Phosphorylation and Agonist-Specific Intracellular Trafficking of an Epitope-Tagged μ-Opioid Receptor Expressed in HEK 293 Cells
Article first published online: 23 NOV 2002
Journal of Neurochemistry
Volume 65, Issue 4, pages 1636–1645, October 1995
How to Cite
Arden, J. R., Segredo, V., Wang, Z., Lameh, J. and Sadée, W. (1995), Phosphorylation and Agonist-Specific Intracellular Trafficking of an Epitope-Tagged μ-Opioid Receptor Expressed in HEK 293 Cells. Journal of Neurochemistry, 65: 1636–1645. doi: 10.1046/j.1471-4159.1995.65041636.x
- Issue published online: 23 NOV 2002
- Article first published online: 23 NOV 2002
- Received January 10, 1995; revised manuscript received May 2, 1995; accepted May 8, 1995.
- Epitope tag;
- μ-Opioid receptor;
Abstract: We expressed the cloned μ-opioid receptor (μR) in high abundance (5.5 × 106 sites/cell) with an amino-terminal epitope tag (EYMPME) in human embryonic kidney 293 cells. The epitope-tagged receptor (EE-μR) was similar to the untagged μR in 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 μ-specific peptide agonist [d-Ala2,MePhe4,Glyol5]enkephalin (DAMGO) caused strong labeling of endocytic vesicles, indicating extensive agonist-induced cellular redistribution of EE-μR. Tracer binding studies suggested partial net internalization and a small degree of down-regulation caused by DAMGO. EE-μR-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 ∼70 kDa with weaker bands at ∼65 kDa. EE-μR was labeled with [γ-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 ∼1.8-fold with the addition of morphine. In conclusion, intracellular trafficking of the μR 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.