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

Abstract

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 (EC50 = 0.1 ± 0.03 nM) and inositol phosphate (EC50 = 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 μM) 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 μM) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor-independent (forskolin (5 μM) or dibutyryl cAMP (1 μM) 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.