1. Elena Tsisanova, Graeme Henderson and Eamonn Kelly
Characteristics of ERK1/2 activation by different μ-opioid receptor agonists
Elena Tsisanova, Graeme Henderson and Eamonn Kelly
School of Physiology & Pharmacology, University of Bristol, Bristol BS8 1TD, UK
Summary
In the current work ability of several μ-opioid receptor agonists with different efficacies (DAMGO, morphine, etorphine and oxycodone) to induce ERK1/2 activation was studied. Effects of kinase inhibitors on ERK1/2 signalling induced by this agonists were also investigated along with internalization and trafficking at the MOPr after acute agonist treatment.
Introduction
The concept of biased agonism has important therapeutical implications, for example, G-protein coupled receptor agonists which can have different effects on β-arrestin mediated signalling, can possess differences in their therapeutical profile in the intact organism [1]. Biased agonism, also called functional selectivity is the phenomenon whereby different agonists acting at the same receptor subtype produce different signalling responses. Previous research indicated that biased agonism can be detected at the µ-opioid receptor (MOPr) [2]. In the current project the ability of MOPr ligands displaying different degrees of bias between G-protein and arrestin pathways as well as different efficacies (DAMGO, morphine, endomorphin-2,etorphine, methadone and oxycodone) to induce ERK activation was investigated.
Aim
To compare the ability of kinase inhibitors (Wortmannin, KN-93 and PP1) to modify ERK1/2 activation induced by MOPr agonists displaying different efficacies and/or degrees of bias. To perform internalization and translocation studies using confocal microscopy to study differences in trafficking at the MOPr after agonist stimulation.
Materials and Methods
For ERK activation HEK293-MOPr-expressing cells were pre-treated for 5 minutes with agonist alone or with a combination of agonist and the CaMKII inhibitor KN93 (10 μM), the PI3 kinase inhibitor Wortmannin (0.1 μM), or the Src inhibitor PP1 (1 μM) in serum free media. Cell lysates were resolved by SDS-gel electrophoresis and Western blotting. The amount of phosphorylated ERK was monitored with a monoclonal antibody for phosphorylated ERK (P-p-44/42 MAPK anti-rabbit Ab) and was normalized to total ERK using an ERK antibody (p-44/42 MAPK anti-mouse Ab) (3). Bands on blots were analysed using ImageJ software. MOPr trafficking experiments were performed using primary monoclonal HA.11 mouse Ab clone 16B12 and anti-mouse AlexaFluor 488 secondary Ab. Phospho-ERK1/2 activation-immunofluorescence experiments were undertaken using an ERK antibody (p-44/42 MAPK anti-mouse Ab) and anti-rabbit AlexaFluor 555 secondary antibody. To visualize the nucleus, Hoechst 33258, pentahydrate (bis-Benzimide) was used.
Results and Discussion A B
Fig. 1 Areas under the curve for time courses of ERK 1/2 activation in control cells and cells pretreated with 1 μM PKC inhibitor Ro , showing different degree of PKC involvement for (A) DAMGO, morphine and endomorphin-2, (B) etorphine, methadone and oxycodone.
Fig. 5 Visualization of DAMGO- (A) and morphine-induced (B) phospho-ERK1/2 translocation. AlexaFluor 555 secondary antibody was used to visualize phospho- or total-Erk 1/2 primary antibody (scale bar = 25/ 50 μm). Total-ERK1/2 levels are shown in (C) C
Fig. 6 Visualization of etorphine- (A) and oxycodone-induced (B) phospho-ERK1/2 translocation. AlexaFluor 555 secondary antibody was used to visualize phospho- or total-Erk 1/2 primary antibody (scale bar = 25/ 50 μm). Total-ERK1/2 levels are shown in (C)
Fig. 2 Effects of PI3 kinase inhibitor Wortmannin and Src inhibitor PP1 on ERK1/2 activation by different agonists. (A) – 10 μM DAMGO; (B) – 30 μM morphine; (C) – 10 μM etorphine; (D) – 30 μM oxycodone D
Figure 4. Immunoflorescent confocal images of HEK-293 cells stably expressing μ-opioid receptor. AlexaFluor 488 secondary antibody was used to visualize HA-tagged MOPr (green) and Hoechst to stain nuclei (blue). Enlarged inserts are from selected regions within the cells, identified by white square boxes in the merged images (scale bar = 25/ 50 μm).
Fig. 7 Quantification of ERK1/2 translocation induced by the MOPr agonists. (A) DAMGO and morphine induced phospho-ERK1/2 nucleus/cytosol ratio. (B) etorphine- and oxycodone-induced phospho-ERK1/2 nucleus/cytosol ratio. (C) ERK1/2 phosphorylation intensity after DAMGO and morphine treatment . (D) ERK1/2 phosphorylation intensity after etorphine and oxycodone treatment.
Fig. 3 Effects of CamKII inhibitor KN-93 on ERK1/2 activation by different agonists. (A) – 10 μM DAMGO and 30 μM morphine (B) – 10 μM etorphine and 30 μM oxycodone; (C) – representative blot of ERK1/2 activation for (A); (D) – representative blot of ERK1/2 activation for (B).
Conclusions
MOPr agonist-induced ERK activation in HEK293 cells is mediated to a large extent by such kinase inhibitors as Src, CaMKII and PI3 kinase;
The extent of ERK 1/2 activation inhibition by PP1 and wormannin did not significantly vary between DAMGO, morphine, etorphine and oxycodone.
CamKII and PI3 kinase seem to have a bigger role in agonist-dependent ERK1/2 activation comparing to Src;
Internalization studies using immunofluorescence imaging revealed that high efficacy agonists DAMGO and etorphine induce more MOPr internalization than morphine and oxycodone which have lower efficacy;
Cell imaging experiments of ERK1/2 activation revealed that following pre-treatment with DAMGO phosphorylated ERK1/2 located more to the nucleus, whereas phosphorylated ERK1/2 following morphine, etorphine or oxycodone treatment localized predominately to the cell cytoplasm;
These differences may in part explain the different functional profiles of these drugs when administered in vivo.
References
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