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Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Swindle CS, Tran KT, Johnson TD, Banerjee P, Mayes AM, Griffith L, Wells A - J. Cell Biol. (2001)

Bottom Line: Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat.Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded.These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

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Inhibition of EGFR activation prevents signaling from EGF-like repeats. (A) WT NR6 cells were treated with the tenascin EGF–like repeats at concentrations that activate ERK MAP kinase (5 uM for 1/2; 1 uM for 11/12/13; and 6 uM for 14). The cells were treated with the EGF-like repeat proteins in the absence (−) or presence (+) of the EGFR-specific pharmacologic inhibitor PD153035. no tx represents no ligand. (B) WT NR6 cells were treated with Ten14 (1 uM) or EGF (0.01 nM) in the presence (+) or absence (−) of an antibody specific for the extracellular domain of EGFR (Clone 528; Calbiochem) under serum-free conditions. (C) B82 cells expressing WT EGFR were challenged with tenascin 14 repeats (2 uM) under serum-free conditions. Numbers below a lane represent relative values of intensity for p42 in each lane for that experiment as determined by densitometry. Shown are one of three experiments.
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fig4: Inhibition of EGFR activation prevents signaling from EGF-like repeats. (A) WT NR6 cells were treated with the tenascin EGF–like repeats at concentrations that activate ERK MAP kinase (5 uM for 1/2; 1 uM for 11/12/13; and 6 uM for 14). The cells were treated with the EGF-like repeat proteins in the absence (−) or presence (+) of the EGFR-specific pharmacologic inhibitor PD153035. no tx represents no ligand. (B) WT NR6 cells were treated with Ten14 (1 uM) or EGF (0.01 nM) in the presence (+) or absence (−) of an antibody specific for the extracellular domain of EGFR (Clone 528; Calbiochem) under serum-free conditions. (C) B82 cells expressing WT EGFR were challenged with tenascin 14 repeats (2 uM) under serum-free conditions. Numbers below a lane represent relative values of intensity for p42 in each lane for that experiment as determined by densitometry. Shown are one of three experiments.

Mentions: That EGFR kinase activity is required for downstream cell responses was corroborated directly by inhibiting EGFR using the pharmacological agent PD153035. This selective inhibitor of EGFR kinase blocked ERK MAP kinase activation by the tenascin EGF–like repeats (Fig. 4 A).


Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor.

Swindle CS, Tran KT, Johnson TD, Banerjee P, Mayes AM, Griffith L, Wells A - J. Cell Biol. (2001)

Inhibition of EGFR activation prevents signaling from EGF-like repeats. (A) WT NR6 cells were treated with the tenascin EGF–like repeats at concentrations that activate ERK MAP kinase (5 uM for 1/2; 1 uM for 11/12/13; and 6 uM for 14). The cells were treated with the EGF-like repeat proteins in the absence (−) or presence (+) of the EGFR-specific pharmacologic inhibitor PD153035. no tx represents no ligand. (B) WT NR6 cells were treated with Ten14 (1 uM) or EGF (0.01 nM) in the presence (+) or absence (−) of an antibody specific for the extracellular domain of EGFR (Clone 528; Calbiochem) under serum-free conditions. (C) B82 cells expressing WT EGFR were challenged with tenascin 14 repeats (2 uM) under serum-free conditions. Numbers below a lane represent relative values of intensity for p42 in each lane for that experiment as determined by densitometry. Shown are one of three experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150768&req=5

fig4: Inhibition of EGFR activation prevents signaling from EGF-like repeats. (A) WT NR6 cells were treated with the tenascin EGF–like repeats at concentrations that activate ERK MAP kinase (5 uM for 1/2; 1 uM for 11/12/13; and 6 uM for 14). The cells were treated with the EGF-like repeat proteins in the absence (−) or presence (+) of the EGFR-specific pharmacologic inhibitor PD153035. no tx represents no ligand. (B) WT NR6 cells were treated with Ten14 (1 uM) or EGF (0.01 nM) in the presence (+) or absence (−) of an antibody specific for the extracellular domain of EGFR (Clone 528; Calbiochem) under serum-free conditions. (C) B82 cells expressing WT EGFR were challenged with tenascin 14 repeats (2 uM) under serum-free conditions. Numbers below a lane represent relative values of intensity for p42 in each lane for that experiment as determined by densitometry. Shown are one of three experiments.
Mentions: That EGFR kinase activity is required for downstream cell responses was corroborated directly by inhibiting EGFR using the pharmacological agent PD153035. This selective inhibitor of EGFR kinase blocked ERK MAP kinase activation by the tenascin EGF–like repeats (Fig. 4 A).

Bottom Line: Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat.Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded.These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.

Show MeSH
Related in: MedlinePlus