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Originally published In Press as doi:10.1074/jbc.M402161200 on May 13, 2004

J. Biol. Chem., Vol. 279, Issue 28, 28889-28895, July 9, 2004
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Competition for Talin Results in Trans-dominant Inhibition of Integrin Activation*

David A. Calderwood{ddagger}§, Vera Tai{ddagger}, Gilbert Di Paolo||, Pietro De Camilli||, and Mark H. Ginsberg{ddagger}**

From the {ddagger}Deptartment of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, the §Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, and the ||Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510

Received for publication, February 26, 2004 , and in revised form, May 10, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin {beta} subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i) {beta} tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii) trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii) expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type I{gamma}-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrin adhesion receptors are essential for the development and survival of multicellular animals. An important feature of integrins is their ability to undergo rapid changes in affinity for extracellular ligand, a process referred to as insideout signaling or integrin activation (1–3). Integrin activation is under the control of signals from within the cell and regulates cell adhesion, migration, and assembly of an extracellular matrix (4, 5). Tight control of integrin activation is required for many biological processes including embryonic development, wound healing, hemostasis, immune response, and tumor cell metastasis (6–8).

Most cells express more than one integrin, and cross-talk between integrins allows ligand binding to one integrin to regulate the function of other integrins on the same cell (9–19). One form of integrin cross-talk occurs when ligation of one integrin inhibits the activation of a second integrin, the socalled trans-dominant inhibition of integrin activation (9). Trans-dominant inhibition mediated by {beta}1, {beta}2, or {beta}3 integrins has been demonstrated in a variety of cell types and shown to regulate cell adhesion, spreading, migration, clot retraction, and differentiation (9, 10, 14, 18, 20). Trans-dominant integrin inhibition may be relevant physiologically and clinically in a number of settings. For example, {alpha}2{beta}1-dependent platelet adhesion to collagen is inhibited following {alpha}IIb{beta}3-mediated fibrinogen binding to ADP-activated platelets (14), and this may contribute to increased platelet adhesion to the subendothelium in Glanzmann's thrombastenia and afibrinogenemia (14). Furthermore, in patients with acute coronary syndrome, transdominant inhibition of {alpha}2{beta}1 may underlie the significant long term reduction of clinical events following short term treatment with pharmacological {alpha}IIb{beta}3 inhibitors (14). Trans-dominant inhibition also regulates adhesion of human T-lymphocytes and thus contributes to the control of leukocyte transmigration (10). In addition, there is now considerable interest in whether {alpha}v{beta}3-mediated trans-dominant inhibition of {alpha}5{beta}1 accounts for the effects of endogenous or pharmaceutical antagonists of {alpha}v{beta}3 on tumor angiogenesis (19, 21, 22).

Despite its potential importance, the mechanism of transdominant integrin inhibition has not been elucidated. However, it is known that inhibition is dependent on the {beta} subunitcytoplasmic tail of the suppressive integrin (9). Furthermore, the expression of isolated integrin {beta} tails fused to an irrelevant type I trans-membrane and extracellular domain in a variety of cell lines, megakaryocytes, and platelets and in the thymus and cardiac tissue in vivo is sufficient to induce trans-dominant integrin inhibition (20, 23–31). One explanation for trans-dominant inhibition is that the suppressive {beta} tail competes with other integrin {beta} tails for limiting cytoplasmic factors necessary for integrin activation (23, 26). Thus, trans-dominant integrin inhibition may be mediated by the titration of essential intracellular protein(s).

