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Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

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tTG is associated with β1 and β3, but not with β2 integrins on the cell surface. (A) Interaction of tTG with β1 and β3 integrins on the cell surface. (Upper panel) TPA-differentiated HEL cells were biotinylated in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates of surface-biotinylated cells. Anti–mouse IgG was used in control immunoprecipitations (cont.). Biotinylated proteins in the immunoprecipitates were visualized on blots by neutravidin-peroxidase. (Lower panel) The same immunoprecipitates as in the top panel were blotted for tTG. Note association of tTG with β1 and β3 but not with β2 integrins. β1, β2, and β3 integrin bands are marked by arrowheads. Asterisks mark IgG heavy chains. Molecular weight markers are shown to the right of the blot. (B) tTG can be cross-linked to β1 and β3 integrins on the cell surface. TPA-differentiated HEL cells were treated for 20 min with 0.5 mM membrane-impermeable reducible cross-linker DTSSP in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates. After immunoprecipitation samples were run on 8% gels under nonreducing (upper panel) or reducing (lower panel) conditions. To avoid the appearance of IgG bands, the blots were probed for tTG using biotinylated anti-tTG mAb TG100 followed by neutravidin-peroxidase. Arrow in A and B points to tTG bands. Molecular weight markers (nonreduced: myosin heavy chain, 200 kD; IgG, 160 kD; and BSA, 68 kD) are shown to the right of the blots.
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Figure 6: tTG is associated with β1 and β3, but not with β2 integrins on the cell surface. (A) Interaction of tTG with β1 and β3 integrins on the cell surface. (Upper panel) TPA-differentiated HEL cells were biotinylated in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates of surface-biotinylated cells. Anti–mouse IgG was used in control immunoprecipitations (cont.). Biotinylated proteins in the immunoprecipitates were visualized on blots by neutravidin-peroxidase. (Lower panel) The same immunoprecipitates as in the top panel were blotted for tTG. Note association of tTG with β1 and β3 but not with β2 integrins. β1, β2, and β3 integrin bands are marked by arrowheads. Asterisks mark IgG heavy chains. Molecular weight markers are shown to the right of the blot. (B) tTG can be cross-linked to β1 and β3 integrins on the cell surface. TPA-differentiated HEL cells were treated for 20 min with 0.5 mM membrane-impermeable reducible cross-linker DTSSP in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates. After immunoprecipitation samples were run on 8% gels under nonreducing (upper panel) or reducing (lower panel) conditions. To avoid the appearance of IgG bands, the blots were probed for tTG using biotinylated anti-tTG mAb TG100 followed by neutravidin-peroxidase. Arrow in A and B points to tTG bands. Molecular weight markers (nonreduced: myosin heavy chain, 200 kD; IgG, 160 kD; and BSA, 68 kD) are shown to the right of the blots.

Mentions: To test association of tTG with other integrins on the cell surface, we biotinylated cell surface proteins on live TPA-differentiated HEL cells that express high levels of β1, β2, and β3 integrins. Immunoprecipitation with antiintegrin antibodies followed by blotting with avidin-peroxidase revealed an 80-kD protein that comigrated with tTG and was associated with β1 and β3, but not with β2 integrins (Fig. 6 A, upper panel, arrow). Furthermore, proteins that comigrated with β1 and β3, but not with β2 integrins, could be detected in tTG immunoprecipitates (Fig. 6 A, upper panel). The identity of the 80-kD protein as tTG and its lack of association with β2 integrins was confirmed by blotting the corresponding immunoprecipitates for tTG (Fig. 6 A, lower panel).


Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

Akimov SS, Krylov D, Fleischman LF, Belkin AM - J. Cell Biol. (2000)

tTG is associated with β1 and β3, but not with β2 integrins on the cell surface. (A) Interaction of tTG with β1 and β3 integrins on the cell surface. (Upper panel) TPA-differentiated HEL cells were biotinylated in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates of surface-biotinylated cells. Anti–mouse IgG was used in control immunoprecipitations (cont.). Biotinylated proteins in the immunoprecipitates were visualized on blots by neutravidin-peroxidase. (Lower panel) The same immunoprecipitates as in the top panel were blotted for tTG. Note association of tTG with β1 and β3 but not with β2 integrins. β1, β2, and β3 integrin bands are marked by arrowheads. Asterisks mark IgG heavy chains. Molecular weight markers are shown to the right of the blot. (B) tTG can be cross-linked to β1 and β3 integrins on the cell surface. TPA-differentiated HEL cells were treated for 20 min with 0.5 mM membrane-impermeable reducible cross-linker DTSSP in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates. After immunoprecipitation samples were run on 8% gels under nonreducing (upper panel) or reducing (lower panel) conditions. To avoid the appearance of IgG bands, the blots were probed for tTG using biotinylated anti-tTG mAb TG100 followed by neutravidin-peroxidase. Arrow in A and B points to tTG bands. Molecular weight markers (nonreduced: myosin heavy chain, 200 kD; IgG, 160 kD; and BSA, 68 kD) are shown to the right of the blots.
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Figure 6: tTG is associated with β1 and β3, but not with β2 integrins on the cell surface. (A) Interaction of tTG with β1 and β3 integrins on the cell surface. (Upper panel) TPA-differentiated HEL cells were biotinylated in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates of surface-biotinylated cells. Anti–mouse IgG was used in control immunoprecipitations (cont.). Biotinylated proteins in the immunoprecipitates were visualized on blots by neutravidin-peroxidase. (Lower panel) The same immunoprecipitates as in the top panel were blotted for tTG. Note association of tTG with β1 and β3 but not with β2 integrins. β1, β2, and β3 integrin bands are marked by arrowheads. Asterisks mark IgG heavy chains. Molecular weight markers are shown to the right of the blot. (B) tTG can be cross-linked to β1 and β3 integrins on the cell surface. TPA-differentiated HEL cells were treated for 20 min with 0.5 mM membrane-impermeable reducible cross-linker DTSSP in suspension and β1, β2, and β3 integrins and tTG were immunoprecipitated from RIPA lysates. After immunoprecipitation samples were run on 8% gels under nonreducing (upper panel) or reducing (lower panel) conditions. To avoid the appearance of IgG bands, the blots were probed for tTG using biotinylated anti-tTG mAb TG100 followed by neutravidin-peroxidase. Arrow in A and B points to tTG bands. Molecular weight markers (nonreduced: myosin heavy chain, 200 kD; IgG, 160 kD; and BSA, 68 kD) are shown to the right of the blots.
Mentions: To test association of tTG with other integrins on the cell surface, we biotinylated cell surface proteins on live TPA-differentiated HEL cells that express high levels of β1, β2, and β3 integrins. Immunoprecipitation with antiintegrin antibodies followed by blotting with avidin-peroxidase revealed an 80-kD protein that comigrated with tTG and was associated with β1 and β3, but not with β2 integrins (Fig. 6 A, upper panel, arrow). Furthermore, proteins that comigrated with β1 and β3, but not with β2 integrins, could be detected in tTG immunoprecipitates (Fig. 6 A, upper panel). The identity of the 80-kD protein as tTG and its lack of association with β2 integrins was confirmed by blotting the corresponding immunoprecipitates for tTG (Fig. 6 A, lower panel).

Bottom Line: These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII.Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins.Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

ABSTRACT
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Show MeSH
Related in: MedlinePlus