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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 mediates association of α5β1 and αIIbβ3 integrins with Fn via its 42-kD fragment. 35S-labeled untreated (A) and TPA-treated (B) HEL cells were extracted with RIPA buffer (lanes 1). Cell extracts were incubated with Sepharose-immobilized Fn (lanes 2), 110-kD (lanes 3), or 42-kD (lanes 4) Fn fragments, or immunoprecipitated with polyclonal anti-tTG antibody (lanes 5), anti–β1 integrin mAb 9EG7 (lanes 6), anti–β3 integrin mAb 25E11 (lanes 7) or control Sepharose beads (lanes 8). 35S-labeled eluates from immobilized Fn and Fn fragments, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Unlabeled RIPA extracts of untreated (C, E, and G) or TPA-treated (D, F, and H) HEL cells, analogous to those in A and B, respectively, were incubated with immobilized Fn and Fn fragments or immunoprecipitated with antibodies against tTG, β1, and β3 integrins. Eluates from immobilized Fn and Fn fragments and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibody to β1A integrin cytodomain (C and D), polyclonal antibody to β3 integrin (E and F), or anti-tTG mAb tTG100 (G and H). (A and B) Protein bands corresponding to α5, αIIb, β1, and β3 integrins and tTG are marked to the right of each gel. Molecular weight markers are shown to the left of the gels.
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Figure 8: tTG mediates association of α5β1 and αIIbβ3 integrins with Fn via its 42-kD fragment. 35S-labeled untreated (A) and TPA-treated (B) HEL cells were extracted with RIPA buffer (lanes 1). Cell extracts were incubated with Sepharose-immobilized Fn (lanes 2), 110-kD (lanes 3), or 42-kD (lanes 4) Fn fragments, or immunoprecipitated with polyclonal anti-tTG antibody (lanes 5), anti–β1 integrin mAb 9EG7 (lanes 6), anti–β3 integrin mAb 25E11 (lanes 7) or control Sepharose beads (lanes 8). 35S-labeled eluates from immobilized Fn and Fn fragments, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Unlabeled RIPA extracts of untreated (C, E, and G) or TPA-treated (D, F, and H) HEL cells, analogous to those in A and B, respectively, were incubated with immobilized Fn and Fn fragments or immunoprecipitated with antibodies against tTG, β1, and β3 integrins. Eluates from immobilized Fn and Fn fragments and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibody to β1A integrin cytodomain (C and D), polyclonal antibody to β3 integrin (E and F), or anti-tTG mAb tTG100 (G and H). (A and B) Protein bands corresponding to α5, αIIb, β1, and β3 integrins and tTG are marked to the right of each gel. Molecular weight markers are shown to the left of the gels.

Mentions: An even more convincing proof of this concept came from experiments with HEL cells, which synthesize essentially no endogenous Fn even when treated with TPA, and express large amounts of α5β1 and αIIbβ3 integrins, which serve as Fn receptors (Jarvinen et al. 1987; data not shown). We found that stimulation of these cells with TPA sharply increased the overall expression of tTG while having no effect on the levels of α5β1 and αIIbβ3 (see below). This provided a convenient inducible model with which to test the ability of tTG to mediate the interaction of integrins with immobilized Fn and its fragments. Affinity chromatography of 35S-labeled RIPA lysates of both untreated and TPA-treated cells (Fig. 8A and Fig. B) showed that tTG was the predominant protein in the eluates from immobilized Fn and its 42-kD fragment, but did not bind the 110-kD Fn fragment (Fig. 8A and Fig. B, lanes 2–4). The identity of tTG was confirmed by immunoprecipitation (Fig. 8A and Fig. B, lanes 5) and by blotting the unlabeled eluates and immunoprecipitates for tTG (Fig. 8G and Fig. H). Consistent with the drastic enhancement of tTG synthesis in HEL cells by TPA, this treatment also greatly elevated the amounts of α5β1 and αIIbβ3 integrins coprecipitating with tTG and vice versa (Fig. 8, A–F, lanes 5; Fig. 8G and Fig. H, lanes 6 and 7). Notably, α5β1 and αIIbβ3 integrins bound to immobilized Fn and its 42-kD fragment, but not to the 110-kD fragment, indicating that binding of these integrins to Fn and its 42-kD fragment in RIPA buffer is indirect and mediated by tTG (Fig. 8, A–F, lanes 2–4). As a consequence of larger amounts of integrin–tTG complexes, more α5β1 and αIIbβ3 integrins were isolated on Fn and the 42-kD fragment, from the TPA-treated than from the untreated cells (Fig. 8, A–F, lanes 2 and 4). Taken together, these data establish that tTG serves as a bridge to link α5β1 and αIIbβ3 integrins with the gelatin-binding domain of Fn.


