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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 migration of Swiss 3T3 cells does not require the EGF receptor but potentiates EGF receptor action. (A) The effect of the EGF receptor kinase inhibitor (AG1478) on thrombin- and EGF-induced cell migration was examined in a transwell migration assay. Cells were labeled with the fluorescent indicator CFSE and placed in an 8-μm transwell insert in the presence or absence of AG1478 (0.5 μM). The insert was then placed in a 24-well plate with DME alone or in wells to which was added thrombin (2.5 U/ml) or EGF (25 nM). Fluorescence measurements to assess migration of CFSE-labeled cells through the transwell membrane were then carried out at the intervals shown. The results are means of duplicate assays from a single experiment, and are representative of the same effect seen in three separate preparations. (B) Cell migration in response to EGF and thrombin was assessed as described above, except that these agonists were added alone or in combination either in the upper transwell insert chamber or in the lower chamber of the 24-well plate. It can be seen that the presence of thrombin in the upper chamber potentiated the migratory response to EGF in the lower chamber. This was highly selective, as other combinations of agonist addition did not have this effect. The results are the mean ± SEM of three samples from a single experiment and are representative of the same qualitative effect seen in two such experiments.
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Figure 8: Thrombin-induced migration of Swiss 3T3 cells does not require the EGF receptor but potentiates EGF receptor action. (A) The effect of the EGF receptor kinase inhibitor (AG1478) on thrombin- and EGF-induced cell migration was examined in a transwell migration assay. Cells were labeled with the fluorescent indicator CFSE and placed in an 8-μm transwell insert in the presence or absence of AG1478 (0.5 μM). The insert was then placed in a 24-well plate with DME alone or in wells to which was added thrombin (2.5 U/ml) or EGF (25 nM). Fluorescence measurements to assess migration of CFSE-labeled cells through the transwell membrane were then carried out at the intervals shown. The results are means of duplicate assays from a single experiment, and are representative of the same effect seen in three separate preparations. (B) Cell migration in response to EGF and thrombin was assessed as described above, except that these agonists were added alone or in combination either in the upper transwell insert chamber or in the lower chamber of the 24-well plate. It can be seen that the presence of thrombin in the upper chamber potentiated the migratory response to EGF in the lower chamber. This was highly selective, as other combinations of agonist addition did not have this effect. The results are the mean ± SEM of three samples from a single experiment and are representative of the same qualitative effect seen in two such experiments.

Mentions: To see if cell migration, as opposed to cell proliferation, stimulated by thrombin may require EGF receptor activity, we have carried out a cell migration assay of cells stimulated by thrombin or EGF in the presence and absence of AG1478 (Fig. 8 A). Both thrombin and EGF induced cell migration when added to the lower chamber of a transwell plate. Unstimulated cells showed little migration. Cells activated with EGF that were pretreated with AG1478 displayed a blockade of migration to levels of unstimulated cells. In contrast, there was no inhibition of migration of thrombin-stimulated cells (Fig. 8 A). Thus, the tyrosine kinase activity of EGF receptors is not required for thrombin-stimulated cell migration.


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 migration of Swiss 3T3 cells does not require the EGF receptor but potentiates EGF receptor action. (A) The effect of the EGF receptor kinase inhibitor (AG1478) on thrombin- and EGF-induced cell migration was examined in a transwell migration assay. Cells were labeled with the fluorescent indicator CFSE and placed in an 8-μm transwell insert in the presence or absence of AG1478 (0.5 μM). The insert was then placed in a 24-well plate with DME alone or in wells to which was added thrombin (2.5 U/ml) or EGF (25 nM). Fluorescence measurements to assess migration of CFSE-labeled cells through the transwell membrane were then carried out at the intervals shown. The results are means of duplicate assays from a single experiment, and are representative of the same effect seen in three separate preparations. (B) Cell migration in response to EGF and thrombin was assessed as described above, except that these agonists were added alone or in combination either in the upper transwell insert chamber or in the lower chamber of the 24-well plate. It can be seen that the presence of thrombin in the upper chamber potentiated the migratory response to EGF in the lower chamber. This was highly selective, as other combinations of agonist addition did not have this effect. The results are the mean ± SEM of three samples from a single experiment and are representative of the same qualitative effect seen in two such experiments.
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Related In: Results  -  Collection

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Figure 8: Thrombin-induced migration of Swiss 3T3 cells does not require the EGF receptor but potentiates EGF receptor action. (A) The effect of the EGF receptor kinase inhibitor (AG1478) on thrombin- and EGF-induced cell migration was examined in a transwell migration assay. Cells were labeled with the fluorescent indicator CFSE and placed in an 8-μm transwell insert in the presence or absence of AG1478 (0.5 μM). The insert was then placed in a 24-well plate with DME alone or in wells to which was added thrombin (2.5 U/ml) or EGF (25 nM). Fluorescence measurements to assess migration of CFSE-labeled cells through the transwell membrane were then carried out at the intervals shown. The results are means of duplicate assays from a single experiment, and are representative of the same effect seen in three separate preparations. (B) Cell migration in response to EGF and thrombin was assessed as described above, except that these agonists were added alone or in combination either in the upper transwell insert chamber or in the lower chamber of the 24-well plate. It can be seen that the presence of thrombin in the upper chamber potentiated the migratory response to EGF in the lower chamber. This was highly selective, as other combinations of agonist addition did not have this effect. The results are the mean ± SEM of three samples from a single experiment and are representative of the same qualitative effect seen in two such experiments.
Mentions: To see if cell migration, as opposed to cell proliferation, stimulated by thrombin may require EGF receptor activity, we have carried out a cell migration assay of cells stimulated by thrombin or EGF in the presence and absence of AG1478 (Fig. 8 A). Both thrombin and EGF induced cell migration when added to the lower chamber of a transwell plate. Unstimulated cells showed little migration. Cells activated with EGF that were pretreated with AG1478 displayed a blockade of migration to levels of unstimulated cells. In contrast, there was no inhibition of migration of thrombin-stimulated cells (Fig. 8 A). Thus, the tyrosine kinase activity of EGF receptors is not required for thrombin-stimulated cell migration.

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