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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 DNA synthesis is not dependent on EGF receptor tyrosine kinase activity in Swiss 3T3 cells. The abilities of thrombin and EGF to induce DNA synthesis of Swiss 3T3 cells in the presence or absence of the EGF receptor kinase inhibitor AG1478 were measured by the incorporation of [3H]thymidine. Cells were left inactive or stimulated with dilutions of thrombin (diluted as indicated from a maximum of 6 U/ml) or EGF (maximum of 60 nM) for 24 h in the presence of [3H]thymidine. Thrombin was a more effective agonist than EGF but EGF was much more sensitive to inhibition by AG1478 (0.5 μM) than thrombin. Results are the mean ± SEM of 8–12 observations from two experiments.
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Figure 1: Thrombin-induced DNA synthesis is not dependent on EGF receptor tyrosine kinase activity in Swiss 3T3 cells. The abilities of thrombin and EGF to induce DNA synthesis of Swiss 3T3 cells in the presence or absence of the EGF receptor kinase inhibitor AG1478 were measured by the incorporation of [3H]thymidine. Cells were left inactive or stimulated with dilutions of thrombin (diluted as indicated from a maximum of 6 U/ml) or EGF (maximum of 60 nM) for 24 h in the presence of [3H]thymidine. Thrombin was a more effective agonist than EGF but EGF was much more sensitive to inhibition by AG1478 (0.5 μM) than thrombin. Results are the mean ± SEM of 8–12 observations from two experiments.

Mentions: Both thrombin and EGF stimulated the synthesis of DNA in Swiss 3T3 cells, but thrombin was a much more effective stimulus than EGF for this response (Fig. 1). To examine the requirement of the EGF receptor tyrosine kinase in each of these responses, cells were preincubated for 1 h with or without the EGF receptor kinase inhibitor AG1478 (0.5 μM). AG1478 completely inhibited DNA synthesis stimulated by EGF. The thrombin-induced response was resistant to inhibition by AG1478, being only partially inhibited.


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 DNA synthesis is not dependent on EGF receptor tyrosine kinase activity in Swiss 3T3 cells. The abilities of thrombin and EGF to induce DNA synthesis of Swiss 3T3 cells in the presence or absence of the EGF receptor kinase inhibitor AG1478 were measured by the incorporation of [3H]thymidine. Cells were left inactive or stimulated with dilutions of thrombin (diluted as indicated from a maximum of 6 U/ml) or EGF (maximum of 60 nM) for 24 h in the presence of [3H]thymidine. Thrombin was a more effective agonist than EGF but EGF was much more sensitive to inhibition by AG1478 (0.5 μM) than thrombin. Results are the mean ± SEM of 8–12 observations from two experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Thrombin-induced DNA synthesis is not dependent on EGF receptor tyrosine kinase activity in Swiss 3T3 cells. The abilities of thrombin and EGF to induce DNA synthesis of Swiss 3T3 cells in the presence or absence of the EGF receptor kinase inhibitor AG1478 were measured by the incorporation of [3H]thymidine. Cells were left inactive or stimulated with dilutions of thrombin (diluted as indicated from a maximum of 6 U/ml) or EGF (maximum of 60 nM) for 24 h in the presence of [3H]thymidine. Thrombin was a more effective agonist than EGF but EGF was much more sensitive to inhibition by AG1478 (0.5 μM) than thrombin. Results are the mean ± SEM of 8–12 observations from two experiments.
Mentions: Both thrombin and EGF stimulated the synthesis of DNA in Swiss 3T3 cells, but thrombin was a much more effective stimulus than EGF for this response (Fig. 1). To examine the requirement of the EGF receptor tyrosine kinase in each of these responses, cells were preincubated for 1 h with or without the EGF receptor kinase inhibitor AG1478 (0.5 μM). AG1478 completely inhibited DNA synthesis stimulated by EGF. The thrombin-induced response was resistant to inhibition by AG1478, being only partially inhibited.

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