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Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.

Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Khokha R, Quaranta V - J. Cell Biol. (2003)

Bottom Line: Therefore, the elucidation of their identities and functions is of great interest.Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5.Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. sschenk@scripps

ABSTRACT
Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

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Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I125-EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125-EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I125-EGF was determined by calculating the specific binding of I125EGF (“specific”) based on total and nonspecific binding of I125-EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I125-EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
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fig4: Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I125-EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125-EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I125-EGF was determined by calculating the specific binding of I125EGF (“specific”) based on total and nonspecific binding of I125-EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I125-EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).

Mentions: To test the specificity of rDIII binding to EGFR, MDA-MB-231 cells were incubated with rDIII in the presence of increasing concentrations of mouse EGF, and analyzed by flow cytometry. As shown in Fig. 4 A, EGF decreased the fluorescence signal of bound rDIII in a dose-dependent fashion. This competitive displacement of rDIII by EGF strongly supports the specificity of rDIII binding to EGFR.


Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution.

Schenk S, Hintermann E, Bilban M, Koshikawa N, Hojilla C, Khokha R, Quaranta V - J. Cell Biol. (2003)

Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I125-EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125-EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I125-EGF was determined by calculating the specific binding of I125EGF (“specific”) based on total and nonspecific binding of I125-EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I125-EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
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fig4: Competitive binding of rDIII and EGF. (A) Flow cytometry. MDA-MB-231 cells were incubated with 2.00 μM rDIII, in the presence of increasing concentrations of EGF (0.45, 0.85, 1.25, and 2.00 μM). rDIII binding to the cell surface was detected with anti-His tag and Alexa® 488 antibodies. The fluorescence signal for rDIII gradually decreases with increasing EGF concentrations. (B) Displacement of cell surface-bound I125-EGF by rDIII. MDA-MB-231 cells were incubated with I0.5 nM 125-EGF and increasing concentrations of cold rDIII (top) or EGF (bottom). The 0.5 nM (≈0.15 μCi) working concentration of I125-EGF was determined by calculating the specific binding of I125EGF (“specific”) based on total and nonspecific binding of I125-EGF to MDA-MB-231 cells (inset in bottom panel). Cells were incubated with increasing concentrations of I125-EGF in the absence (total binding; “total”) or presence of an excess amount (330 nM) of unlabeled EGF (nonspecific binding; “nonspecific”).
Mentions: To test the specificity of rDIII binding to EGFR, MDA-MB-231 cells were incubated with rDIII in the presence of increasing concentrations of mouse EGF, and analyzed by flow cytometry. As shown in Fig. 4 A, EGF decreased the fluorescence signal of bound rDIII in a dose-dependent fashion. This competitive displacement of rDIII by EGF strongly supports the specificity of rDIII binding to EGFR.

Bottom Line: Therefore, the elucidation of their identities and functions is of great interest.Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5.Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. sschenk@scripps

ABSTRACT
Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

Show MeSH
Related in: MedlinePlus