Requirement of receptor internalization for opioid stimulation of mitogen-activated protein kinase: Biochemical and immunofluorescence confocal microscopic evidence

Previously, we implicated the opioid receptor (OR), G(beta gamma) subunits, and Ras in the opioid activation of extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family involved in mitogenic signaling. We now report that OR endocytosis also plays a role in the opioid stimulation of ERK activity. COS-7 and HEK-293 cells were cotransfected with the cDNA of delta-, mu-, or kappa-OR, dynamin wild-type (D-wr), or the dominant suppressor mutant dynamin K44A, which blocks receptor endocytosis. The activation of ERK by opioid agonists in the presence of D-WT was detected. In contrast, parallel ectopic coexpression of the K44A mutant with OR, followed by agonist treatment, resulted in a time-dependent attenuation of ERK activation. Immunofluorescence confocal microscopy of delta-OR and D-WT-cotransfected COS-7 cells revealed that agonist exposure for 10 min resulted in an ablation of cell surface delta-OR immunoreactivity (IR) and an intensification of cytoplasmic (presumably endosomal) staining as seen in the absence of overexpressed D-WT. After 1 hr of delta-agonist exposure the cells displayed substantial internalization of delta-OR IR. If the cells were cotransfected with delta-OR and dynamin mutant K44A, which was retained on the cell surface even after 1 hr of delta-agonist treatment. Parallel immunofluorescence confocal microscopy, using an anti-ERK antibody, showed that agonist-induced time-dependent ERK IR trafficking into perinuclear and nuclear loci was impaired in the internalization-defective cells. Thus, both biochemical and immunofluorescence confocal microscopic evidence supports the hypothesis that the opioid activation of ERK requires receptor internalization in transfected mammalian cells.