Phosphorylation is not required for dynamin-dependent endocytosis of a truncated mutant opioid receptor.
Opioid receptors are regulated within minutes after activation by G protein-coupled receptor kinase-mediated phosphorylation and dynamin-dependent endocytosis. We addressed the question of whether phosphorylation is required for opioid receptor endocytosis by examining a functional, truncated mutant delta opioid receptor (DOR344T), which is missing phosphorylation sites located in the carboxyl-terminal cytoplasmic domain. DOR344T receptors expressed in Chinese hamster ovary cells remained predominantly in the plasma membrane, even in the presence of saturating concentrations of agonist, consistent with previous studies demonstrating strongly inhibited endocytosis of truncated receptors in this cell type. In marked contrast, DOR344T receptors expressed at similar levels in human embryonal kidney (HEK) 293 cells exhibited rapid, ligand-induced internalization either in the presence of peptide (DADLE) or alkaloid (etorphine) agonist. Quantitative assays using ELISA and flow cytometric techniques indicated that DOR344T receptors were endocytosed in HEK293 cells with similarly rapid kinetics as full-length DOR (t1/2 < 10 min), and both full-length DOR and DOR344T mutant receptors were endocytosed by a dynamin-dependent mechanism involving clathrin-coated pits. Nevertheless, DOR344T receptors failed to undergo any detectable constitutive or agonist-induced phosphorylation in the same cells in which dynamin-dependent endocytosis was observed. These findings establish the first example of a G protein-coupled receptor that does not require phosphorylation to undergo dynamin-dependent endocytosis, and they suggest that significant cell type-specific differences exist in the biochemical requirements for ligand-induced concentration of opioid receptors in clathrin-coated pits.
Murray
S R
SR
Departments of Psychiatry and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0984, USA.
Evans
C J
CJ
von Zastrow
M
M
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D006224:Cricetinae; D034281:Dynamins; D004705:Endocytosis; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D011957:Receptors, Opioid / Q000235:genetics / Q000378:metabolism*
D002966:Clathrin; D011957:Receptors, Opioid; D020558:GTP Phosphohydrolases; D034281:Dynamins