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

Abstract

Previously, we implicated the opioid receptor (OR), G(βγ) sub, units, 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 δ-, μ, or κ-OR, dynamin wild-type (D(WT)), or the dominant suppressor mutant dynamin K44A, which blocks receptor endocytosis. The activation of ERK by opioid agonists in the presence of DWT 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 δ-OR and D(WT)-cotransfected COS-7 cells revealed that agonist exposure for 10 min resulted in an ablation of cell surface δ-OR immunoreactivity (IR) and an intensification of cytoplasmic (presumably endosomal) staining as seen in the absence of overexpressed DWT. After 1 hr of δ-agonist exposure the cells displayed substantial internalization of δ- OR IR. If the cells were cotransfected with δ-OR and dynamin mutant K44A, OR IR was retained on the cell surface even after 1 hr of δ-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.