Agonist-dependent phosphorylation of an epitope-tagged human prostacyclin receptor

An epitope-tagged human prostacyclin receptor (HA-hIP) was constructed and stably transfected into human embryonic kidney 293 cells. The receptor exhibited high (K-d = 0.4 +/- 0.08 nM, B-max = 0.7 +/- 0.2 pmol/mg protein; n = 4) and low (K-d = 75 +/- 27.4 nM, B-max = 7.1 +/- 3.6 pmol/mg protein; n = 4) affinity for iloprost and coupled to both cAMP (EC(50) = 0.1 +/- 0.03 nM) and inositol phosphate (EC(50) = 43.1 +/- 10 nM) production. The receptor resolved on SDS-polyacrylamide gel electrophoresis as a broad complex with a molecular mass of 44-62 kDa and is glycosylated and phosphorylated. Stimulation of transfected cells with iloprost induced a rapid time- and concentration-dependent phosphorylation of HAhIP. Pretreatment of cells with a protein kinase C (PKC) inhibitor (GF109203X; 5 mu M) abolished basal phosphorylation and dramatically reduced iloprost-induced HAhIP phosphorylation. A protein kinase A (PKA) inhibitor (H89) was largely ineffective under the same conditions. HAhIP phosphorylation was stimulated by receptor dependent (thrombin, 2 units/ml) or receptor-independent (phorbol 12-myristate 13-acetate, 5 mu M) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor independent (forskolin (5 mu M) or dibutyryl cAMP (1 mu M)) activation of PKA did not induce HAhIP phosphorylation. These results indicate that the human prostacyclin receptor may be regulated by agonist-dependent phosphorylation. This appears to be mediated, in part, by activation of PKC but not by PKA.