Limits...
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.

Show MeSH

Related in: MedlinePlus

EGF receptors at the leading edge of thrombin-stimulated cells are responsive to EGF. Swiss 3T3 cells were grown to confluency, serum deprived overnight, and the monolayer was “wounded” with a 1-mm-wide blunt scraper. Cells were reactivated with thrombin (Thr, 1 U/ml) for 4 h and then fixed or were subsequently activated for 5 min with EGF (10 nM) and then fixed. Cells were processed for immunohistochemistry and then incubated with EGF receptor antibodies (EGFR) and phosphotyrosine antibodies (P-Y). As seen previously, thrombin induced the accumulation of EGF receptors at the leading edge of cells beginning migration and that there was only a small amount of tyrosine phosphorylation associated with the actin arc (top right). However, activation of these cells for 5 min with EGF induced a large increase in tyrosine phosphorylation both at the actin arc and at focal adhesions (bottom right).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199612&req=5

Figure 7: EGF receptors at the leading edge of thrombin-stimulated cells are responsive to EGF. Swiss 3T3 cells were grown to confluency, serum deprived overnight, and the monolayer was “wounded” with a 1-mm-wide blunt scraper. Cells were reactivated with thrombin (Thr, 1 U/ml) for 4 h and then fixed or were subsequently activated for 5 min with EGF (10 nM) and then fixed. Cells were processed for immunohistochemistry and then incubated with EGF receptor antibodies (EGFR) and phosphotyrosine antibodies (P-Y). As seen previously, thrombin induced the accumulation of EGF receptors at the leading edge of cells beginning migration and that there was only a small amount of tyrosine phosphorylation associated with the actin arc (top right). However, activation of these cells for 5 min with EGF induced a large increase in tyrosine phosphorylation both at the actin arc and at focal adhesions (bottom right).

Mentions: To examine the functionality of aggregated EGF receptors, migration of Swiss 3T3 cells was determined in cells with or without prior stimulation with thrombin for 7 h, after which cells were left with no further addition or were activated secondarily for 5 min with EGF. Staining for the EGF receptor revealed the characteristic accumulation of EGF receptors at the leading edge of cells activated by thrombin (Fig. 7). In the absence of further additions, there was only a relatively small amount of tyrosine phosphorylation of proteins at the actin arc (Fig. 7). However, cells treated additionally for 5 min with EGF showed a marked tyrosine phosphorylation not only at the actin arc but also at focal adhesions (Fig. 7). As we could not observe EGF receptors at focal adhesions, this indicates that signaling has occurred from the actin arc and potentially other EGF receptors so as to induce tyrosine phosphorylation some distance from the site of receptor activation. The results strongly suggest that EGF receptors aggregated at the actin arc by thrombin are functional.


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)

EGF receptors at the leading edge of thrombin-stimulated cells are responsive to EGF. Swiss 3T3 cells were grown to confluency, serum deprived overnight, and the monolayer was “wounded” with a 1-mm-wide blunt scraper. Cells were reactivated with thrombin (Thr, 1 U/ml) for 4 h and then fixed or were subsequently activated for 5 min with EGF (10 nM) and then fixed. Cells were processed for immunohistochemistry and then incubated with EGF receptor antibodies (EGFR) and phosphotyrosine antibodies (P-Y). As seen previously, thrombin induced the accumulation of EGF receptors at the leading edge of cells beginning migration and that there was only a small amount of tyrosine phosphorylation associated with the actin arc (top right). However, activation of these cells for 5 min with EGF induced a large increase in tyrosine phosphorylation both at the actin arc and at focal adhesions (bottom right).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: EGF receptors at the leading edge of thrombin-stimulated cells are responsive to EGF. Swiss 3T3 cells were grown to confluency, serum deprived overnight, and the monolayer was “wounded” with a 1-mm-wide blunt scraper. Cells were reactivated with thrombin (Thr, 1 U/ml) for 4 h and then fixed or were subsequently activated for 5 min with EGF (10 nM) and then fixed. Cells were processed for immunohistochemistry and then incubated with EGF receptor antibodies (EGFR) and phosphotyrosine antibodies (P-Y). As seen previously, thrombin induced the accumulation of EGF receptors at the leading edge of cells beginning migration and that there was only a small amount of tyrosine phosphorylation associated with the actin arc (top right). However, activation of these cells for 5 min with EGF induced a large increase in tyrosine phosphorylation both at the actin arc and at focal adhesions (bottom right).
Mentions: To examine the functionality of aggregated EGF receptors, migration of Swiss 3T3 cells was determined in cells with or without prior stimulation with thrombin for 7 h, after which cells were left with no further addition or were activated secondarily for 5 min with EGF. Staining for the EGF receptor revealed the characteristic accumulation of EGF receptors at the leading edge of cells activated by thrombin (Fig. 7). In the absence of further additions, there was only a relatively small amount of tyrosine phosphorylation of proteins at the actin arc (Fig. 7). However, cells treated additionally for 5 min with EGF showed a marked tyrosine phosphorylation not only at the actin arc but also at focal adhesions (Fig. 7). As we could not observe EGF receptors at focal adhesions, this indicates that signaling has occurred from the actin arc and potentially other EGF receptors so as to induce tyrosine phosphorylation some distance from the site of receptor activation. The results strongly suggest that EGF receptors aggregated at the actin arc by thrombin are functional.

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