Agonist-dependent phosphorylation of an epitope-tagged human prostacyclin receptor.
An epitope-tagged human prostacyclin receptor (HAhIP) was constructed and stably transfected into human embryonic kidney 293 cells. The receptor exhibited high (Kd = 0.4 +/- 0.08 nM, Bmax = 0.7 +/- 0.2 pmol/mg protein; n = 4) and low (Kd = 75 +/- 27.4 nM, Bmax = 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 microM) 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 microM) PKC activation; both were abolished by pretreatment of cells with GF109203X. In contrast, receptor-independent (forskolin (5 microM) or dibutyryl cAMP (1 microM)) 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.
Smyth
E M
EM
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. garret@spirit.gcrc.upenn.edu
Nestor
P V
PV
FitzGerald
G A
GA
D000595:Amino Acid Sequence; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D000939:Epitopes; D011464:Epoprostenol / Q000378:metabolism*; D006801:Humans; D016285:Iloprost / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017433:Protein Structure, Secondary; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000378:metabolism*
D000939:Epitopes; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011464:Epoprostenol; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D016285:Iloprost