PHOSPHORYLATION AND AGONIST-SPECIFIC INTRACELLULAR TRAFFICKING OF AN EPITOPE-TAGGED MU-OPIOID RECEPTOR EXPRESSED IN HEK-293 CELLS

We expressed the cloned mu-opioid receptor (mu R) in high abundance (5 .5 x 10(6) sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-mu R ) was similar to the untagged mu R in ligand binding and agonist-depen dent inhibition of cyclic AMP accumulation. By confocal microscopy, th e labeled receptor was shown to be largely confined to the plasma memb rane. Pretreatment with morphine failed to affect the cellular distrib ution of the receptor as judged by immunofluorescence and tracer bindi ng studies. In contrast, exposure to the mu-specific peptide agonist [ D-Ala(2),MePhe(4),Gly-ol(5)] enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-mu R. Tracer binding studies suggested partial ne t internalization and a small degree of down-regulation caused by DAMG O. EE-mu R-containing membranes were solubilized in detergent olamidop ropyl)dimethylammonio]-1-propanesulfonate] and immunoprecipitated by a n anti-epitope monoclonal antibody. Immunoblotting revealed a prominen t band at similar to 70 kDa with weaker bands at similar to 65 kDa. EE -mu R was labeled with [gamma-P-32]ATP in permeabilized cells, immunop recipitated, and analyzed by polyacrylamide gel electrophoresis autora diography. A prominent band at 65-70 kDa indicated the presence of bas al receptor phosphorylation occurring in the absence of agonist, which was enhanced similar to 1.8-fold with the addition of morphine. In co nclusion, intracellular trafficking of the mu R appears to depend on t he agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is su bstantial, even in the absence of agonist.