(A) The sequence alignment of amino acids composing the Cmpd-6 binding site between the human and murine β₂ARs. Shaded (yellow) amino acids represent the most essential ones, phenylalanine-133 (F-133), substituted to valine (V) in the murine receptor, and lysine-141 (K-141), for Cmpd-6 binding to the β₂AR. (B) The Cmpd-6 binding site in the human (left) and modeled murine (right) β₂ARs shows the topographical molecular surface of F-133 (purple) and V-133 (cyan) on the transmembrane-3 (TM-3). Illustrations were created with the previously reported structure PDB-6N48 using the PyMOL program. (C) Radioligand competition binding was performed with isolated membranes from 293ExpiF cells transiently expressing either the WT or V133F mutant murine β₂AR as described in Figure 1, C and D. Curve fits were plotted with data sets obtained from 4 independent experiments done in duplicate. The shift of curves was expressed as fold changes in IC₅₀ values between Cmpd-6– and DMSO-treated conditions. Statistical analyses for the shift in each of the WT and mutant receptor were performed using paired 2-tailed Student’s t tests. ***P_adj < 0.001 compared with the DMSO-treated condition. (D) HEK293 cells stably expressing the GloSensor reporter were transiently transfected with one of the human WT, murine WT, and murine V133F mutant β₂AR. After incubation with Cmpd-6 at 5 μM or DMSO vehicle, the cAMP level was monitored as described in Methods. Values were expressed as percentage of the isoproterenol-stimulated (ISO-stimulated) maximal response obtained as a control for comparable receptor expression in each transfection condition and represent mean ± SEM obtained from 4 independent experiments performed in duplicate. Statistical analyses were performed using 1-way ANOVA, repeated (related) measures with Tukey’s posthoc test. Adjusted **P_adj < 0.01.