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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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Characterization of DIII of Ln-5 γ2-chain. (A) Schematic depiction of DIII. rDIII encompasses the most COOH-terminal 3.5 EGF-like repeats (γ2III5, 4, 3, and COOH-terminal part of 2) of rat Ln-5 γ2-chain DIII. Six cysteines (filled circles) highlight the EGF-like domain signature, and the LE repeat signature is characterized by eight cysteines and an additional loop. The position of rDIII within the Ln-5 cruciform structure and the products of MMP cleavage are shown. (B) Characterization of rDIII using SDS-PAGE and WB. In SDS-PAGE (two left panels), rDIII resolved as a single band under both nonreducing and reducing conditions. Purified rDIII was judged to be better than 95% homogenous by Coomassie blue staining. In WB of reducing PAGE (two right panels), the rDIII band was recognized by both 2778 and anti-His-tag, as expected. A much fainter, higher mol wt band was also visible, which is likely dimerized rDIII. The apparent mol wt was calculated based on pre-stained mol wt standard SeeBlue® (Invitrogen).
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fig1: Characterization of DIII of Ln-5 γ2-chain. (A) Schematic depiction of DIII. rDIII encompasses the most COOH-terminal 3.5 EGF-like repeats (γ2III5, 4, 3, and COOH-terminal part of 2) of rat Ln-5 γ2-chain DIII. Six cysteines (filled circles) highlight the EGF-like domain signature, and the LE repeat signature is characterized by eight cysteines and an additional loop. The position of rDIII within the Ln-5 cruciform structure and the products of MMP cleavage are shown. (B) Characterization of rDIII using SDS-PAGE and WB. In SDS-PAGE (two left panels), rDIII resolved as a single band under both nonreducing and reducing conditions. Purified rDIII was judged to be better than 95% homogenous by Coomassie blue staining. In WB of reducing PAGE (two right panels), the rDIII band was recognized by both 2778 and anti-His-tag, as expected. A much fainter, higher mol wt band was also visible, which is likely dimerized rDIII. The apparent mol wt was calculated based on pre-stained mol wt standard SeeBlue® (Invitrogen).

Mentions: Previously, we showed that MMP-2 (Giannelli et al., 1997) and MT1-MMP cleave the γ2 subunit of Ln-5 (Koshikawa et al., 2000). The positions of the MT1-MMP cleavage sites have now been identified (Fig. 1 A; 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)

Characterization of DIII of Ln-5 γ2-chain. (A) Schematic depiction of DIII. rDIII encompasses the most COOH-terminal 3.5 EGF-like repeats (γ2III5, 4, 3, and COOH-terminal part of 2) of rat Ln-5 γ2-chain DIII. Six cysteines (filled circles) highlight the EGF-like domain signature, and the LE repeat signature is characterized by eight cysteines and an additional loop. The position of rDIII within the Ln-5 cruciform structure and the products of MMP cleavage are shown. (B) Characterization of rDIII using SDS-PAGE and WB. In SDS-PAGE (two left panels), rDIII resolved as a single band under both nonreducing and reducing conditions. Purified rDIII was judged to be better than 95% homogenous by Coomassie blue staining. In WB of reducing PAGE (two right panels), the rDIII band was recognized by both 2778 and anti-His-tag, as expected. A much fainter, higher mol wt band was also visible, which is likely dimerized rDIII. The apparent mol wt was calculated based on pre-stained mol wt standard SeeBlue® (Invitrogen).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172889&req=5

fig1: Characterization of DIII of Ln-5 γ2-chain. (A) Schematic depiction of DIII. rDIII encompasses the most COOH-terminal 3.5 EGF-like repeats (γ2III5, 4, 3, and COOH-terminal part of 2) of rat Ln-5 γ2-chain DIII. Six cysteines (filled circles) highlight the EGF-like domain signature, and the LE repeat signature is characterized by eight cysteines and an additional loop. The position of rDIII within the Ln-5 cruciform structure and the products of MMP cleavage are shown. (B) Characterization of rDIII using SDS-PAGE and WB. In SDS-PAGE (two left panels), rDIII resolved as a single band under both nonreducing and reducing conditions. Purified rDIII was judged to be better than 95% homogenous by Coomassie blue staining. In WB of reducing PAGE (two right panels), the rDIII band was recognized by both 2778 and anti-His-tag, as expected. A much fainter, higher mol wt band was also visible, which is likely dimerized rDIII. The apparent mol wt was calculated based on pre-stained mol wt standard SeeBlue® (Invitrogen).
Mentions: Previously, we showed that MMP-2 (Giannelli et al., 1997) and MT1-MMP cleave the γ2 subunit of Ln-5 (Koshikawa et al., 2000). The positions of the MT1-MMP cleavage sites have now been identified (Fig. 1 A; 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