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

The Journal of Biological Chemistry
Emer M SmythGarret A FitzGerald

Abstract

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 (f...Continue Reading

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Citations

Nov 24, 2005·Prostaglandins & Other Lipid Mediators·Devashish KothapalliRichard K Assoian
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Dec 14, 2006·Prostaglandins & Other Lipid Mediators·Jeremiah StithamJohn Hwa
Jan 27, 2015·Prostaglandins & Other Lipid Mediators·Stephanie M KnebelAlan H Stephenson
Jul 13, 2000·The Journal of Biological Chemistry·E M SmythG A FitzGerald
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Sep 12, 2001·The Journal of Biological Chemistry·T HataeT Tanabe
Mar 6, 2015·The Journal of Pharmacology and Experimental Therapeutics·Hitoshi KashiwagiFumitaka Ushikubi
Sep 25, 2020·Journal of Cell Communication and Signaling·Roger G Biringer
Jul 22, 2019·International Journal of Environmental Research and Public Health·Roger G Biringer
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Nov 7, 2003·European Journal of Vascular and Endovascular Surgery : the Official Journal of the European Society for Vascular Surgery·J Vane, R E Corin

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