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 (muR) 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-muR) was similar to the untagged mu R ligand binding and agonist-dependent inhibition of cyclic AMP accumulation. By confocal microscopy, the labeled receptor was shown to be largely confined to the plasma membrane. Pretreatment with morphine failed to affect the cellular distribution of the receptor as judged by immunofluorescence and tracer binding studies. In contrast, exposure to the mu-specific peptide agonist [D-Ala2, MePhe4, Gly-ol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-muR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-muR-containing membranes were solubilized in detergent [3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate] and immunoprecipitated by an anti-epitope monoclonal antibody. Immunoblotting revealed a prominent band at approximately 70 kDa with weaker bands at approximately 65 kDa. EE-muR was labeled with [gamma-32P]ATP in permeabilized cells, immunoprecipitated, and analyzed by polyacrylamide gel electrophoresis autoradiography. A prominent band at 65-70 kDa indicated the presence of basal receptor phosphorylation occurring in the absence of agonist, which was enhanced approximately 1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the muR appears to depend on the agonist, with morphine and DAMGO having markedly different effects. Unlike other G protein-coupled receptors, basal phosphorylation is substantial, even in the absence of agonist.
Arden
J R
JR
Department of Pharmacy and Pharmaceutical Chemistry, University of California, San Francisco 94143, USA.
Segredo
V
V
Wang
Z
Z
Lameh
J
J
Sadée
W
W
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000939:Epitopes; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D018613:Microscopy, Confocal; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D017450:Receptors, Opioid, mu / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D016324:Sequence Tagged Sites
D000939:Epitopes; D015335:Molecular Probes; D017450:Receptors, Opioid, mu