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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 does not activate the EGF receptor in Swiss 3T3 cells. Swiss 3T3 cells were left inactive (0) or were stimulated for 5 min with EGF (E5, 10 nM) or thrombin (1 U/ml) for 1 min (T1) or 5 min (T5) in the presence or absence of AG1478 (0.5 μM). Cells were lysed and extracted proteins were immunoprecipitated with (A) EGF receptor antibodies or (B) phosphotyrosine antibodies. Proteins were separated by SDS-PAGE and Western blotted for phosphotyrosine (A) and Shc (B). It can be seen that the EGF receptor was tyrosine phosphorylated when cells were acutely activated with EGF (5 min). Thrombin had no effect on the phosphorylation state of the EGF receptor (A). In all lanes there were equal amounts of EGF receptor immunoprecipitated (not shown). Similarly, EGF brought about the association of 52- and 66-kD forms of Shc with phosphotyrosine immunoprecipitates, but thrombin had little such effect (B). The heavy chain (HC) of the immunoprecipitating antibodies is indicated, as are positions of molecular weight standards (shown in kD) in B. Qualitatively identical results were seen in four separate preparations.
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Figure 2: Thrombin does not activate the EGF receptor in Swiss 3T3 cells. Swiss 3T3 cells were left inactive (0) or were stimulated for 5 min with EGF (E5, 10 nM) or thrombin (1 U/ml) for 1 min (T1) or 5 min (T5) in the presence or absence of AG1478 (0.5 μM). Cells were lysed and extracted proteins were immunoprecipitated with (A) EGF receptor antibodies or (B) phosphotyrosine antibodies. Proteins were separated by SDS-PAGE and Western blotted for phosphotyrosine (A) and Shc (B). It can be seen that the EGF receptor was tyrosine phosphorylated when cells were acutely activated with EGF (5 min). Thrombin had no effect on the phosphorylation state of the EGF receptor (A). In all lanes there were equal amounts of EGF receptor immunoprecipitated (not shown). Similarly, EGF brought about the association of 52- and 66-kD forms of Shc with phosphotyrosine immunoprecipitates, but thrombin had little such effect (B). The heavy chain (HC) of the immunoprecipitating antibodies is indicated, as are positions of molecular weight standards (shown in kD) in B. Qualitatively identical results were seen in four separate preparations.

Mentions: Cells stimulated for 5 min with EGF showed enhanced tyrosine phosphorylation of the EGF receptor, as seen by blotting EGF receptor immunoprecipitates with phosphotyrosine antibodies (Fig. 2 A). This was completely inhibited by preincubation of cells with AG1478 (0.5 μM). Thrombin, in contrast, at either 1 or 5 min of stimulation, had no influence on the tyrosine phosphorylation state of the EGF receptor (Fig. 2 A), or after longer time periods (not shown). Consistent with these observations, EGF and not thrombin induced the association of Shc with phosphotyrosine immunoprecipitates (Fig. 2 B). Both the 66- and 52-kD forms of Shc were found in these immunoprecipitates, and this EGF-induced association was blocked in cells preincubated with AG1478 (Fig. 2 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 does not activate the EGF receptor in Swiss 3T3 cells. Swiss 3T3 cells were left inactive (0) or were stimulated for 5 min with EGF (E5, 10 nM) or thrombin (1 U/ml) for 1 min (T1) or 5 min (T5) in the presence or absence of AG1478 (0.5 μM). Cells were lysed and extracted proteins were immunoprecipitated with (A) EGF receptor antibodies or (B) phosphotyrosine antibodies. Proteins were separated by SDS-PAGE and Western blotted for phosphotyrosine (A) and Shc (B). It can be seen that the EGF receptor was tyrosine phosphorylated when cells were acutely activated with EGF (5 min). Thrombin had no effect on the phosphorylation state of the EGF receptor (A). In all lanes there were equal amounts of EGF receptor immunoprecipitated (not shown). Similarly, EGF brought about the association of 52- and 66-kD forms of Shc with phosphotyrosine immunoprecipitates, but thrombin had little such effect (B). The heavy chain (HC) of the immunoprecipitating antibodies is indicated, as are positions of molecular weight standards (shown in kD) in B. Qualitatively identical results were seen in four separate preparations.
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Related In: Results  -  Collection

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Figure 2: Thrombin does not activate the EGF receptor in Swiss 3T3 cells. Swiss 3T3 cells were left inactive (0) or were stimulated for 5 min with EGF (E5, 10 nM) or thrombin (1 U/ml) for 1 min (T1) or 5 min (T5) in the presence or absence of AG1478 (0.5 μM). Cells were lysed and extracted proteins were immunoprecipitated with (A) EGF receptor antibodies or (B) phosphotyrosine antibodies. Proteins were separated by SDS-PAGE and Western blotted for phosphotyrosine (A) and Shc (B). It can be seen that the EGF receptor was tyrosine phosphorylated when cells were acutely activated with EGF (5 min). Thrombin had no effect on the phosphorylation state of the EGF receptor (A). In all lanes there were equal amounts of EGF receptor immunoprecipitated (not shown). Similarly, EGF brought about the association of 52- and 66-kD forms of Shc with phosphotyrosine immunoprecipitates, but thrombin had little such effect (B). The heavy chain (HC) of the immunoprecipitating antibodies is indicated, as are positions of molecular weight standards (shown in kD) in B. Qualitatively identical results were seen in four separate preparations.
Mentions: Cells stimulated for 5 min with EGF showed enhanced tyrosine phosphorylation of the EGF receptor, as seen by blotting EGF receptor immunoprecipitates with phosphotyrosine antibodies (Fig. 2 A). This was completely inhibited by preincubation of cells with AG1478 (0.5 μM). Thrombin, in contrast, at either 1 or 5 min of stimulation, had no influence on the tyrosine phosphorylation state of the EGF receptor (Fig. 2 A), or after longer time periods (not shown). Consistent with these observations, EGF and not thrombin induced the association of Shc with phosphotyrosine immunoprecipitates (Fig. 2 B). Both the 66- and 52-kD forms of Shc were found in these immunoprecipitates, and this EGF-induced association was blocked in cells preincubated with AG1478 (Fig. 2 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