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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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Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa®-conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS3. After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS3, and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).
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fig2: Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa®-conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS3. After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS3, and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).

Mentions: To determine whether rDIII binds to the cell surface, MDA-MB-231 breast cancer cells were incubated with rDIII, followed by excess 2778 and fluorescently labeled anti-IgG secondary antibody. Flow cytometry showed dose-dependent staining of rDIII-treated cells. Saturation of rDIII–cell surface interactions occurred at ∼4.5 μM rDIII, resulting in fluorescence ∼12-fold higher than the control (Fig. 2 A). Detection of rDIII with anti-His antibody gave similar results (unpublished data). Another recombinant His-tagged γ2 fragment, encompassing DIII to DV (rDIII-V), did not bind to MDA-MB-231 cell surfaces (unpublished data).


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)

Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa®-conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS3. After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS3, and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).
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Related In: Results  -  Collection

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fig2: Binding of rDIII to EGFR. (A) rDIII binding to cell surfaces detected by flow cytometry. MDA-MB-231 cells were incubated with 4.5 (open black histogram) or 2 μM (open gray) rDIII or control rabbit IgG (filled), followed by 2778, and the appropriate Alexa®-conjugated secondary antibody. (B) Recovery of biotin–rDIII–EGFR complexes with streptavidin-coated beads. 1.5 μM biotinylated rDIII or 0.75 μM EGF was incubated with MDA-MB-231 cells, followed by cross-linking with BS3. After detergent solubilization, cell lysates were precipitated with streptavidin-coated beads. WB of adsorbed material with EGFR pAb (top) detected a distinct band of 175 kD in samples containing rDIII (lane 3) or EGF (lane 2), but not in control samples (lane 1, no ligand). To control for EGFR expression and specificity of cross-linking to EGFR, total MDA-MB-231 cell lysates were loaded in lane 4 and stripped blots were treated with anti-insulin receptor β antibody (bottom), respectively. (C) Immunoprecipitation of biotin–rDIII–EGFR complexes with antibodies to EGFR. Cells were treated with biotinylated rDIII or EGF and BS3, and cell lysates were immunoprecipitated with EGFR mAb. Samples were analyzed by WB using streptavidin-HRP and ECL. A distinct band at 175 kD was visible for samples containing EGF (lane 2, 0.75 μM) or rDIII (lane 3, 1.0 μM, and lane 4, 1.5 μM; top). There is no corresponding band in the control lane (lane 1; no ligand). Note, the resolution of the gradient gels used is not sufficient to distinguish between EGF or rDIII bound to EGFR, where the former would be expected to run at ∼180 kD and the latter at ∼195 kD. To ensure equal loading in each lane, the filter was stripped and reprobed with EGFR pAb (bottom).
Mentions: To determine whether rDIII binds to the cell surface, MDA-MB-231 breast cancer cells were incubated with rDIII, followed by excess 2778 and fluorescently labeled anti-IgG secondary antibody. Flow cytometry showed dose-dependent staining of rDIII-treated cells. Saturation of rDIII–cell surface interactions occurred at ∼4.5 μM rDIII, resulting in fluorescence ∼12-fold higher than the control (Fig. 2 A). Detection of rDIII with anti-His antibody gave similar results (unpublished data). Another recombinant His-tagged γ2 fragment, encompassing DIII to DV (rDIII-V), did not bind to MDA-MB-231 cell surfaces (unpublished data).

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