Talin, a cytoskeletal protein, binds to most integrin {beta} tails (32). Talin binding to the integrin {beta} tail activates integrins (33–36), and disruption of the integrin-talin interaction prevents integrin activation (35). Thus, talin binding to integrin {beta} tails is a final step in integrin activation. Therefore, we examined whether competition for talin mediates trans-dominant inhibition of integrin activation. We report that {beta} tails containing mutations that selectively inhibit talin binding are unable to mediate trans-dominant integrin inhibition and that overexpression of the integrin {beta} tail-binding domain of talin can reverse trans-dominant inhibition. Furthermore, phosphatidylinositol phosphate kinase type I{gamma}-90 (PIPKI{gamma}-90)1 binds talin and competes for talin binding to integrin {beta} tails. The overexpression of PIPKI{gamma}-90 also inhibits integrin activation. In contrast, PIPKI{gamma}-87, a variant that lacks the talin-binding site, fails to inhibit integrin activation. Thus, the sequestration of talin causes this form of trans-dominant integrin inhibition.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and cDNAs—Monoclonal antibodies, anti-talin 8d4 (Sigma), anti-hemagglutinin 12CA5 (American Type Culture Collection), anti-Tac 7G7B6 (American Type Culture Collection), anti-{beta}1 integrin 9EG7 (BD Biosciences), and anti-GFP (Clontech), were obtained commercially. The anti-{alpha}IIb{beta}3 monoclonal antibodies PAC-1 and anti-LIBS6, the anti-{beta}3 C-terminal peptide antiserum 8275, and the {alpha}IIb{beta}-specific antagonist Ro43-5054 have been described previously (37–39). cDNAs encoding Tac-{alpha}5, Tac-{beta}3, mouse talin F2F3 (residues 206–305; SwissProt entry TALI_MOUSE (P26039 [GenBank] )), {alpha}IIb and {beta}3 integrin tail model proteins, GFP-PIPKI{gamma}-87, GFP-PIPKI{gamma}-90, GFP-PIPKI{gamma}-90(W647A), and GST-fibronectin, type III repeats 9–11 (FN9–11) have been described previously (26, 33, 40–42). Point mutations were introduced using the QuikChange site-directed-mutagenesis kit (Stratagene) and confirmed by DNA sequencing.

Affinity Chromatography with Recombinant Integrin Tails—Recombinant integrin tail model proteins were produced and purified as described previously (43). Talin was purified from outdated platelets, and cell lysates were prepared as described previously (34, 43). Affinity chromatography was performed using recombinant integrin tails bound to the His-bind resin (Novagen) as described previously (34, 43, 44). 5 µl of coated beads and 1 µg of purified talin were used routinely. Bound proteins were fractionated by SDS-PAGE and analyzed by Western blotting.

FACS Analysis of the Activation State of {alpha}IIb{beta}3—Chinese hamster ovary (CHO) cells expressing {alpha}IIb{beta}3, {alpha}IIb{alpha}5{beta}3, or {alpha}IIb{alpha}6A{beta}3{beta}1A (45, 46) were transfected transiently with cDNAs encoding Tac-{alpha}5, Tac-{beta}3, or Tac-{beta}3 mutants (2 µg) or GFP or GFP-PIPK{gamma} variants (2 µg) using LipofectAMINE PLUS (Invitrogen). Twenty-four h later, a three-color flow cytometry was performed as described previously (34, 35, 45). Tac-transfected cells were suspended and stained with PAC-1 and then washed and stained with biotinylated anti-Tac mAb 7G7B6. Bound PAC-1 and 7G7B6 were detected with fluorescein isothiocyanate-conjugated goat anti-mouse IgM (BioSource) and streptavidin-conjugated R-phycoerythrin (Molecular Probes) respectively. GFP-transfected cells were suspended and stained with PAC-1 and then washed and stained with R-phycoerythrin-conjugated goat anti-mouse IgM (Biomeda). Five min prior to analysis, propidium iodide (PI; 2 µg/ml final) was added. Cells were washed and analyzed on a FACSCalibur instrument (BD Biosciences). PAC-1 binding to live (PI-negative) transfected (7G7B6- or GFP-positive) cells was assessed. Activation was quantified as an activation index defined as AI = (F – Fo)/(Fmax – Fo), where F is the mean fluorescence intensity (MFI) of PAC-1 binding, Fo is the MFI of PAC-1 binding in presence of the {alpha}IIb{beta}3 antagonist Ro43-5054 (1 µM), and Fmax is the MFI of PAC-1 binding in the presence of {alpha}{beta}3-activating mAb anti-LIBS6 (2 µM). Percentage inhibitions were calculated as 100x (AI0 – AI)/(AI0) where AI0 = AI in cells transfected with the control vector (Tac-{alpha}5 or GFP).

FACS Analysis of the Activation State of {alpha}5{beta}1—CHO cells were transfected transiently as described above, and 24 h later, they were analyzed by flow cytometry as described previously (35, 42). Cells were suspended and incubated with biotinylated recombinant GST-FN9–11. Cells were washed and, where necessary, stained with anti-Tac mAb 7G7B6. Bound GST-FN9–11 and 7G7B6 were detected with streptavidin-conjugated R-phycoerythrin and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (BioSource) respectively. Five min prior to analysis, PI (2 µg/ml final) was added. GST-FN9–11 binding to live (PI-negative) transfected (7G7B6- or GFP-positive) cells was assessed. The activation index was defined as (F – Fo)/(Fmax – Fo), where F is the MFI of GST-FN9–11 binding, Fo is the MFI of GST-FN9–11 binding in presence of EDTA (10 mM), and Fmax is the MFI of GST-FN9–11 binding in the presence of the {beta}1-activating mAb 9EG7 (0.01 µg/ml). Percentage inhibition was calculated as for PAC-1.

