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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 stimulates the localization of EGF receptors to the actin arc in migrating Swiss 3T3 cells. Subconfluent Swiss 3T3 cells were serum starved overnight and then reactivated for 5 h with thrombin (1 U/ml). Cells were fixed and incubated with antibodies to the EGF receptor (EGFR) followed by secondary FITC-labeled antibodies and Texas red-X–labeled phalloidin to show F-actin structures. Cells were then visualized by confocal microscopy. The top panels show that the EGF receptor colocalizes with the phalloidin-stained actin arc on the dorsal aspect of the cell. The bottom panels show that this EGF receptor staining is maintained ventrally on the actin arc but that there is little staining of actin stress fibers. Such association of EGF receptors with the actin arc was seen in all thrombin-activated migrating cells examined.
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Figure 5: Thrombin stimulates the localization of EGF receptors to the actin arc in migrating Swiss 3T3 cells. Subconfluent Swiss 3T3 cells were serum starved overnight and then reactivated for 5 h with thrombin (1 U/ml). Cells were fixed and incubated with antibodies to the EGF receptor (EGFR) followed by secondary FITC-labeled antibodies and Texas red-X–labeled phalloidin to show F-actin structures. Cells were then visualized by confocal microscopy. The top panels show that the EGF receptor colocalizes with the phalloidin-stained actin arc on the dorsal aspect of the cell. The bottom panels show that this EGF receptor staining is maintained ventrally on the actin arc but that there is little staining of actin stress fibers. Such association of EGF receptors with the actin arc was seen in all thrombin-activated migrating cells examined.

Mentions: To confirm the site of accumulation of the EGF receptor in thrombin-stimulated cells as the actin arc, cells were double-labeled with EGF receptor antibodies and with fluorescent phalloidin to label F-actin (Fig. 5). It can be clearly seen that staining with these two agents was coincident on the actin arc. Both dorsal and ventral views of a representative cell are shown. There is enrichment of EGF receptor dorsally on the actin arc, less staining ventrally, and only weak staining on stress fibers. Thus, there is a degree of selectivity with regard to the actin-based structures with which the EGF receptor associates.


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 stimulates the localization of EGF receptors to the actin arc in migrating Swiss 3T3 cells. Subconfluent Swiss 3T3 cells were serum starved overnight and then reactivated for 5 h with thrombin (1 U/ml). Cells were fixed and incubated with antibodies to the EGF receptor (EGFR) followed by secondary FITC-labeled antibodies and Texas red-X–labeled phalloidin to show F-actin structures. Cells were then visualized by confocal microscopy. The top panels show that the EGF receptor colocalizes with the phalloidin-stained actin arc on the dorsal aspect of the cell. The bottom panels show that this EGF receptor staining is maintained ventrally on the actin arc but that there is little staining of actin stress fibers. Such association of EGF receptors with the actin arc was seen in all thrombin-activated migrating cells examined.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Thrombin stimulates the localization of EGF receptors to the actin arc in migrating Swiss 3T3 cells. Subconfluent Swiss 3T3 cells were serum starved overnight and then reactivated for 5 h with thrombin (1 U/ml). Cells were fixed and incubated with antibodies to the EGF receptor (EGFR) followed by secondary FITC-labeled antibodies and Texas red-X–labeled phalloidin to show F-actin structures. Cells were then visualized by confocal microscopy. The top panels show that the EGF receptor colocalizes with the phalloidin-stained actin arc on the dorsal aspect of the cell. The bottom panels show that this EGF receptor staining is maintained ventrally on the actin arc but that there is little staining of actin stress fibers. Such association of EGF receptors with the actin arc was seen in all thrombin-activated migrating cells examined.
Mentions: To confirm the site of accumulation of the EGF receptor in thrombin-stimulated cells as the actin arc, cells were double-labeled with EGF receptor antibodies and with fluorescent phalloidin to label F-actin (Fig. 5). It can be clearly seen that staining with these two agents was coincident on the actin arc. Both dorsal and ventral views of a representative cell are shown. There is enrichment of EGF receptor dorsally on the actin arc, less staining ventrally, and only weak staining on stress fibers. Thus, there is a degree of selectivity with regard to the actin-based structures with which the EGF receptor associates.

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