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. 2000 Feb 21;148(4):825-38.
doi: 10.1083/jcb.148.4.825.

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

S S Akimov  1 , D Krylov, L F Fleischman, A M Belkin
Affiliations

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

S S Akimov et al. J Cell Biol. .

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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Figures

Figure 1
Figure 1
Surface tTG mediates adhesion and spreading of WI-38 fibroblasts on 42-kD Fn fragment. (A) A scheme of modular structure of Fn and its 42-kD and 110-kD proteolytic fragments. (B and C) Spreading assays with WI-38 fibroblasts. (B) Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments either untreated or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (C) Cells were plated for 4 h on 42-kD Fn fragment in the presence of 10 μM recombinant NH2-terminal tTG domain tTG1-165, 200 μM GRGDSP or GRGESP peptides, or 10 μg/ml function-blocking mAb JB1A against human β1 integrins. Bar, 20 μM. (D) Quantitative adhesion assays with WI-38 fibroblasts plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments without treatment or in the presence of 10 μM tTG1-165, 10 μg/ml mAb CUB7402, 10 μg/ml polyclonal anti-tTG antibody, or 10 μg/ml blocking anti-β1 integrin mAb JB1A. Shown are the means of quadruplicate measurements. (E) Quantitative spreading assays with WI-38 fibroblasts plated on Fn or 42-kD Fn fragment for 30–180 min either without treatment or in the presence of 10 μg/ml polyclonal anti-tTG antibody. Shown are the average areas on substrate for 120 cells. (B–E) Adhesion and spreading assays were performed with cells in serum-free medium in the presence of cycloheximide.
Figure 2
Figure 2
TPA-induced adhesion and spreading of HEL cells on 42-kD Fn fragment is mediated by surface tTG. (A) Expression levels of tTG on the surface of live untreated and TPA-treated cells were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Transglutaminase activity on the surface of live untreated and TPA-treated cells was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars show the means of triplicate measurements. (C) Quantitative adhesion assays with untreated and TPA-treated cells plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments either without or in the presence of 10 μg/ml polyclonal anti-tTG antibody. (D) Spreading assays with untreated and TPA-treated cells. Cells were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments without any treatment or after TPA treatment either in the absence or with 10 μg/ml polyclonal anti-tTG antibody. Bar, 20 μM. (C and D) For adhesion and spreading assays HEL cells were plated in serum-free medium in the presence of cycloheximide.
Figure 3
Figure 3
Expression of exogenous tTG or its enzymatically inactive mutant tTG[C277→S], but not of FXIIIa promotes cell spreading. (A–E) REF52 cells were transfected with vector (vect.), wild-type tTG (tTG), tTG noncatalytic mutant C277→S (tTG[C277-S]), or FXIIIa (FXIIIa). (A) Expression levels of tTG on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal anti-tTG antibody and flow cytometry. (B) Expression levels of FXIIIa on the surface of live transfectants were determined by immunostaining with 10 μg/ml polyclonal antibody against FXIIIa and flow cytometry. (C) Transglutaminase activity in the cytosolic fractions of the transfectants was quantified by incorporation of [3H]putrescine into N,N-dimethylcaseine by transamidating enzymes present in cell lysates (Lorand et al. 1972). Shown are the results of triplicate determinations. (D) Transglutaminase activity on the surface of live transfectants was quantified by measuring cell-mediated incorporation of [3H]putrescine into N,N-dimethylcaseine. Bars represent the means of triplicate measurements. (E) Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were photographed in regular culture. Bar, 20 μM.
Figure 4
Figure 4
tTG-dependent stimulation of cell adhesion and spreading on Fn and its 42-kD fragment does not require the cross-linking activity. (A) Spreading assays with REF52 transfectants. Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were plated for 4 h on Fn, 110-kD, or 42-kD Fn fragments. Bar, 20 μM. (B) Quantitative adhesion assays with REF52 transfectants plated for 1 h on Fn (open bars), 110-kD (crossed bars), and 42-kD (filled bars) fragments. Shown are the means of quadruplicate measurements. (C and D) Quantitative spreading assays with REF52 transfectants. Cells expressing vector, tTG, tTG[C277→S], or FXIIIa were plated for 30–180 min on Fn (C) or the 42-kD Fn fragment (D). Shown are the average areas on the substrates for 90 cells. (A–D) Cells were plated for adhesion and spreading assays in serum-free cycloheximide-containing medium. (E) Analysis of tTG association with β1 integrins. FGF receptor, β1 integrins, and tTG were immunoprecipitated with polyclonal antibody Flg(H-76) or HMβ1-1 and CUB7402 mAbs, respectively, from REF52 cells expressing vector, tTG or tTG[C277→S]. The resulting immunoprecipitates were blotted for tTG. Large arrow marks the transfected human tTG and small arrow indicates the endogenous rat tTG. (F) FXIIIa is not associated with β1 integrins. FGF receptor, β1 integrins, and FXIIIa were immunoprecipitated from FXIIIa-expressing REF52 cells and the resulting immunoprecipitates were probed for FXIIIa with a polyclonal antibody.
