßAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the ßAR is Gßγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated ßAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced ßAR desensitization is GRK2 dependent. CONCLUSIONS: TNFα-induced ßAR desensitization is mediated by GRK2 and is independent of Gßγ, uncovering a hitherto unknown cross-talk between TNFα and ßAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.
Subject(s)
G-Protein-Coupled Receptor Kinase 2/metabolism , Heart Failure/metabolism , Myocytes, Cardiac/enzymology , Receptors, Adrenergic, beta/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Disease Models, Animal , HEK293 Cells , Heart Failure/pathology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Transgenic , Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/physiology , Propranolol/pharmacology , Receptors, Tumor Necrosis Factor, Type II/metabolism , Sympathetic Nervous System/physiology , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Phosphoinositide 3-kinase γ (PI3Kγ) is activated by G protein-coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the ß-adrenergic receptor (ßAR, a GPCR) complex altering G protein coupling. PI3Kγ inhibition results in significant increase of ßAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the ßAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 and 93, resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of ß2ARs is observed with a phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in ß2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ-mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.
