Internalization and sequestration of the human prostacyclin receptor

Prostacyclin (PGI(2)), the major product of cyolooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G pr otein-coupled receptor, the IF. PKC-mediated phosphorylation of human (h) I P is a critical determinant of agonist-induced desensitization (Smyth, E. M ., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-2326 6). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IF, tagged at the N te rminus with hemagglutinin (HA) and fused at the C terminus to the green flu orescent protein (GFP), was coupled to increased cAMP (EC50 = 0.39 +/- 0.09 nM) and inositol phosphate (EC50 = 86.6 +/- 18.3 nM) generation when overe xpressed in HEK 293 cells. Iloprost-induced Sequestration of HA-hIP-GFP, fo llowed in real time by confocal microscopy, was partially colocalized to cl athrin-coated vesicles. Iloprost induced a time- and concentration-dependen t loss of cell surface HA, indicating receptor internalization, which was p revented by inhibitors of clathrin-mediated trafficking and partially reduc ed by cotransfection of cells with a dynamin dominant negative mutant. Sequ estration (EC50 = 27.6 +/- 5.7 nM) was evident at those concentrations of i loprost that induce PKC-dependent desensitization. Neither the PKC inhibito r GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, alte red receptor sequestration indicating that, unlike desensitization, interna lization is PKC-independent. Deletion of the C terminus prevented iloprost- induced internalization, demonstrating the critical nature of this region f or sequestration. Internalization was unaltered by cotransfection of cells with G;protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hip is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from d esensitization. Trafficking is dependent on determinants located in the C t erminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-de pendent clathrin-coated vesicular endocytotic pathway although other dynami n-independent pathways may also be involved.