Figure 5
Figure 5
tTG associates with multiple β1 and β3 integrins in different cell types. (A) Immunodepletion. An 80-kD protein associated with β1 integrins is immunodepleted with anti-tTG antibody. β1 integrins and tTG were immunoprecipitated from RIPA lysates of TPA-treated 35S-labeled HEL cells with mAb 9EG7 or polyclonal anti-tTG antibody, respectively. Note a comigration of 80-kD protein coprecipitating with β1 integrins, with tTG (arrow). Preadsorbtion of 35S-labeled RIPA lysates with polyclonal anti-tTG antibody followed by immunoprecipitation of β1 integrins with mAb 9EG7 caused a disappearance of 80-kD protein from the β1 integrin immunoprecipitates. Arrows in A–E point to tTG bands. Brackets in A and B mark α5β1 integrin. (A–C) Molecular weight markers are shown to the right of the gels. (B) Reprecipitation. An 80-kD protein associated with β1 integrins is reprecipitated by three antibodies against tTG. β1 integrins were immunoprecipitated from RIPA lysates of TPA-treated 35S-labeled HEL cells with 9EG7 mAb. The 35S-labeled β1 integrin immune complexes were treated with 0.4% SDS (left panel) or boiled in 1% SDS (right panel). 35S-labeled eluates from the β1 integrin immune complexes were divided into four equal aliquots and subjected to reprecipitation in RIPA buffer with antibody against β1A integrin cytodomain (lane 1), anti-tTG polyclonal antibody (lane 2), mAb CUB7402 (lane 3), or mAb TG100 (lane 4) against tTG. (C) tTG interacts with β1 integrins inside the cell during biosynthesis. TPA-treated HEL cells were labeled with [35S]Translabel for 1 h, then chased with regular medium for 0, 2, 6, or 18 h. β1 integrins were immunoprecipitated from 35S-labeled RIPA lysates with 9EG7 mAb. The resulting immunoprecipitates were divided into halves. Half of each sample was run on the gel (upper panel), whereas another half was boiled in 1% SDS and tTG was reprecipitated from these samples using anti-tTG polyclonal antibody (lower panel). Large and small arrowheads point to mature β1 integrin and its underglycosylated precursor, respectively. (D) Association of tTG with multiple integrins. Immunoprecipitates from PAC-1 smooth muscle cells with antibodies against α1, α3, α5, αv, β1, and β3 integrins, FGF receptor, tTG, or without primary antibody (cont.) were blotted for tTG. (E) β1 integrin cytodomain is not required for binding tTG. Transfected human and endogenous hamster β1 integrins were immunoprecipitated with mAbs TS2/16 and 7E2, respectively, from CHO cells expressing exogenous β1A, β1D, or β1 integrin with deleted cytodomain. No primary antibody was used in control immunoprecipitations (cont.). The immunoprecipitates were blotted for tTG.
Figure 6
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.
Figure 7
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.
Figure 8
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.
Figure 9
Figure 9
tTG amplifies integrin-mediated tyrosine phosphorylation of FAK and colocalizes with β1 integrins at focal adhesions. (A and B) tTG potentiates FAK phosphorylation. (A) Time course of FAK phosphorylation in REF52 cells expressing vector (vect.) or wild-type tTG (tTG), plated on Fn. Cells were either kept in suspension (susp.) or plated on Fn for 45, 90, or 180 min. (B) REF52 cells expressing vector (vect.) or wild-type tTG (tTG), were plated for 3 h on dishes coated with Fn, 110-kD, or 42-kD Fn fragments, polyclonal anti-tTG antibody or laminin (Ln). (A and B) The transfectants were plated on ECM proteins or anti-tTG antibody in serum-free medium in the presence of cycloheximide. The cells were lysed and FAK was immunoprecipitated from cell lysates followed by SDS-PAGE and immunoblotting of the immune complexes for phosphotyrosine with PY20 mAb (Belkin et al. 1996). (C–E) tTG colocalizes with β1 integrins on the cell surface of REF52 fibroblasts and causes enlargement of focal adhesions. (C) Live, nonpermeabilized cells transfected with vector (vect.) or tTG (tTG) were double stained for cell surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Note codistribution of these proteins at focal adhesions and much larger size of these structures in tTG transfectants. (D) Formaldehyde-fixed, permeabilized cells transfected with vector or tTG, were double stained for vinculin and actin with mAb 7F9 and rhodamine-phalloidin. Note the increased size of focal adhesions and altered organization of actin bundles in the tTG transfectants. (E) Cells overexpressing tTG were plated for 1 or 2 h on 42-kD Fn fragment, fixed, and then double stained for surface tTG with mAb CUB7402 and β1 integrins with hamster mAb HMβ1-1. Arrows indicate focal adhesion sites. Bar, 20 μM.
Figure 10
Figure 10
A model proposing the role of tTG in cell adhesion. Association of integrins with tTG promotes cell adhesion and spreading due to formation of ternary adhesion complexes with Fn. (A) Integrin-mediated adhesion to Fn in the absence of tTG. (B) tTG enhances adhesion acting as a bridge between integrins and Fn. (C) tTG enhances adhesion by mediating the formation of ternary complexes where all three proteins interact with each other.
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