Fura-2–treated HEK-293–derived cells in which the genes encoding the endogenous Gα_q and Gα₁₁ were knocked out were cotransfected with plasmids encoding either Gα₁₁^R60C (A) or Gα₁₁^WT (B), along with the PTHR1. Forty-eight hours after transfection, cells were pretreated for 45 minutes with Fura-2,AM at a concentration of 5 × 10^–6 M, together with either DMSO (0.1%) or DMSO containing YM-254890 at the indicated concentration and then treated with vehicle (Veh.) or PTH(1-34) (1 × 10^–7 M) and analyzed for changes in Ca²⁺_i signaling: blue line, PTH + DMSO; green line, PTH + 1 × 10^-8 M YM-254890; red line, PTH + 1 × 10^-7 M YM-254890; purple line, PTH + 1 × 10^–6 M YM-254890; orange line, PTH + 1 × 10^–5 M YM-254890; aqua line, PTH + 1 × 10^–4.5 M YM-254890; black line, Vehicle + DMSO. Ratiometric Fura-2 fluorescence was measured using an Envision plate reader. Data are means (± SEM) of duplicate wells from a single experiment; 2 other replicate experiments yielded comparable results. (C) Dose-response curves for YM-254890 generated by plotting the AUC for each PTH-induced response observed in the absence or presence of inhibitor in the experiments of panels A and B. (D) Model of Gα_q in complex with YM-254890. The orientation on the left shows the close proximity of Arg60, mutated in ADH2, with YM-254890 (red), and the orientation on the right shows the close proximity of Arg183, at which more strongly activating oncogenic mutations occur, with bound guanine nucleotide GDP (cyan). The side chain nitrogens of Arg60 and Arg180 are colored blue; the RAS and helical portions of the G protein are shaded dark and light gray, respectively, and helix-5 of the RAS domain, which participates in GPCR interaction, is shaded aqua. The model was prepared using the x-ray coordinate file PDB.3AH8 (23).