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Delta opioid activation of the mitogen-activated protein kinase cascade does not require transphosphorylation of receptor tyrosine kinases.

Kramer HK, Onoprishvili I, Andria ML, Hanna K, Sheinkman K, Haddad LB, Simon EJ - BMC Pharmacol. (2002)

Bottom Line: Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed delta-ORs are stimulated by the delta-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET).Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET.Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated delta-OR, itself, is likely to act as its own signalling scaffold.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Science, Fordham University, Larkin Hall-Room 160, 440 East Fordham Road, Bronx, NY 14440, USA. hakramer@fordham.edu

ABSTRACT

Background: In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned delta-opioid receptor (delta-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive.

Results: Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed delta-ORs are stimulated by the delta-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the delta-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In delta-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation.

Conclusion: Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated delta-OR, itself, is likely to act as its own signalling scaffold.

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ERK phosphorylation by DSLET (filled squares) or EGF (open triangles) in C6 glioma cells stably-expressing the δ-OR. C6-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 nM – 1 μM for 5 minutes) or DSLET (0.1 nM – 1 μM for 5 minutes). In some instances, DSLET-mediated ERK phosphorylation was performed in cells pre-treated with AG1478 (1 μM; filled circles) 60 minutes prior to DSLET. After agonist exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. PhosphoERK -immunopositive bands were quantified using the MCID system. Band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments.
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Figure 7: ERK phosphorylation by DSLET (filled squares) or EGF (open triangles) in C6 glioma cells stably-expressing the δ-OR. C6-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 nM – 1 μM for 5 minutes) or DSLET (0.1 nM – 1 μM for 5 minutes). In some instances, DSLET-mediated ERK phosphorylation was performed in cells pre-treated with AG1478 (1 μM; filled circles) 60 minutes prior to DSLET. After agonist exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. PhosphoERK -immunopositive bands were quantified using the MCID system. Band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments.

Mentions: C6 glioma cells have been used as host cells for the transfection of several GPCR types, and have the advantage of expressing high levels of ERK, while being devoid of the EGFR. In C6 glioma cells that express the mouse δ-OR, DSLET produced a concentration-dependent increase in the appearance of phospho-ERK 1 and 2. DSLET-mediated ERK activation in C6-δ-OR cells was completely inhibited by either pre-treatment with PTX or naltrindole (data not shown). In contrast, EGF was unable to elicit ERK activation, at any concentration tested, up to 1 μM (Figure 7). Furthermore, the EGFR antagonist, AG1478, had no effect on DSLET-mediated ERK activation, which eliminated the possibility that these antagonists produced non-specific, inhibitory effects (Figure 7).


Delta opioid activation of the mitogen-activated protein kinase cascade does not require transphosphorylation of receptor tyrosine kinases.

Kramer HK, Onoprishvili I, Andria ML, Hanna K, Sheinkman K, Haddad LB, Simon EJ - BMC Pharmacol. (2002)

ERK phosphorylation by DSLET (filled squares) or EGF (open triangles) in C6 glioma cells stably-expressing the δ-OR. C6-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 nM – 1 μM for 5 minutes) or DSLET (0.1 nM – 1 μM for 5 minutes). In some instances, DSLET-mediated ERK phosphorylation was performed in cells pre-treated with AG1478 (1 μM; filled circles) 60 minutes prior to DSLET. After agonist exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. PhosphoERK -immunopositive bands were quantified using the MCID system. Band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC88976&req=5

Figure 7: ERK phosphorylation by DSLET (filled squares) or EGF (open triangles) in C6 glioma cells stably-expressing the δ-OR. C6-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 nM – 1 μM for 5 minutes) or DSLET (0.1 nM – 1 μM for 5 minutes). In some instances, DSLET-mediated ERK phosphorylation was performed in cells pre-treated with AG1478 (1 μM; filled circles) 60 minutes prior to DSLET. After agonist exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. PhosphoERK -immunopositive bands were quantified using the MCID system. Band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments.
Mentions: C6 glioma cells have been used as host cells for the transfection of several GPCR types, and have the advantage of expressing high levels of ERK, while being devoid of the EGFR. In C6 glioma cells that express the mouse δ-OR, DSLET produced a concentration-dependent increase in the appearance of phospho-ERK 1 and 2. DSLET-mediated ERK activation in C6-δ-OR cells was completely inhibited by either pre-treatment with PTX or naltrindole (data not shown). In contrast, EGF was unable to elicit ERK activation, at any concentration tested, up to 1 μM (Figure 7). Furthermore, the EGFR antagonist, AG1478, had no effect on DSLET-mediated ERK activation, which eliminated the possibility that these antagonists produced non-specific, inhibitory effects (Figure 7).

Bottom Line: Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed delta-ORs are stimulated by the delta-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET).Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET.Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated delta-OR, itself, is likely to act as its own signalling scaffold.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Science, Fordham University, Larkin Hall-Room 160, 440 East Fordham Road, Bronx, NY 14440, USA. hakramer@fordham.edu

ABSTRACT

Background: In this study, we investigated the mechanism(s) by which delta opioids induce their potent activation of extracellular signal-regulated protein kinases (ERKs) in different cell lines expressing the cloned delta-opioid receptor (delta-OR). While it has been known for some time that OR stimulation leads to the phosphorylation of both ERK isoforms, the exact progression of events has remained elusive.

Results: Our results indicate that the transphosphorylation of an endogenous epidermal growth factor receptor (EGFR) in the human embryonic kidney (HEK-293) cell line does not occur when co-expressed delta-ORs are stimulated by the delta-opioid agonist, D-Ser-Leu-enkephalin-Thr (DSLET). Moreover, neither pre-incubation of cultures with the selective EGFR antagonist, AG1478, nor down-regulation of the EGFR to a point where EGF could no longer activate ERKs had an inhibitory effect on ERK activation by DSLET. These results appear to rule out any structural or catalytic role for the EGFR in the delta-opioid-mediated MAPK cascade. To confirm these results, we used C6 glioma cells, a cell line devoid of the EGFR. In delta-OR-expressing C6 glioma cells, opioids produce a robust phosphorylation of ERK 1 and 2, whereas EGF has no stimulatory effect. Furthermore, antagonists to the RTKs that are endogenously expressed in C6 glioma cells (insulin receptor (IR) and platelet-derived growth factor receptor (PDGFR)) were unable to reduce opioid-mediated ERK activation.

Conclusion: Taken together, these data suggest that the transactivation of resident RTKs does not appear to be required for OR-mediated ERK phosphorylation and that the tyrosine-phosphorylated delta-OR, itself, is likely to act as its own signalling scaffold.

Show MeSH
Related in: MedlinePlus