Cell-spreading Assays—CHO cells expressing {alpha}IIb{beta}3 were transfected transiently as described above with cDNAs encoding Tac-{alpha}5, Tac-{beta}3, or Tac-{beta}3 mutants (2 µg) and enhanced GFP (1 µg) as a marker of transfection. Twenty-four h later, cells were plated onto tissue culture dishes coated with 15 µg/ml fibrinogen. Cells were allowed to spread for 1 h and were viewed using a Zeiss Axiovert 100 epifluorescence microscope with a x10 objective. Bright-field and GFP images were collected on a SPOT charge-coupled device (CCD) camera (Diagnostic Instruments Inc., Sterling Heights, MI). Images were processed, and the area of GFP-positive cells was calculated using Scion Image Beta 4.0.2 software (Scion Corporation, Frederick, MD, www.scioncorp.com).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolated {beta} Tails Require an Intact Talin-binding Site to Mediate Trans-dominant Inhibition of Integrin {alpha}IIb{beta}3 Activation— Integrin activation is inhibited by overexpression of integrin {beta}3 tails fused to the trans-membrane and extracellular domains of the Tac subunit of the interleukin-2 receptor (Tac-{beta}3) (26). To examine whether competition for talin mediates trans-dominant inhibition, we generated Tac-{beta}3 constructs with impaired talin binding activity and assessed their effect on integrin activation. We previously identified point mutations at Trp739 and Leu746 in the {beta}3 tail that inhibit talin binding from cell lysates without detectably inhibiting the binding of other proteins from platelets (35) or CHO cells (data not shown). Furthermore, these mutations inhibited the direct interaction between recombinant integrin {beta}3 tails and purified talin (Fig. 1, B and C). Substitution of Tyr747 with Ala also inhibited the binding of talin; however, this mutation is not specific for talin as the binding of many other proteins also is inhibited (35). Thus, W739A or L746A substitutions in the {beta}3 tail selectively inhibit direct talin binding to integrin {beta}3 tails in vitro, whereas D740A or D740K mutations do not inhibit direct binding. When introduced into full-length integrins, W739A or L746A substitutions disrupt integrin activation, a process dependent on talin binding (35), indicating that these mutations also inhibit talin binding in vivo.



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  		FIG. 1.
Point mutations in the {beta}3 tail inhibit talin binding. A, sequence of the -cytoplasmic domain and the point mutations tested are shown. B, affinity chromatography using wild type and mutant {beta}3 model proteins was performed using purified talin. Talin binding was assessed by Western blotting using an antibody that recognizes the talin rod domain. The faster migrating species in the input lane corresponds to the talin rod domain generated by proteolysis of full-length talin (34). As reported previously (44), integrin {beta} tails bind more strongly to full-length talin than to the rod domain. The bottom row shows the equal loading of each model tail protein as judged by Coomassie Blue staining. C, talin binding was quantified by densitometry and normalized for binding to wild type {beta}3 (mean ± S.E.; n = 4).

 
The introduction of mutations that selectively disrupt talin binding into Tac-{beta}3 allowed us to investigate the role of talin in trans-dominant inhibition of integrin activation. The activation state of {alpha}IIb{beta}3 integrins was assessed by measuring the binding of the ligand-mimetic mAb, PAC-1 (47). CHO cells stably expressing {alpha}IIb{beta}3 were transfected with vector encoding Tac-{beta}3 mutants, Tac-{beta}3, or Tac-{alpha}5 (a negative control) (26). 24 h later, the cells were harvested, and the binding of anti-Tac (to quantify Tac expression levels) and PAC-1 (to assess integrin affinity state) to live (PI-negative) cells was analyzed by three-color flow cytometry. As reported previously (26), the cells expressing Tac-{beta}3, but not those expressing Tac-{alpha}5, exhibited inhibition of integrin activation that correlated with Tac expression as evidenced by a diagonal slope of the leftward shift of the FACS dot plot (Fig. 2A). Inhibition was cell-autonomous because only transfected cells showed a reduction in activation. Furthermore, the addition of the activating monoclonal antibody, anti-LIBS6, stimulated PAC-1 binding to similar levels in all of the cells (data not shown), showing that the inhibition of PAC-1 binding was not the result of a reduction in the expression of {alpha}IIb{beta}3 following transfection with Tac-{beta}3. Notably, Tac-{beta}3W739A or L746A, mutants that are selectively deficient in talin binding, failed to suppress PAC-1 binding (Fig. 2A). Thus, the mutations that disrupt talin binding inhibit trans-dominant suppression of {alpha}IIb{beta}3 by isolated {beta} tails.



