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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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Western blots showing the effect of AG 1478 on ERK phosphorylation by EGF (2A) or DSLET (2B). (2A) HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (5 ng/ml for 5 minutes) in the presence or absence of PTX pretreatment (100 ng/ml; overnight) or AG1478 (1 μM; 60 minutes prior) . After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1, and ERK 2. (2B) AG1478 has no effect on DSLET-stimulated ERK phosphorylation. AG1478 (1 μM) was added 60 minutes prior to DSLET (1 pM – 1 μM for 5 minutes). After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1 and ERK 2. The immunoblots are representative of an experiment that was repeated three times.
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Figure 2: Western blots showing the effect of AG 1478 on ERK phosphorylation by EGF (2A) or DSLET (2B). (2A) HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (5 ng/ml for 5 minutes) in the presence or absence of PTX pretreatment (100 ng/ml; overnight) or AG1478 (1 μM; 60 minutes prior) . After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1, and ERK 2. (2B) AG1478 has no effect on DSLET-stimulated ERK phosphorylation. AG1478 (1 μM) was added 60 minutes prior to DSLET (1 pM – 1 μM for 5 minutes). After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1 and ERK 2. The immunoblots are representative of an experiment that was repeated three times.

Mentions: We have shown that δ-opioids increase the phosphorylation of ERKl/2 in HEK-293 cells when the mouse wild type (WT) δ-OR is stably expressed [11,13]. The concentrations required to observe DSLET-mediated ERK phosphorylation (EC50 = 10 nM; Figure 1a) are consistent with those reported for other δ-OR-mediated events, including G protein activation and adenylyl cyclase inhibition [16,17]. Moreover, no change in total ERK expression was observed, concomitantly, with ERK phosphorylation in any of our experiments (data not shown). Pretreating the cells with PTX (100 ng/ml), or the specific δ-OR antagonist, naltrindole (10 μM), completely blocked the DSEET-induced increase in ERK phosphorylation (Figure 1b). These results suggest that DSLET utilizes a Gi-coupled δ-OR to stimulate ERK 1 and 2. We then took advantage of the HEK-293 cell line's endogenous expression of the EGFR to examine this receptor's ability to activate ERKs. Similar to DSLET, EGF (5 ng/ml) produces a robust activation of ERKs in δ-OR-expressing HEK-293 cells after a 5 minute treatment (Figure 2a). PTX did not attenuate EGF-mediated ERK activation (Figure 2a), although this response was completely blocked by the EGFR kinase inhibitor, AG1478 (1 μM) (Figure 2a). Conversely, DSLET-induced ERK activation was unaffected by AG1478, suggesting that different tyrosine kinases are involved in opioid- and EGF-dependent MAPK activity (Figure 2b). DSLET-mediated ERK activation was significantly inhibited by the Src kinase inhibitor, PP1 (10 μM) (Figure 3). PP1 also had a significant inhibitory (75% reduction) effect on MAPK activation by EGF (Figure 3).


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)

Western blots showing the effect of AG 1478 on ERK phosphorylation by EGF (2A) or DSLET (2B). (2A) HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (5 ng/ml for 5 minutes) in the presence or absence of PTX pretreatment (100 ng/ml; overnight) or AG1478 (1 μM; 60 minutes prior) . After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1, and ERK 2. (2B) AG1478 has no effect on DSLET-stimulated ERK phosphorylation. AG1478 (1 μM) was added 60 minutes prior to DSLET (1 pM – 1 μM for 5 minutes). After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1 and ERK 2. The immunoblots are representative of an experiment that was repeated three times.
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Figure 2: Western blots showing the effect of AG 1478 on ERK phosphorylation by EGF (2A) or DSLET (2B). (2A) HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (5 ng/ml for 5 minutes) in the presence or absence of PTX pretreatment (100 ng/ml; overnight) or AG1478 (1 μM; 60 minutes prior) . After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1, and ERK 2. (2B) AG1478 has no effect on DSLET-stimulated ERK phosphorylation. AG1478 (1 μM) was added 60 minutes prior to DSLET (1 pM – 1 μM for 5 minutes). After DSLET exposure, cell lysates were prepared and ERK phosphorylation was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to α-tubulin, ERK 1 and ERK 2. The immunoblots are representative of an experiment that was repeated three times.
Mentions: We have shown that δ-opioids increase the phosphorylation of ERKl/2 in HEK-293 cells when the mouse wild type (WT) δ-OR is stably expressed [11,13]. The concentrations required to observe DSLET-mediated ERK phosphorylation (EC50 = 10 nM; Figure 1a) are consistent with those reported for other δ-OR-mediated events, including G protein activation and adenylyl cyclase inhibition [16,17]. Moreover, no change in total ERK expression was observed, concomitantly, with ERK phosphorylation in any of our experiments (data not shown). Pretreating the cells with PTX (100 ng/ml), or the specific δ-OR antagonist, naltrindole (10 μM), completely blocked the DSEET-induced increase in ERK phosphorylation (Figure 1b). These results suggest that DSLET utilizes a Gi-coupled δ-OR to stimulate ERK 1 and 2. We then took advantage of the HEK-293 cell line's endogenous expression of the EGFR to examine this receptor's ability to activate ERKs. Similar to DSLET, EGF (5 ng/ml) produces a robust activation of ERKs in δ-OR-expressing HEK-293 cells after a 5 minute treatment (Figure 2a). PTX did not attenuate EGF-mediated ERK activation (Figure 2a), although this response was completely blocked by the EGFR kinase inhibitor, AG1478 (1 μM) (Figure 2a). Conversely, DSLET-induced ERK activation was unaffected by AG1478, suggesting that different tyrosine kinases are involved in opioid- and EGF-dependent MAPK activity (Figure 2b). DSLET-mediated ERK activation was significantly inhibited by the Src kinase inhibitor, PP1 (10 μM) (Figure 3). PP1 also had a significant inhibitory (75% reduction) effect on MAPK activation by EGF (Figure 3).

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