G Protein independent phosphorylation and internalization of the δ‐opioid receptor

Agonist activation of the delta-opioid receptor leads to internalization via G betagamma recruitment of G protein coupled receptor kinase-2, which phosphorylates the receptor at several sites, including Ser363, allowing beta-arrestin binding and localization to clathrin coated pits. Using human embryonic kidney cells expressing a delta-opioid receptor we tested the hypothesis that prevention of receptor coupling to G protein by treatment with pertussis toxin (PTX) will block these processes. PTX treatment did not reduce phosphorylation of delta-opioid receptor Ser363 in response to the agonist [D-Pen2, D-Pen5]enkephalin, or recruitment of beta-arrestin 2-green fluorescent protein to the membrane and only slowed, but did not prevent, [D-Pen2, D-Pen5]enkephalin-induced internalization. Similarly, PTX treatment only partially prevented the ability of the delta-opioid peptide agonists deltorphin II and [Met5]enkephalin and the non-peptide agonist BW373U86 to induce receptor internalization. No internalization was seen with morphine, oxymorphindole or the putative delta(1)-opioid agonist TAN-67 in the presence or absence of PTX, even though TAN-67 showed a strong activation of G protein, as measured by guanosine-5'-O-(3-[(35)S]thio)triphosphate binding. The ability of an agonist to stimulate phosphorylation at Ser363 was predictive of its capacity to induce internalization. The results suggest a role for G protein in delta-opioid receptor internalization, but show that alternative G protein independent pathways exist.