Pharmacological characterization of protease activated receptor-1 by a serum responsive element-dependent reporter gene assay: Major role of calmodulin

Abstract We studied the protease activated receptor-1 coupling to a serum response element (SRE)-dependent luciferase activity readout in transfected COS-7 cells. Thrombin, with a pEC 50 of 10.5, was 3000-fold more potent than the peptide agonists SFLLR and its derived compound C721-40 in stimulating luciferase activity, although the three agonists exhibited similar efficacy at the maximal concentration tested. Interestingly, SFLLR- and C721-40-induced luciferase activity was biphasic, suggesting that at least two populations of G proteins couple to the receptor. Further pharmacological characterization of this system was performed using selective protease activated receptor-1 antagonists. SCH203099 and ER-112787 blocked SFLLR-induced luciferase activity with similar potencies (p K B of 7), slightly higher than that exhibited by an arylisoxazole derivative compound from Merck (p K B of 6.1). These values correlated with their affinities established by competition binding experiments using [ 3 H]-C721-40 as radioligand for protease activated receptor-1. Transduction mechanisms of protease activated receptor-1 coupling to SRE-dependent luciferase activity were examined using specific inhibitors. The Ca 2+ chelator BAPTA-AM, as well as the calmodulin inhibitors W-7 and ophiobolin A, robustly inhibited SFLLR-induced SRE activation. Overexpression of RGS2 and a dominant negative rhoA protein abolished the SFLLR signal in an additive manner, suggesting a major role of Gq and G12/13 proteins. Furthermore, inhibition of phospholipase C, MAP-kinases, phosphatidyl inositol-3 kinase, rho-kinase and Ca 2+ /calmodulin-dependent protein kinases, all downstream effectors of Gq and G12/13, partially blocked the SFLLR-induced luciferase signal. Taken together, this SRE-luciferase assay reveals a complex network of transduction pathways of protease activated receptor-1 in accordance with the pleiotrophic action of thrombin.