Differential Regulation of Phosphorylation of the cAMP Response Element-Binding Protein after Activation of EP2and EP4Prostanoid Receptors by Prostaglandin E2

Abstract

The EP₂ and EP₄ prostanoid receptors are G-protein-coupled receptors whose activation by their endogenous ligand, prostaglandin (PG) E₂, stimulates the formation of intracellular cAMP. We have previously reported that the stimulation of cAMP formation in EP₄-expressing cells is significantly less than in EP₂-expressing cells, despite nearly identical levels of receptor expression (J Biol Chem 277:2614–2619, 2002). In addition, a component of EP₄ receptor signaling, but not of EP₂ receptor signaling, was found to involve the activation of phosphatidylinositol 3-kinase (PI3K). In this study, we report that PGE₂ stimulation of cells expressing either the EP₂ or EP₄ receptor results in the phosphorylation of the cAMP response element binding protein (CREB) at serine-133. Pretreatment of cells with N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H-89), an inhibitor of protein kinase A (PKA), attenuated the PGE₂-mediated phosphorylation of CREB in EP₂-expressing cells, but not in EP₄-expressing cells. Pretreatment of cells with wortmannin, an inhibitor of PI3K, had no effects on the PGE₂-mediated phosphorylation of CREB in either EP₂- or EP₄-expressing cells, although it significantly increased the PGE₂-mediated activation of PKA in EP₄-expressing cells. However, combined pretreatment with H-89 and wortmannin blocked PGE₂-mediated phosphorylation in EP₂-expressing cells as well as in EP₂-expressing cells. PGE₂-mediated intracellular cAMP formation was not affected by pretreatment with wortmannin, or combined treatment with wortmannin and H-89, in either the EP₂- or EP₄-expressing cells. These findings suggest that PGE₂ stimulation of EP₄ receptors, but not EP₂ receptors, results in the activation of a PI3K signaling pathway that inhibits the activity of PKA and that the PGE₂-mediated phosphorylation of CREB by these receptors occurs through different signaling pathways.