Markedly impaired signaling and binding to P2Y₁₂ K174E. (A) Agonist (ADP; 0.01 pM to 10 μM)–dependent inhibition of forskolin (1 μM; 10 minutes)–stimulated adenylyl cyclase activity was assessed in CHO cells stably expressing wild-type and K174E P2Y₁₂ receptor. Data are expressed as percentage inhibition of forskolin-stimulated adenylyl cyclase and represent means (± SEM) of 3 independent experiments. (B) Receptor levels were measured in CHO cells stably expressing wild-type or K174E receptor using [³H]2MeS-ADP (0.1-10 μM) in the presence of the P2Y₁₂ receptor antagonist AR-C69931MX (1 μM) to determine specific binding. Data are expressed as specific binding of [³H]2MeS-ADP (cpm) per milligram of protein and represent means (± SEM) of 3 independent experiments.