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

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

tTG mediates association of α5β1 and αIIbβ3 integrins with Fn via its 42-kD fragment. 35S-labeled untreated (A) and TPA-treated (B) HEL cells were extracted with RIPA buffer (lanes 1). Cell extracts were incubated with Sepharose-immobilized Fn (lanes 2), 110-kD (lanes 3), or 42-kD (lanes 4) Fn fragments, or immunoprecipitated with polyclonal anti-tTG antibody (lanes 5), anti–β1 integrin mAb 9EG7 (lanes 6), anti–β3 integrin mAb 25E11 (lanes 7) or control Sepharose beads (lanes 8). 35S-labeled eluates from immobilized Fn and Fn fragments, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Unlabeled RIPA extracts of untreated (C, E, and G) or TPA-treated (D, F, and H) HEL cells, analogous to those in A and B, respectively, were incubated with immobilized Fn and Fn fragments or immunoprecipitated with antibodies against tTG, β1, and β3 integrins. Eluates from immobilized Fn and Fn fragments and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibody to β1A integrin cytodomain (C and D), polyclonal antibody to β3 integrin (E and F), or anti-tTG mAb tTG100 (G and H). (A and B) Protein bands corresponding to α5, αIIb, β1, and β3 integrins and tTG are marked to the right of each gel. Molecular weight markers are shown to the left of the gels.
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Related In: Results  -  Collection

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Figure 8: tTG mediates association of α5β1 and αIIbβ3 integrins with Fn via its 42-kD fragment. 35S-labeled untreated (A) and TPA-treated (B) HEL cells were extracted with RIPA buffer (lanes 1). Cell extracts were incubated with Sepharose-immobilized Fn (lanes 2), 110-kD (lanes 3), or 42-kD (lanes 4) Fn fragments, or immunoprecipitated with polyclonal anti-tTG antibody (lanes 5), anti–β1 integrin mAb 9EG7 (lanes 6), anti–β3 integrin mAb 25E11 (lanes 7) or control Sepharose beads (lanes 8). 35S-labeled eluates from immobilized Fn and Fn fragments, and immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Unlabeled RIPA extracts of untreated (C, E, and G) or TPA-treated (D, F, and H) HEL cells, analogous to those in A and B, respectively, were incubated with immobilized Fn and Fn fragments or immunoprecipitated with antibodies against tTG, β1, and β3 integrins. Eluates from immobilized Fn and Fn fragments and immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with polyclonal antibody to β1A integrin cytodomain (C and D), polyclonal antibody to β3 integrin (E and F), or anti-tTG mAb tTG100 (G and H). (A and B) Protein bands corresponding to α5, αIIb, β1, and β3 integrins and tTG are marked to the right of each gel. Molecular weight markers are shown to the left of the gels.
Mentions: An even more convincing proof of this concept came from experiments with HEL cells, which synthesize essentially no endogenous Fn even when treated with TPA, and express large amounts of α5β1 and αIIbβ3 integrins, which serve as Fn receptors (Jarvinen et al. 1987; data not shown). We found that stimulation of these cells with TPA sharply increased the overall expression of tTG while having no effect on the levels of α5β1 and αIIbβ3 (see below). This provided a convenient inducible model with which to test the ability of tTG to mediate the interaction of integrins with immobilized Fn and its fragments. Affinity chromatography of 35S-labeled RIPA lysates of both untreated and TPA-treated cells (Fig. 8A and Fig. B) showed that tTG was the predominant protein in the eluates from immobilized Fn and its 42-kD fragment, but did not bind the 110-kD Fn fragment (Fig. 8A and Fig. B, lanes 2–4). The identity of tTG was confirmed by immunoprecipitation (Fig. 8A and Fig. B, lanes 5) and by blotting the unlabeled eluates and immunoprecipitates for tTG (Fig. 8G and Fig. H). Consistent with the drastic enhancement of tTG synthesis in HEL cells by TPA, this treatment also greatly elevated the amounts of α5β1 and αIIbβ3 integrins coprecipitating with tTG and vice versa (Fig. 8, A–F, lanes 5; Fig. 8G and Fig. H, lanes 6 and 7). Notably, α5β1 and αIIbβ3 integrins bound to immobilized Fn and its 42-kD fragment, but not to the 110-kD fragment, indicating that binding of these integrins to Fn and its 42-kD fragment in RIPA buffer is indirect and mediated by tTG (Fig. 8, A–F, lanes 2–4). As a consequence of larger amounts of integrin–tTG complexes, more α5β1 and αIIbβ3 integrins were isolated on Fn and the 42-kD fragment, from the TPA-treated than from the untreated cells (Fig. 8, A–F, lanes 2 and 4). Taken together, these data establish that tTG serves as a bridge to link α5β1 and αIIbβ3 integrins with the gelatin-binding domain of Fn.

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