GαoA dissociation BRET assay of MS1 (A) and Comp5 (B) and β-arrestin recruitment BRET assays of MS1 (C) and Comp5 (D) were performed to assess MS1 and Comp5 allosteric regulation of L-methadone, morphine and oxycodone, respectively, through MOR. Data are mean +/− SEM of a dataset composed of three independent experiments, each one carried out in duplicate. KB is the PAM binding affinity to free receptor, α is the binding cooperativity factor, and β is the efficacy cooperativity factor.