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-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type delta opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization 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, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.
Trapaidze
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Department of Pharmacology, New York University School of Medicine, New York, New York 10016, USA. Lakshmi.Devi@med.nyu.edu
Keith
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DE
Cvejic
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Evans
C J
CJ
Devi
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LA
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018076:DNA, Complementary; D015536:Down-Regulation; D004705:Endocytosis; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D017465:Receptors, Opioid, delta; D016308:Enkephalin, Leucine-2-Alanine