Agonists at the δ‐opioid receptor modify the binding of µ‐receptor agonists to the µ–δ receptor hetero‐oligomer

BACKGROUND AND PURPOSE µ‐ and δ‐opioid receptors form heteromeric complexes with unique ligand binding and G protein‐coupling profiles linked to G protein α z‐subunit (Gα z ) activation. However, the mechanism of action of agonists and their regulation of the µ–δ receptor heteromer are not well understood. EXPERIMENTAL APPROACH Competition radioligand binding, cell surface receptor internalization in intact cells, confocal microscopy and receptor immunofluorescence techniques were employed to study the regulation of the µ–δ receptor heteromer in heterologous cells with and without agonist exposure. KEY RESULTS Gα z enhanced affinity of some agonists at µ–δ receptor heteromers, independent of agonist chemical structure. δ‐Opioid agonists displaced µ‐agonist binding with high affinity from µ–δ heteromers, but not µ receptor homomers, suggestive of δ‐agonists occupying a novel µ‐receptor ligand binding pocket within the heteromers. Also, δ‐agonists induced internalization of µ‐opioid receptors in cells co‐expressing µ‐ and δ‐receptors, but not those expressing µ‐receptors alone, indicative of µ–δ heteromer internalization. This dose‐dependent, Pertussis toxin‐resistant and clathrin‐ and dynamin‐dependent effect required agonist occupancy of both µ‐ and δ‐opioid receptors. In contrast to µ‐receptor homomers, agonist‐induced internalization of µ–δ heteromers persisted following chronic morphine exposure. CONCLUSIONS AND IMPLICATIONS The µ–δ receptor heteromer may contain a novel δ‐agonist‐detected, high‐affinity, µ‐receptor ligand binding pocket and is regulated differently from the µ‐receptor homomer following chronic morphine exposure. Occupancy of both µ‐ and δ‐receptor binding pockets is required for δ‐agonist‐induced endocytosis of µ–δ receptor heteromers. δ‐Opioid agonists target µ–δ receptor heteromers, and thus have a broader pharmacological specificity than previously identified.