Internalization and sequestration of the human prostacyclin receptor.
Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for 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 desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.
Smyth
E M
EM
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Austin
S C
SC
Reilly
M P
MP
FitzGerald
G A
GA
D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D034281:Dynamins; D004705:Endocytosis / Q000187:drug effects; D011464:Epoprostenol / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014162:Transfection
D019390:Arrestins; D007211:Indoles; D007295:Inositol Phosphates; D008301:Maleimides; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D011993:Recombinant Fusion Proteins; D011464:Epoprostenol; D000242:Cyclic AMP; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020558:GTP Phosphohydrolases; D020962:Heterotrimeric GTP-Binding Proteins; D034281:Dynamins; D016285:Iloprost; C070515:bisindolylmaleimide I