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Activation of endogenous thrombin receptors causes clustering and sensitization of epidermal growth factor receptors of swiss 3T3 cells without transactivation.

Crouch MF, Davy DA, Willard FS, Berven LA - J. Cell Biol. (2001)

Bottom Line: Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors.Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation.Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Signaling Group, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia. michael.crouch@anu.edu.au

ABSTRACT
The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cgamma1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.

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Thrombin-induced MAP kinase activation is resistant to inhibition by AG1478. Swiss 3T3 cells were activated with (A) thrombin (T, 1 U/ml) or (B) EGF (E, 10 nM) for 1, 2, 5 or 15 min, as indicated, in the presence or absence of AG1478 (0.5 μM). Cells were lysed with SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE, Western blotted, and probed with phospho-specific MAP kinase (MAPK) antibodies. It was found that EGF was a much more potent agonist for MAP kinase activation than thrombin, and that the EGF response was abolished by AG1478 treatment. While a weaker agonist, the thrombin stimulation of MAP kinase activation was only partially inhibited by AG1478. The time courses of activation of MAPK were different for thrombin and EGF. Whereas thrombin desensitized after 15 min (A), the response to EGF was maintained at this time point (B). The positions of 42- and 44-kD forms of MAP kinases are shown. The same effects were observed in two separate cell preparations.
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Figure 3: Thrombin-induced MAP kinase activation is resistant to inhibition by AG1478. Swiss 3T3 cells were activated with (A) thrombin (T, 1 U/ml) or (B) EGF (E, 10 nM) for 1, 2, 5 or 15 min, as indicated, in the presence or absence of AG1478 (0.5 μM). Cells were lysed with SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE, Western blotted, and probed with phospho-specific MAP kinase (MAPK) antibodies. It was found that EGF was a much more potent agonist for MAP kinase activation than thrombin, and that the EGF response was abolished by AG1478 treatment. While a weaker agonist, the thrombin stimulation of MAP kinase activation was only partially inhibited by AG1478. The time courses of activation of MAPK were different for thrombin and EGF. Whereas thrombin desensitized after 15 min (A), the response to EGF was maintained at this time point (B). The positions of 42- and 44-kD forms of MAP kinases are shown. The same effects were observed in two separate cell preparations.

Mentions: We have examined the effect of thrombin and EGF on the MAP kinase pathway by the use of phospho-MAP kinase (42- and 44-kD MAP kinase) antibodies. Stimulation of cells for 5 min with EGF induced the phosphorylation of both the MAP kinases extracellular signal–regulated kinase (ERK)1 and ERK2 (Fig. 3 A). This was inhibited by AG1478 (0.5 μM) pretreatment of cells. Thrombin was a weaker stimulus of activation of phosphorylation of either kinase and this stimulation was only partially inhibited by AG1478 (Fig. 3 A). The time courses of activation of MAP kinase were also different for thrombin and EGF. While thrombin stimulation of MAP kinase was fully desensitized after 15 min of activation, the EGF MAP kinase response was sustained at this time point (Fig. 3 B).


Activation of endogenous thrombin receptors causes clustering and sensitization of epidermal growth factor receptors of swiss 3T3 cells without transactivation.

Crouch MF, Davy DA, Willard FS, Berven LA - J. Cell Biol. (2001)

Thrombin-induced MAP kinase activation is resistant to inhibition by AG1478. Swiss 3T3 cells were activated with (A) thrombin (T, 1 U/ml) or (B) EGF (E, 10 nM) for 1, 2, 5 or 15 min, as indicated, in the presence or absence of AG1478 (0.5 μM). Cells were lysed with SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE, Western blotted, and probed with phospho-specific MAP kinase (MAPK) antibodies. It was found that EGF was a much more potent agonist for MAP kinase activation than thrombin, and that the EGF response was abolished by AG1478 treatment. While a weaker agonist, the thrombin stimulation of MAP kinase activation was only partially inhibited by AG1478. The time courses of activation of MAPK were different for thrombin and EGF. Whereas thrombin desensitized after 15 min (A), the response to EGF was maintained at this time point (B). The positions of 42- and 44-kD forms of MAP kinases are shown. The same effects were observed in two separate cell preparations.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199612&req=5

Figure 3: Thrombin-induced MAP kinase activation is resistant to inhibition by AG1478. Swiss 3T3 cells were activated with (A) thrombin (T, 1 U/ml) or (B) EGF (E, 10 nM) for 1, 2, 5 or 15 min, as indicated, in the presence or absence of AG1478 (0.5 μM). Cells were lysed with SDS-PAGE sample buffer, and proteins were separated by SDS-PAGE, Western blotted, and probed with phospho-specific MAP kinase (MAPK) antibodies. It was found that EGF was a much more potent agonist for MAP kinase activation than thrombin, and that the EGF response was abolished by AG1478 treatment. While a weaker agonist, the thrombin stimulation of MAP kinase activation was only partially inhibited by AG1478. The time courses of activation of MAPK were different for thrombin and EGF. Whereas thrombin desensitized after 15 min (A), the response to EGF was maintained at this time point (B). The positions of 42- and 44-kD forms of MAP kinases are shown. The same effects were observed in two separate cell preparations.
Mentions: We have examined the effect of thrombin and EGF on the MAP kinase pathway by the use of phospho-MAP kinase (42- and 44-kD MAP kinase) antibodies. Stimulation of cells for 5 min with EGF induced the phosphorylation of both the MAP kinases extracellular signal–regulated kinase (ERK)1 and ERK2 (Fig. 3 A). This was inhibited by AG1478 (0.5 μM) pretreatment of cells. Thrombin was a weaker stimulus of activation of phosphorylation of either kinase and this stimulation was only partially inhibited by AG1478 (Fig. 3 A). The time courses of activation of MAP kinase were also different for thrombin and EGF. While thrombin stimulation of MAP kinase was fully desensitized after 15 min of activation, the EGF MAP kinase response was sustained at this time point (Fig. 3 B).

Bottom Line: Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors.Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation.Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Signaling Group, John Curtin School of Medical Research, Australian National University, Canberra, A.C.T. 2601, Australia. michael.crouch@anu.edu.au

ABSTRACT
The G protein-coupled thrombin receptor can induce cellular responses in some systems by transactivating the epidermal growth factor (EGF) receptor. This is in part due to the stimulation of ectoproteases that generate EGF receptor ligands. We show here that this cannot account for the stimulation of proliferation or migration by thrombin of Swiss 3T3 cells. Thrombin has no direct effect on the activation state of the EGF receptor or of its downstream effectors. However, thrombin induces the subcellular clustering of the EGF receptor at filamentous actin-containing structures at the leading edge and actin arcs of migrating cells in association with other signaling molecules, including Shc and phospholipase Cgamma1. In these thrombin-primed cells, the subsequent migratory response to EGF is potentiated. Thrombin did not potentiate the EGF-stimulated EGF receptor phosphorylation. Thus, in Swiss 3T3 cells the G protein-coupled thrombin receptor can potentiate the EGF tyrosine kinase receptor response when activated by EGF, and this appears to be due to the subcellular concentration of the receptor with downstream effectors and not to the overall ability of EGF to induce receptor transphosphorylation. Thus, the EGF receptor subcellular localization which is altered by thrombin appears to be an important determinant of the efficacy of downstream EGF receptor signaling in cell migration.

Show MeSH
Related in: MedlinePlus