LAUSR

More than 100 people die daily in the United States from opioid-related drug overdoses. µ-Opioid receptor (MOR) antagonists, such as naltrexone (Ntx) and naloxone, partially suppress overdose effects, but also induce supersensitivity to MOR activation. Opioid receptors are folded in the endoplasmic reticulum (ER), undergo transport to the Golgi, eventually reach the plasma membrane, and may also later undergo endocytosis. We examined the effects of opioid ligands on MOR trafficking during the earlier processes. Properly folded protein cargo recruits ER exit sites (ERES) and then enters coated vesicles for delivery to the Golgi. We transfected SH-SY5Y cells with fluorescently tagged Sec24 variants to visualize and quantify ERES, and with the strongly ER-retained MOR mutant, MOR[N190K]. We attained sub-µm resolution, for information on ligand effects on ERES levels in live cells. Forster resonance energy transfer (FRET) showed that MOR closely interacts with both Sec24C and Sec24D. Using Sec24D-eGFP to fluorescently mark ERES, we observed that the antagonists increase the fraction of the cytoplasm occupied by ERES. We also found that SH-SY5Y cells overexpressing wild-type δ-opioid receptors have increased ERES levels after exposure to naloxone. These effects most likely occur when an antagonist acts as a pharmacological chaperone of opioid receptors. We rule out the alternative hypothesis that antagonists affect ERES levels via changes in [cAMP] because Ntx did not detectably change [cAMP]. In contrast to these effects of antagonists, several opioid agonists (morphine, fentanyl, buprenorphine, and methadone) lacked detectable effects on ERES levels. Full or partial agonists, but not antagonists, phosphorylate MOR at S375, but SH-SY5Y cells overexpressing MOR[N190K][S375A] showed no change in ERES density in response to agonists. The possibility that antagonists induce supersensitivity by pharmacologically chaperoning opioid receptors could suggest innovative approaches for opioid abuse disorder.