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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 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.
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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.

Mentions: Immunoprecipitation in RIPA buffer, which disrupts integrin–ligand interactions (data not shown), was used to isolate integrin–tTG complexes from cell lysates. We initiated the analysis with HEL cells because they do not synthesize any detectable Fn (Jarvinen et al. 1987). A predominant 80-kD protein that comigrated with tTG and coprecipitated with β1 integrins from lysates of 35S-labeled TPA-treated HEL cells was immunodepleted with polyclonal antibody against tTG, strongly suggesting that it is tTG (Fig. 5 A, arrow). Additionally, after preadsoption of tTG, the intensity of the β1 integrin band slightly decreased, whereas a detectable amount of β1 integrin appeared in tTG immunoprecipitates (Fig. 5 A). The identity of the 80-kD protein was further proved by reprecipitation experiments (Berditchevski et al. 1996). β1 integrins were first immunoprecipitated from 35S-labeled HEL cells using mAb 9EG7 (Fig. 5 B). The resulting immune complexes were then eluted with either 0.4% SDS at 25°C or 1% SDS with boiling and reprecipitated with antibody against the cytoplasmic domain of β1A integrin (Fig. 5 B, lane 1) or three anti-tTG antibodies (Fig. 5 B, lanes 2–4). The 80-kD protein appeared in all cases, proving its identity as tTG (Fig. 5 B, arrow). Notably, β1 integrins coprecipitated with tTG even after treatment with 0.4% SDS, showing extremely stable association between these proteins (Fig. 5 B, left panel). However, after boiling in 1% SDS, integrins no longer coprecipitated with tTG (Fig. 5 B, right panel), demonstrating that their association is noncovalent. The absence of other 35S-labeled proteins in β1 integrin or tTG immunoprecipitates after reprecipitation proves that this association is highly specific and is not mediated by a third protein.


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

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

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.
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Related In: Results  -  Collection

Show All Figures
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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.
Mentions: Immunoprecipitation in RIPA buffer, which disrupts integrin–ligand interactions (data not shown), was used to isolate integrin–tTG complexes from cell lysates. We initiated the analysis with HEL cells because they do not synthesize any detectable Fn (Jarvinen et al. 1987). A predominant 80-kD protein that comigrated with tTG and coprecipitated with β1 integrins from lysates of 35S-labeled TPA-treated HEL cells was immunodepleted with polyclonal antibody against tTG, strongly suggesting that it is tTG (Fig. 5 A, arrow). Additionally, after preadsoption of tTG, the intensity of the β1 integrin band slightly decreased, whereas a detectable amount of β1 integrin appeared in tTG immunoprecipitates (Fig. 5 A). The identity of the 80-kD protein was further proved by reprecipitation experiments (Berditchevski et al. 1996). β1 integrins were first immunoprecipitated from 35S-labeled HEL cells using mAb 9EG7 (Fig. 5 B). The resulting immune complexes were then eluted with either 0.4% SDS at 25°C or 1% SDS with boiling and reprecipitated with antibody against the cytoplasmic domain of β1A integrin (Fig. 5 B, lane 1) or three anti-tTG antibodies (Fig. 5 B, lanes 2–4). The 80-kD protein appeared in all cases, proving its identity as tTG (Fig. 5 B, arrow). Notably, β1 integrins coprecipitated with tTG even after treatment with 0.4% SDS, showing extremely stable association between these proteins (Fig. 5 B, left panel). However, after boiling in 1% SDS, integrins no longer coprecipitated with tTG (Fig. 5 B, right panel), demonstrating that their association is noncovalent. The absence of other 35S-labeled proteins in β1 integrin or tTG immunoprecipitates after reprecipitation proves that this association is highly specific and is not mediated by a third protein.

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