Competition binding constant values (K_i) were determined by HTRF binding assay. The values of potency (EC₅₀) and efficacy (E_max, normalized to DAMGO) come from cAMP assays performed on CHO-MOR (WT & W6.48L) cells and β-arrestin2 recruitment performed on CHO-MOR (WT & W6.48L)-β-arrestin2 cells. Data were presented as mean ± SEM of three or more assays run in duplicate or triplicate. The difference between K_i, EC₅₀, and E_max values of wild-type versus W6.48L mutant MOR was acquired via unpaired, two-tailed t-test. The corresponding binding curves were shown in Figure 2 , and corresponding dose-response curves were shown in Figure 3 .