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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 induces the clustering of EGF receptors but not associated tyrosine phosphorylation. Swiss 3T3 cells were activated with thrombin for 5 h and then fixed and incubated with antibodies to the EGF receptor (EGFR) and/or phosphotyrosine (P-Y). Cells were then incubated with secondary fluorescent antibodies and visualized by confocal microscopy. A and B show the same cell which was double-labeled with P-Y and EGF receptor antibodies. It can be seen that the EGF receptor localized to ribbed structures at the anterior of the migrating cell, but that there was little tyrosine phosphorylation associated with the EGF receptor. C shows that in unstimulated cells there is little polarized accumulation of EGF receptor. Cells treated with AG1478 (D) still showed EGF receptor accumulation on actin arcs in thrombin-stimulated cells.
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Figure 4: Thrombin induces the clustering of EGF receptors but not associated tyrosine phosphorylation. Swiss 3T3 cells were activated with thrombin for 5 h and then fixed and incubated with antibodies to the EGF receptor (EGFR) and/or phosphotyrosine (P-Y). Cells were then incubated with secondary fluorescent antibodies and visualized by confocal microscopy. A and B show the same cell which was double-labeled with P-Y and EGF receptor antibodies. It can be seen that the EGF receptor localized to ribbed structures at the anterior of the migrating cell, but that there was little tyrosine phosphorylation associated with the EGF receptor. C shows that in unstimulated cells there is little polarized accumulation of EGF receptor. Cells treated with AG1478 (D) still showed EGF receptor accumulation on actin arcs in thrombin-stimulated cells.

Mentions: Confocal microscopy of cells labeled with antibodies to the EGF receptor showed a differential distribution of the receptor depending on activation status of subconfluent migrating cells. Migrating cells were identified by their characteristic fan shape and polarized distribution of F-actin. In unstimulated cells there was little enrichment of EGF receptor staining within nonnuclear regions of the cell (Fig. 4 C). In contrast, cells activated with thrombin for 5 h and induced to migrate showed EGF receptor immunoreactivity toward the leading edge and on structures similar in appearance to the actin arc (Fig. 4 B). Double labeling of these cells with phosphotyrosine antibodies showed there to be only weak tyrosine phosphorylation coincident with the EGF receptor (Fig. 4 A), with the predominant phosphotyrosine staining on punctate structures (Fig. 4 A) that were coincident with the localization of focal adhesion kinase (FAK, not shown; see Fig. 6). This thrombin-stimulated clustering of EGF receptors was not inhibited by AG1478 (Fig. 4 D), showing a lack of requirement for EGF receptor kinase activity. This distribution of the EGF receptor was only seen in nonconfluent cells, and not in nonmigrating, contact-inhibited cells.


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 induces the clustering of EGF receptors but not associated tyrosine phosphorylation. Swiss 3T3 cells were activated with thrombin for 5 h and then fixed and incubated with antibodies to the EGF receptor (EGFR) and/or phosphotyrosine (P-Y). Cells were then incubated with secondary fluorescent antibodies and visualized by confocal microscopy. A and B show the same cell which was double-labeled with P-Y and EGF receptor antibodies. It can be seen that the EGF receptor localized to ribbed structures at the anterior of the migrating cell, but that there was little tyrosine phosphorylation associated with the EGF receptor. C shows that in unstimulated cells there is little polarized accumulation of EGF receptor. Cells treated with AG1478 (D) still showed EGF receptor accumulation on actin arcs in thrombin-stimulated cells.
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Related In: Results  -  Collection

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Figure 4: Thrombin induces the clustering of EGF receptors but not associated tyrosine phosphorylation. Swiss 3T3 cells were activated with thrombin for 5 h and then fixed and incubated with antibodies to the EGF receptor (EGFR) and/or phosphotyrosine (P-Y). Cells were then incubated with secondary fluorescent antibodies and visualized by confocal microscopy. A and B show the same cell which was double-labeled with P-Y and EGF receptor antibodies. It can be seen that the EGF receptor localized to ribbed structures at the anterior of the migrating cell, but that there was little tyrosine phosphorylation associated with the EGF receptor. C shows that in unstimulated cells there is little polarized accumulation of EGF receptor. Cells treated with AG1478 (D) still showed EGF receptor accumulation on actin arcs in thrombin-stimulated cells.
Mentions: Confocal microscopy of cells labeled with antibodies to the EGF receptor showed a differential distribution of the receptor depending on activation status of subconfluent migrating cells. Migrating cells were identified by their characteristic fan shape and polarized distribution of F-actin. In unstimulated cells there was little enrichment of EGF receptor staining within nonnuclear regions of the cell (Fig. 4 C). In contrast, cells activated with thrombin for 5 h and induced to migrate showed EGF receptor immunoreactivity toward the leading edge and on structures similar in appearance to the actin arc (Fig. 4 B). Double labeling of these cells with phosphotyrosine antibodies showed there to be only weak tyrosine phosphorylation coincident with the EGF receptor (Fig. 4 A), with the predominant phosphotyrosine staining on punctate structures (Fig. 4 A) that were coincident with the localization of focal adhesion kinase (FAK, not shown; see Fig. 6). This thrombin-stimulated clustering of EGF receptors was not inhibited by AG1478 (Fig. 4 D), showing a lack of requirement for EGF receptor kinase activity. This distribution of the EGF receptor was only seen in nonconfluent cells, and not in nonmigrating, contact-inhibited cells.

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