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Opioid modulation of extracellular signal-regulated protein kinase activity is ras-dependent and involves Gbetagamma
M M Belcheva1, Z Vogel, E Ignatova
1E. A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104-1079, USA.
Abstract:
Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.
Insights
Opioid agonists activate extracellular signal-regulated protein kinase (ERK) pathways through G protein-coupled receptors. This study reveals opioid receptor involvement in ERK activation and modulation of growth factor signaling.
Area of Science:
- Molecular Cell Biology
- Neuropharmacology
- Signal Transduction
Background:
- G protein-coupled receptors (GPCRs) and tyrosine kinase receptors (TKRs) interact, but convergence points are unclear, especially for opioids.
- Understanding opioid receptor signaling networks is crucial for elucidating complex cellular responses.
Purpose of the Study:
- To investigate the mechanism of opioid agonist-induced activation of extracellular signal-regulated protein kinase (ERK).
- To explore the role of opioid receptors in modulating TKR signaling, specifically epidermal growth factor (EGF)-induced ERK activity.
Main Methods:
- Transient cotransfection of COS-7 cells with opioid receptors (mu, delta, kappa) and ERK1/ERK2.
- Stimulation with selective opioid agonists and measurement of ERK activity.
- Inhibition studies using antagonists, pertussis toxin, dominant-negative Ras, and a beta-gamma scavenger.
Main Results:
- Opioid agonists rapidly and dose-dependently stimulated ERK1 and ERK2 activity via opioid receptors.
- Activation was dependent on Gi/o proteins, Ras, and beta-gamma subunits.
- Chronic opioid treatment attenuated EGF-induced ERK activation, mediated by opioid receptors and involving beta-gamma subunits.
Conclusions:
- Opioid agonists activate ERK1 and ERK2 through a mechanism involving Ras and Gi/o protein beta-gamma subunits.
- Opioid receptors modulate TKR signaling, impacting growth factor-induced ERK activity.
- These findings delineate a key convergence point between GPCR and TKR pathways in cellular signaling.