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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 DSLET or EGF on tyrosine phosphorylation of the δ-OR or the EGFR. HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 – 100 ng/ml for 5 minutes) or DSLET (10 nM-10 μM for 5 minutes). After agonist exposure, cell lysates were prepared and the δ-OR or the EGFR 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 δ-OR (upper panel) or the EGFR (lower panel). The immunoblots are representative of an experiment that was repeated three times.
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Figure 4: Western blots showing the effect of DSLET or EGF on tyrosine phosphorylation of the δ-OR or the EGFR. HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 – 100 ng/ml for 5 minutes) or DSLET (10 nM-10 μM for 5 minutes). After agonist exposure, cell lysates were prepared and the δ-OR or the EGFR 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 δ-OR (upper panel) or the EGFR (lower panel). The immunoblots are representative of an experiment that was repeated three times.

Mentions: Our next experiments were designed to examine whether the EGFR becomes tyrosine phosphorylated during ERK activation by DSLET. As stated in the introduction, several GPCRs (including the EPAR, β2-AR, and substance P (SP-R)) [14,18-20] increase EGFR phosphorylation during the course of ERK activation, and it is believed, that in these instances, this event is an initial step in the progression of the MAPK cascade. Our laboratory has previously reported [11,13] that 5-opioids induce tyrosine phosphorylation of the δ-OR, and it appears that this is catalyzed by the non-receptor tyrosine kinase Src. However, transphosphorylation of the EGFR after OR activation has not been reported. WT-δ-OR-expressing HEK-293 cells were exposed to increasing concentrations of either DSLET (1–1000 nM) or EGF (0.1–100 ng/ml) for five minutes to induce ERK activation. As stated earlier, either DSLET or EGF increased the appearance of phosphorylated ERK in a time and concentration dependent manner. A portion of the lysates used to measure ERK activation was used to immunoprecipitate the EGFR or δ-OR to determine their degree of tyrosine phosphorylation. After immunoprecipitation of the FLAG-tagged δ-OR or the EGFR with the appropriate antibodies, we found that only DSLET induced tyrosine phosphorylation of the δ-OR, whereas only EGF potentiated EGFR phosphorylation (Figure 4). 1 μM DSLET-induced tyrosine phosphorylation of the δ-OR was inhibited by naltrindole, PP1, but not by AG1478 (Figure 5). These results suggest that transactivation of the EGFR does not play a role in δ-OR phosphorylation or ERK activation by δ-opioids.


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 DSLET or EGF on tyrosine phosphorylation of the δ-OR or the EGFR. HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 – 100 ng/ml for 5 minutes) or DSLET (10 nM-10 μM for 5 minutes). After agonist exposure, cell lysates were prepared and the δ-OR or the EGFR 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 δ-OR (upper panel) or the EGFR (lower panel). The immunoblots are representative of an experiment that was repeated three times.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC88976&req=5

Figure 4: Western blots showing the effect of DSLET or EGF on tyrosine phosphorylation of the δ-OR or the EGFR. HEK-δ-OR cells were serum-starved overnight prior to exposure to EGF (0.1 – 100 ng/ml for 5 minutes) or DSLET (10 nM-10 μM for 5 minutes). After agonist exposure, cell lysates were prepared and the δ-OR or the EGFR 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 δ-OR (upper panel) or the EGFR (lower panel). The immunoblots are representative of an experiment that was repeated three times.
Mentions: Our next experiments were designed to examine whether the EGFR becomes tyrosine phosphorylated during ERK activation by DSLET. As stated in the introduction, several GPCRs (including the EPAR, β2-AR, and substance P (SP-R)) [14,18-20] increase EGFR phosphorylation during the course of ERK activation, and it is believed, that in these instances, this event is an initial step in the progression of the MAPK cascade. Our laboratory has previously reported [11,13] that 5-opioids induce tyrosine phosphorylation of the δ-OR, and it appears that this is catalyzed by the non-receptor tyrosine kinase Src. However, transphosphorylation of the EGFR after OR activation has not been reported. WT-δ-OR-expressing HEK-293 cells were exposed to increasing concentrations of either DSLET (1–1000 nM) or EGF (0.1–100 ng/ml) for five minutes to induce ERK activation. As stated earlier, either DSLET or EGF increased the appearance of phosphorylated ERK in a time and concentration dependent manner. A portion of the lysates used to measure ERK activation was used to immunoprecipitate the EGFR or δ-OR to determine their degree of tyrosine phosphorylation. After immunoprecipitation of the FLAG-tagged δ-OR or the EGFR with the appropriate antibodies, we found that only DSLET induced tyrosine phosphorylation of the δ-OR, whereas only EGF potentiated EGFR phosphorylation (Figure 4). 1 μM DSLET-induced tyrosine phosphorylation of the δ-OR was inhibited by naltrindole, PP1, but not by AG1478 (Figure 5). These results suggest that transactivation of the EGFR does not play a role in δ-OR phosphorylation or ERK activation by δ-opioids.

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