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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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Effect of δ-OR agonists, insulin, or PDGF on tyrosine phosphorylation of the insulin receptor or the PDGF receptor. C6-δ-OR cells were serum-starved overnight prior to exposure to DADEE (10 mM), DPDPE (10 mM), DSLET (10 mM), insulin (100 ng/ml), or PDGF (50 mg/ml) for 5 minutes. After agonist exposure, cell lysates were prepared and the IR or the PDGFR was immunoprecipitated as described in the methods section using the appropriate antibodies. Tyrosine phosphorylation of these receptors was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to the tyrosine-phosphorylated forms of the IR (open bars) or the PDGF (hatched bars). Phosphotyrosine bands were quantified using the MCID system. Phosphotyrosine band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments. *p ≤ 0.05 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
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Figure 8: Effect of δ-OR agonists, insulin, or PDGF on tyrosine phosphorylation of the insulin receptor or the PDGF receptor. C6-δ-OR cells were serum-starved overnight prior to exposure to DADEE (10 mM), DPDPE (10 mM), DSLET (10 mM), insulin (100 ng/ml), or PDGF (50 mg/ml) for 5 minutes. After agonist exposure, cell lysates were prepared and the IR or the PDGFR was immunoprecipitated as described in the methods section using the appropriate antibodies. Tyrosine phosphorylation of these receptors was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to the tyrosine-phosphorylated forms of the IR (open bars) or the PDGF (hatched bars). Phosphotyrosine bands were quantified using the MCID system. Phosphotyrosine band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments. *p ≤ 0.05 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.

Mentions: However, C6 glioma cells express additional RTKs including insulin (IR) and platelet-derived growth factor (PDGFR) receptors. It is possible that these sites can be used as surrogate scaffold proteins during ERK activation, and we explored the possibility that δ-opioids induce the transphosphorylation of these two RTKs. At concentrations of 10 μM, neither DSLET, DADLE, nor DPDPE were found to increase the tyrosine phosphorylation of the IR or the PDGFR, whereas insulin (100 ng/ml) or PDGF (50 ng/ml) induced significant tyrosine phosphorylation of these sites (Figure 8). Moreover, we pre-treated C6-δ-OR cells with the IR antagonist, hydroxy-2-napthalenylmethylphosphonic acid-tris acetoxymethylester (HNMPA-(AM)3; 100 μM), or the PDGFR antagonist, (tyrosphostin 9; 100 μM) before the addition of DSLET (1 μM). Despite the presence of these catalytic RTK inhibitors, δ-opioid-mediated ERK activation was not significantly altered in comparison to untreated cultures (Figure 9). Furthermore, immunoprecipitation of the δ-OR, followed by immunoblotting using anti-phosphotyrosine antibodies, revealed that these inhibitors did not attenuate tyrosine phosphorylation of δ-ORs after DSLET treatment (Figure 9). The results presented, herein, strongly suggest that transactivation of RTKs does not make a significant contribution to DSLET-induced ERK activation in at least two different cell lines.


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)

Effect of δ-OR agonists, insulin, or PDGF on tyrosine phosphorylation of the insulin receptor or the PDGF receptor. C6-δ-OR cells were serum-starved overnight prior to exposure to DADEE (10 mM), DPDPE (10 mM), DSLET (10 mM), insulin (100 ng/ml), or PDGF (50 mg/ml) for 5 minutes. After agonist exposure, cell lysates were prepared and the IR or the PDGFR was immunoprecipitated as described in the methods section using the appropriate antibodies. Tyrosine phosphorylation of these receptors was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to the tyrosine-phosphorylated forms of the IR (open bars) or the PDGF (hatched bars). Phosphotyrosine bands were quantified using the MCID system. Phosphotyrosine band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments. *p ≤ 0.05 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
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Related In: Results  -  Collection

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Figure 8: Effect of δ-OR agonists, insulin, or PDGF on tyrosine phosphorylation of the insulin receptor or the PDGF receptor. C6-δ-OR cells were serum-starved overnight prior to exposure to DADEE (10 mM), DPDPE (10 mM), DSLET (10 mM), insulin (100 ng/ml), or PDGF (50 mg/ml) for 5 minutes. After agonist exposure, cell lysates were prepared and the IR or the PDGFR was immunoprecipitated as described in the methods section using the appropriate antibodies. Tyrosine phosphorylation of these receptors was determined by SDS PAGE, followed by immunoblotting of proteins onto nitrocellulose. Immunopositive bands correspond to the tyrosine-phosphorylated forms of the IR (open bars) or the PDGF (hatched bars). Phosphotyrosine bands were quantified using the MCID system. Phosphotyrosine band intensities are expressed graphically as: grain density × band area - [region of detection (ROD)] × pixel. Data points represent the mean ± SEM of three experiments. *p ≤ 0.05 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
Mentions: However, C6 glioma cells express additional RTKs including insulin (IR) and platelet-derived growth factor (PDGFR) receptors. It is possible that these sites can be used as surrogate scaffold proteins during ERK activation, and we explored the possibility that δ-opioids induce the transphosphorylation of these two RTKs. At concentrations of 10 μM, neither DSLET, DADLE, nor DPDPE were found to increase the tyrosine phosphorylation of the IR or the PDGFR, whereas insulin (100 ng/ml) or PDGF (50 ng/ml) induced significant tyrosine phosphorylation of these sites (Figure 8). Moreover, we pre-treated C6-δ-OR cells with the IR antagonist, hydroxy-2-napthalenylmethylphosphonic acid-tris acetoxymethylester (HNMPA-(AM)3; 100 μM), or the PDGFR antagonist, (tyrosphostin 9; 100 μM) before the addition of DSLET (1 μM). Despite the presence of these catalytic RTK inhibitors, δ-opioid-mediated ERK activation was not significantly altered in comparison to untreated cultures (Figure 9). Furthermore, immunoprecipitation of the δ-OR, followed by immunoblotting using anti-phosphotyrosine antibodies, revealed that these inhibitors did not attenuate tyrosine phosphorylation of δ-ORs after DSLET treatment (Figure 9). The results presented, herein, strongly suggest that transactivation of RTKs does not make a significant contribution to DSLET-induced ERK activation in at least two different cell lines.

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