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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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Interaction of tTG with β1 integrins allows formation of ternary complexes with Fn. (A) tTG (left lane) or β1 integrins (all other lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the presence of 1 μM unlabeled Fn, its 42-kD fragment, its 110-kD fragment or without any of these proteins added. (B) tTG (left two lanes) or β1 integrins (right two lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the absence or with 5 μM unlabeled NH2-terminal tTG fragment tTG1-165. After immunoprecipitation half of each sample shown in A and B was boiled in 1% SDS, reconstituted with 10 volumes of 1% Triton X-100 in TBS and subjected to reprecipitation with polyclonal antibody against Fn (C and D). Note a disappearance of 35S-labeled Fn bands in the samples treated with excess unlabeled Fn, 42-kD Fn fragment, or tTG1-165, but not with excess unlabeled 110-kD Fn fragment. Arrowheads indicate Fn bands. Brackets mark α5β1 integrin. Arrows point to tTG bands. Molecular weight markers are shown to the right of the gels.
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Figure 7: Interaction of tTG with β1 integrins allows formation of ternary complexes with Fn. (A) tTG (left lane) or β1 integrins (all other lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the presence of 1 μM unlabeled Fn, its 42-kD fragment, its 110-kD fragment or without any of these proteins added. (B) tTG (left two lanes) or β1 integrins (right two lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the absence or with 5 μM unlabeled NH2-terminal tTG fragment tTG1-165. After immunoprecipitation half of each sample shown in A and B was boiled in 1% SDS, reconstituted with 10 volumes of 1% Triton X-100 in TBS and subjected to reprecipitation with polyclonal antibody against Fn (C and D). Note a disappearance of 35S-labeled Fn bands in the samples treated with excess unlabeled Fn, 42-kD Fn fragment, or tTG1-165, but not with excess unlabeled 110-kD Fn fragment. Arrowheads indicate Fn bands. Brackets mark α5β1 integrin. Arrows point to tTG bands. Molecular weight markers are shown to the right of the gels.

Mentions: The foregoing data demonstrate that tTG functions as a cell adhesion receptor for the gelatin-binding region of Fn and interacts with β1 and β3 integrins. We next examined biochemically whether tTG by itself can mediate association of integrins with this part of Fn, which lacks integrin-binding motifs. First, a protein corresponding to Fn was observed in both tTG and β1 integrin immunoprecipitates from 35S-labeled WI-38 human fibroblasts (Fig. 7 A, arrowhead). Addition of unlabeled Fn or its 42-kD fragment to the 35S-labeled RIPA lysates effectively displaced 35S-labeled Fn from the β1 integrin immunoprecipitates, whereas unlabeled 110-kD fragment was unable to do so. None of these treatments decreased the amounts of tTG associated with β1 integrins (Fig. 7 A, arrow). In parallel experiments, an NH2-terminal fragment tTG1-165 strongly inhibited association of Fn with tTG and completely displaced 35S-labeled Fn from both the tTG and β1 integrin immune complexes (Fig. 7 B, arrowhead). The presence of Fn in the immunoprecipitates was confirmed by using half of each sample for reprecipitation with anti-Fn antibody after boiling the immune complexes in 1% SDS (Fig. 7C and Fig. D). The use of GRGDSP peptide in coimmunoprecipitation assays did not cause any decrease in the amounts of 35S-labeled Fn (data not shown). These data indicate that in the RIPA lysates Fn interacts indirectly with β1 integrins due to association of its 42-kD fragment with integrin-bound tTG.


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

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

Interaction of tTG with β1 integrins allows formation of ternary complexes with Fn. (A) tTG (left lane) or β1 integrins (all other lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the presence of 1 μM unlabeled Fn, its 42-kD fragment, its 110-kD fragment or without any of these proteins added. (B) tTG (left two lanes) or β1 integrins (right two lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the absence or with 5 μM unlabeled NH2-terminal tTG fragment tTG1-165. After immunoprecipitation half of each sample shown in A and B was boiled in 1% SDS, reconstituted with 10 volumes of 1% Triton X-100 in TBS and subjected to reprecipitation with polyclonal antibody against Fn (C and D). Note a disappearance of 35S-labeled Fn bands in the samples treated with excess unlabeled Fn, 42-kD Fn fragment, or tTG1-165, but not with excess unlabeled 110-kD Fn fragment. Arrowheads indicate Fn bands. Brackets mark α5β1 integrin. Arrows point to tTG bands. Molecular weight markers are shown to the right of the gels.
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Related In: Results  -  Collection

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Figure 7: Interaction of tTG with β1 integrins allows formation of ternary complexes with Fn. (A) tTG (left lane) or β1 integrins (all other lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the presence of 1 μM unlabeled Fn, its 42-kD fragment, its 110-kD fragment or without any of these proteins added. (B) tTG (left two lanes) or β1 integrins (right two lanes) were immunoprecipitated from RIPA lysates of 35S-labeled WI-38 fibroblasts either in the absence or with 5 μM unlabeled NH2-terminal tTG fragment tTG1-165. After immunoprecipitation half of each sample shown in A and B was boiled in 1% SDS, reconstituted with 10 volumes of 1% Triton X-100 in TBS and subjected to reprecipitation with polyclonal antibody against Fn (C and D). Note a disappearance of 35S-labeled Fn bands in the samples treated with excess unlabeled Fn, 42-kD Fn fragment, or tTG1-165, but not with excess unlabeled 110-kD Fn fragment. Arrowheads indicate Fn bands. Brackets mark α5β1 integrin. Arrows point to tTG bands. Molecular weight markers are shown to the right of the gels.
Mentions: The foregoing data demonstrate that tTG functions as a cell adhesion receptor for the gelatin-binding region of Fn and interacts with β1 and β3 integrins. We next examined biochemically whether tTG by itself can mediate association of integrins with this part of Fn, which lacks integrin-binding motifs. First, a protein corresponding to Fn was observed in both tTG and β1 integrin immunoprecipitates from 35S-labeled WI-38 human fibroblasts (Fig. 7 A, arrowhead). Addition of unlabeled Fn or its 42-kD fragment to the 35S-labeled RIPA lysates effectively displaced 35S-labeled Fn from the β1 integrin immunoprecipitates, whereas unlabeled 110-kD fragment was unable to do so. None of these treatments decreased the amounts of tTG associated with β1 integrins (Fig. 7 A, arrow). In parallel experiments, an NH2-terminal fragment tTG1-165 strongly inhibited association of Fn with tTG and completely displaced 35S-labeled Fn from both the tTG and β1 integrin immune complexes (Fig. 7 B, arrowhead). The presence of Fn in the immunoprecipitates was confirmed by using half of each sample for reprecipitation with anti-Fn antibody after boiling the immune complexes in 1% SDS (Fig. 7C and Fig. D). The use of GRGDSP peptide in coimmunoprecipitation assays did not cause any decrease in the amounts of 35S-labeled Fn (data not shown). These data indicate that in the RIPA lysates Fn interacts indirectly with β1 integrins due to association of its 42-kD fragment with integrin-bound tTG.

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