PHOSPHORYLATION IS NOT REQUIRED FOR DYNAMIN-DEPENDENT ENDOCYTOSIS OF A TRUNCATED MUTANT OPIOID RECEPTOR

Opioid receptors are regulated within minutes after activation by G pr otein-coupled receptor kinase-mediated phosphorylation and dynamin-dep endent endocytosis, We addressed the question of whether phosphorylati on is required for opioid receptor endocytosis by examining a function al, truncated mutant delta opioid receptor (DOR344T), which is missing phosphorylation sites located in the carboxyl-terminal cytoplasmic do main, DOR344T receptors expressed in Chinese hamster ovary cells remai ned predominantly in the plasma membrane, even in the presence of satu rating concentrations of agonist, consistent with previous studies dem onstrating strongly inhibited endocytosis of truncated receptors in th is cell type. In marked contrast, DOR344T receptors expressed at simil ar levels in human embryonal kidney (HEK) 293 cells exhibited rapid, L igand-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 endo cytosed in HEK293 cells with similarly rapid kinetics as full-length D OR (t(1/2) < 10 min), and both full-length DOR and DOR344T mutant rece ptors were endocytosed by a dynamin-dependent mechanism involving clat hrin-coated pits. Nevertheless, DOR344T receptors failed to undergo an y detectable constitutive or agonist-induced phosphorylation in the sa me cells in which dynamin-dependent endocytosis was observed. These fi ndings establish the first example of a G protein-coupled receptor tha t does not require phosphorylation to undergo dynamin-dependent endocy tosis, and they suggest that significant cell type-specific difference s exist in the biochemical requirements for ligand-induced concentrati on of opioid receptors in clathrin-coated pits.