Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor.

Human platelets express two P2Y receptors: Gq-coupled P2Y1 and Gi-coupled P2Y12. Both P2Y1 and P2Y12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of human platelet ADP receptor we constructed a chimeric HA-tagged human P2Y12 receptor with its C-terminus replaced by the corresponding part of human P2Y1 receptor and stably expressed it in CHO-K1 cells. Interestingly, the chimeric P2Y12 mutant exhibited a high level of constitutive activity as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y12 mutant was abolished by pertussis toxin, a Gi inhibitor. The constitutively active P2Y12 mutant retained normal responses to 2-MeSADP, with an EC50 of 0.15 ± 0.04 nM. The constitutively active P2Y12 mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y12 antagonists revealed AR-C78511 as a potent P2Y12 inverse agonist whereas AR-C69931MX as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y12 receptor and the identification of potent inverse agonist.