SEQUESTRATION OF THE DELTA-OPIOID RECEPTOR - ROLE OF THE C-TERMINUS IN AGONIST-MEDIATED INTERNALIZATION

The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupl ed receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, wh ereas other structurally homologous G protein-coupled receptors do not . It is not known whether opioid receptors are regulated by rapid endo cytosis. In transfected Chinese hamster ovary cells expressing the epi tope-tagged wild type delta opioid receptor, exposure to 100 nM [D-Ala (2),D-Leu(5)]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy, The rate of internalizati on of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, th e cells expressing receptors lacking the C-terminal 15 or 37 amino aci ds exhibit a substantially slower rate of internalization. Furthermore , the cells expressing receptors with point mutations of any of the Se r/Thr between Ser(344) and Ser(363) in the C-terminal tail exhibit a s ignificant reduction in the rate of receptor internalization. These re sults suggest that a portion of the C-terminal tail is involved in rec eptor internalization. Agents that block the formation of clathrin-coa ted pits considerably reduce the extent of agonist-mediated internaliz ation of the wild type receptor. Taken together, these results suggest that the mild type opioid receptor undergoes rapid agonist-mediated i nternalization via a classic endocytic pathway and that a portion of t he C-terminal tail plays an important role in this internalization pro cess.