Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of δ-OR or EGFR down-regulation on ERK phosphorylation by acute exposure to DSLET or EGF. HEK-δ-OR cells were serum-starved overnight prior to exposure to DSLET (10 μM for 24 hours) or EGF (100 ng/ml for 24 hours) to induce receptor down-regulation. After chronic agonist pre-treatment, cell monolayers were washed once with fresh media and exposed, acutely, to either DSLET (1 μM for 5 minutes) or EGF (10 ng/ml for 5 minutes). After acute 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 (hatched bars) 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 and **p ≤ 0.01 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC88976&req=5

Figure 6: Effect of δ-OR or EGFR down-regulation on ERK phosphorylation by acute exposure to DSLET or EGF. HEK-δ-OR cells were serum-starved overnight prior to exposure to DSLET (10 μM for 24 hours) or EGF (100 ng/ml for 24 hours) to induce receptor down-regulation. After chronic agonist pre-treatment, cell monolayers were washed once with fresh media and exposed, acutely, to either DSLET (1 μM for 5 minutes) or EGF (10 ng/ml for 5 minutes). After acute 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 (hatched bars) 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 and **p ≤ 0.01 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.

Mentions: Despite the lack of direct evidence that DSLET induces the transactivation of co-expressed RTKs, it is possible that the EGFR, in some way, contributes to ERK activation by opioids. It is possible that RTKs and GPCRs share important protein intermediates that are required during ERK activation. Therefore, we wished to determine what effects the down-regulation of the EGFR would have on ERK signalling via the δ-OR. HEK-293 cells expressing the WT-δ-OR were exposed to EGF (100 ng/ml) for 24 hours in order to down-regulate and desensitize the endogenous EGFR. After chronic EGF treatment, the cultures were washed with serum-free media and then exposed acutely (5 minutes) to either EGF (10 ng/ml) or DSLET (1 μM) to stimulate ERK phosphorylation. In EGF pre-treated cultures, acute EGF (10 ng/ml) was unable to increase the expression of phospho-ERKs compared to media-exposed controls (Figure 6). This lack of response appears to be due to the down-regulation of EGFRs from these cells as confirmed by immunoblots using the anti-EGFR antibody (data not shown). However, DSLET-mediated ERK activation was unaltered in identically pretreated cultures.


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 or EGFR down-regulation on ERK phosphorylation by acute exposure to DSLET or EGF. HEK-δ-OR cells were serum-starved overnight prior to exposure to DSLET (10 μM for 24 hours) or EGF (100 ng/ml for 24 hours) to induce receptor down-regulation. After chronic agonist pre-treatment, cell monolayers were washed once with fresh media and exposed, acutely, to either DSLET (1 μM for 5 minutes) or EGF (10 ng/ml for 5 minutes). After acute 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 (hatched bars) 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 and **p ≤ 0.01 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC88976&req=5

Figure 6: Effect of δ-OR or EGFR down-regulation on ERK phosphorylation by acute exposure to DSLET or EGF. HEK-δ-OR cells were serum-starved overnight prior to exposure to DSLET (10 μM for 24 hours) or EGF (100 ng/ml for 24 hours) to induce receptor down-regulation. After chronic agonist pre-treatment, cell monolayers were washed once with fresh media and exposed, acutely, to either DSLET (1 μM for 5 minutes) or EGF (10 ng/ml for 5 minutes). After acute 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 (hatched bars) 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 and **p ≤ 0.01 compared to untreated samples as determined by ANOVA and the Tukey post-hoc test.
Mentions: Despite the lack of direct evidence that DSLET induces the transactivation of co-expressed RTKs, it is possible that the EGFR, in some way, contributes to ERK activation by opioids. It is possible that RTKs and GPCRs share important protein intermediates that are required during ERK activation. Therefore, we wished to determine what effects the down-regulation of the EGFR would have on ERK signalling via the δ-OR. HEK-293 cells expressing the WT-δ-OR were exposed to EGF (100 ng/ml) for 24 hours in order to down-regulate and desensitize the endogenous EGFR. After chronic EGF treatment, the cultures were washed with serum-free media and then exposed acutely (5 minutes) to either EGF (10 ng/ml) or DSLET (1 μM) to stimulate ERK phosphorylation. In EGF pre-treated cultures, acute EGF (10 ng/ml) was unable to increase the expression of phospho-ERKs compared to media-exposed controls (Figure 6). This lack of response appears to be due to the down-regulation of EGFRs from these cells as confirmed by immunoblots using the anti-EGFR antibody (data not shown). However, DSLET-mediated ERK activation was unaltered in identically pretreated cultures.

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