Regulation of PP2A(C) Carboxylmethylation and Cellular Localisation by Inhibitory Class G-Protein Coupled Receptors in Cardiomyocytes

The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2A(C)). PP2A(C) activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1. Rs) induces PP2A(C) carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2A(C), leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of Gi-coupled A1. Rs with N 6 -cyclopentyladenosine (CPA), and of other Gi-coupled receptors such as muscarinic M-2 receptors (stimulated with carbachol) and angiotensin II AT2 receptors (stimulated with CGP42112) in ARVM, induced PP2A(C) carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 mM CPA increased the cellular association between PP2A(C) and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1. Rs with 10 mu M CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor LY294002 (20 mu M), thereby confirming that PI3K activity is upregulated in response to A1. R stimulation by CPA in ARVM. A1. R-induced PP2A(C) translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (G(alpha t1)) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1. R-induced PP2A(C) translocation was also seen with LY294002 (20 mu M). These data suggest that in ARVM, A1. R-induced PP2A(C) translocation to the particulate fraction occurs through a GiPCR-G beta gamma-PI3K mediated intracellular signalling pathway, which may involve elevated PP2A(C) carboxylmethylation at leu309.