title: Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor.
abstract: Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) 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 the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) 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 P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (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 P2Y(12) receptor and the identification of potent inverse agonist.
authors.affiliation: Department of Physiology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140, USA.
authors.forename: Zhongren
authors.initials: Z
authors.lastname: Ding
authors.forename: Soochong
authors.initials: S
authors.lastname: Kim
authors.forename: Satya P
authors.initials: SP
authors.lastname: Kunapuli
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000819:agonists* / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000235:genetics; D058925:Receptors, Purinergic P2Y12; D011994:Recombinant Proteins / Q000819:agonists / Q000235:genetics
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; C548576:P2RY12 protein, human; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D011994:Recombinant Proteins; D000242:Cyclic AMP; D051057:Proto-Oncogene Proteins c-akt