QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: mol.105.014654v1 69/1/338    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ding, Z. Articles by Kunapuli, S. P. Search for Related Content PubMed PubMed Citation Articles by Ding, Z. Articles by Kunapuli, S. P.

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

Departments of Physiology (Z.D., S.K., S.P.K.) and Pharmacology (S.P.K.) and the Sol Sherry Thrombosis Research Center (Z.D., S.P.K.), Temple University School of Medicine, Philadelphia, Pennsylvania

Human platelets express two P2Y receptors: G_q-coupled P2Y₁, and G_i-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 the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y₁₂ receptor with its C terminus replaced by the corresponding part of human P2Y₁ receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that 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 dramatically inhibited by pertussis toxin, a G_i inhibitor. The constitutively active P2Y₁₂ mutant retained normal responses to 2-methylthio-ADP, 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 (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy--D-ribo-hept-5-enofuranuronoyl]-L-aspartic acid (AR-C78511) as a potent P2Y₁₂ 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 P2Y₁₂ receptor and the identification of potent inverse agonist.

Address correspondence to: Dr. Satya P. Kunapuli, Department of Physiology, Temple University School of Medicine, 3420 N. Broad Street, Philadelphia, PA 19140. E-mail: spk{at}temple.edu