Mitragynine and 7-OH do not recruit β-arrestin. (A) Conceptual representation of the β-arrestin BRET assay employing atomistic cartoons derived from available x-ray crystal structures. To measure β-arrestin recruitment, hMOR37 (brown) was co-expressed with β-arrestin-250 (purple) fused to RLuc839 (teal) and Sp1, GAP43 fused to citrine40 (yellow-green) and SH3, and G protein-coupled receptor kinase 2 (GRK2). On β-arrestin recruitment, association of the Sp1 and SH3 domains results in an increase in the BRET signal between RLuc8 and citrine. (B) β-arrestin recruitment at hMOR, positive control = DAMGO; curves represent the average of n≥3, with error bars representing ±SEM.