Agonist-promoted internalization (endocytosis) of G-protein-coupled receptors (GPCRs), including all three opioid receptor types (μ, δ and κ), has been shown to occur via the clathrin endosomal pathway in response to receptor phosphorylation and the actions of the proteins, β-arrestin and dynamin. Many members of the GPCR family stimulate mitogen-activated protein kinases (MAPK or ERK) activity and, in several cases, it appears that MAPK activation is dependent on receptor internalization. We have reinvestigated the question of whether internalization is obligatory for MAPK activation by opioid receptors, using cell lines expressing the cloned μ or δ receptor. Morphine, which is known to activate both μ and δ receptors, does not induce their rapid internalization into clathrin-coated endosomes. However, morphine produced a robust stimulation of MAPK in both cell lines, as demonstrated by the appearance of phosphorylated MAPK. Moreover, pre-exposure of cells to the internalization inhibitors, concanavalin A or hypertonic sucrose, totally blocked DAMGO (μ-selective agonist) and DTLET (δ-selective agonist)-mediated receptor internalization, yet neither treatment affected MAPK phosphorylation induced by these peptides. Our results provide evidence that receptor internalization is not an obligatory requirement for MAPK activation by μ and δ opioid receptors. Hypotheses are presented to explain the seemingly contradictory results obtained from different laboratories.
