FIGURE 3.

Shifts in binding affinities at AT₁R-G_q and AT₁R-βarr2 fusion proteins quantify the transducer-specific molecular efficacies of a series of AT₁R ligands. Ligands were grouped according to their previously reported efficacies in cells (17, 18, 46, 47). Plotted for each ligand are ¹²⁵I-Sar¹,Ile⁸-AngII competition radioligand binding isotherms performed on purified HEK 293 cell membranes overexpressing the unfused AT₁R (●, ■), the AT₁R-G_q fusion protein (○), or the AT₁R-βarr2 fusion protein (□). Both the x axis and y axis were normalized for simultaneous nonlinear regression curve fitting (see “Curve Fitting and Computation of α” under “Experimental Procedures”), yielding an average value of 40% for AT₁R in the high affinity state. The plotted isotherms represent the global fits of at least three independent experiments, and data points represent the mean of one representative experiment performed in duplicate. The highlighted area between the curves (Δ integral, see “Calculating the Area between Two Binding Curves” under “Experimental Procedures”) represents the magnitude of the curve-shift and is proportional to the molecular efficacy for G_q (blue) or βarr2 (red). A, ¹²⁵I-Sar¹,Ile⁸-AngII competition binding isotherms for the balanced agonist AngII. Similar shifts were observed at each transducer fusion protein. B, ¹²⁵I-Sar¹,Ile⁸-AngII competition binding isotherms for the βarr2-biased agonists SII, TRV120023, TRV120026, TRV120034, TRV120044, and TRV120045. Shifts at the βarr2 fusion protein were larger than those detected at the G_q fusion protein. C, ¹²⁵I-Sar¹,Ile⁸-AngII competition binding isotherms for the G_q-biased agonists TRV120055 and TRV120056. Large affinity shifts were observed at the G_q fusion protein compared with more modest shifts observed at the βarr2 fusion protein. D, ¹²⁵I-Sar¹,Ile⁸-AngII competition binding isotherms for the antagonist telmisartan. Affinity shifts were undetectable at either transducer fusion protein.