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  		FIG. 2.
Free {beta} tails require an intact talin-binding site to mediate trans-dominant inhibition of {alpha}IIb{beta}3 activation. CHO cells expressing {alpha}IIb{beta}3 were transfected transiently with vectors encoding Tac-{alpha}5, Tac-{beta}3, or Tac-{beta}3 mutants, and 24 h later, the binding of PAC-1 (activation-specific anti-{alpha}IIb{beta}3 mAb) and 7G7B6 (anti-Tac) was assessed by FACS analysis. A, FACS dot plots of live (PI-negative) cells transfected with Tac-{alpha}5, Tac-{beta}3, or Tac-{beta}3(L746A) showing staining for Tac expression and PAC-1 binding. Specific PAC-1 binding was inhibited by the addition of the {alpha}IIb{beta}3 antagonist Ro43-5054. B, the percentage inhibition in the activation index of cells transfected with Tac-{beta}3 or Tac-{beta}3 mutants was calculated as described under "Experimental Procedures." Results represent the mean ± S.E. (n ≥ 3), *, p < 0.005 when compared with Tac-{beta}3. C, CHO cells transfected with the indicated Tac constructs were lysed, and Tac protein was immunoprecipitated with the anti-Tac antibody, 7G7B6. Immunoprecipitated protein was fractionated by SDS-PAGE and immunoblotted with an antiserum raised against the C-terminal portion of the {beta}3 tail. The presence of two bands probably represents a mixture of the intracellular and mature glycosylated protein.

 
The activation index (the ratio of specific PAC-1 binding: maximal anti-LIBS6-stimulated specific PAC-1 binding) allows the quantification of {alpha}IIb{beta}3 activation (45). Tac-{beta}3 or Tac-{beta}3 containing mutations that maintain talin binding (D740A or D740K) produced a >60% inhibition of {alpha}IIb{beta}3 activation, whereas Tac-{beta}3 containing mutations that disrupt talin binding (W739A,L746A,K748A, or L746A) had little effect on integrin activation (Fig. 2B). The differing effects of the Tac-{beta}3 mutants were not attributed to differing expression levels, because the analyses were performed on cells gated for similar Tac expression. Furthermore, Western blotting of immunoprecipitated Tac-{beta}3 revealed that all of the Tac-{beta}3 constructs reacted with an antiserum raised against the C-terminal portion of the {beta}3 tail (Fig. 2C), suggesting that differential proteolysis of the Tac-{beta}3 mutants does not account for their differing effects on integrin activation. Hence, free {beta}3 tails require an intact talin-binding site to mediate trans-dominant inhibition of {alpha}IIb{beta}3.

Trans-dominant Inhibition of Activated {beta}1 and {beta}3 Integrins Requires an Intact Talin-binding Site in the Suppressive-free {beta} Tail—When expressed in CHO cells, {alpha}IIb{beta}3 is activated weakly (activation index 0.22 ± 0.02; mean ± S.E.) (38). To test whether competition for talin also mediated the trans-dominant inhibition of more active integrins, we examined chimeric {alpha}IIb{beta}3 integrins. The substitution of the {alpha}5-cytoplasmic tail for that of {alpha}IIb generates a more active integrin, {alpha}IIb{alpha}5{beta}3 (activation index 0.45 ± 0.03; mean ± S.E.) (37). Tac-{beta}3 inhibited the activation of this active chimera, and this inhibition was impaired by mutations that reduce talin binding (Fig. 3A). Therefore, talin binding to Tac-{beta}3 is required for trans-dominant inhibition of {beta}3 integrins, regardless of their activation state. Furthermore, this effect is not limited to {beta}3 integrins, because similar results were obtained with active (activation index 0.40 ± 0.03) chimeric {alpha}IIb{beta}3 integrins containing the {alpha}6A{beta}1A-cytoplasmic domains ({alpha}IIb{alpha}6A{beta}3{beta}1A) (Fig. 3B).



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  		FIG. 3.
Free {beta} tails require an intact talin-binding site to mediate trans-dominant inhibition of active {beta}3 and {beta}1 integrins. CHO cells expressing the chimeric integrins {alpha}IIb{alpha}5{beta}3 (A) or {alpha}IIb{alpha}6A{beta}3{beta}1A (B) were transfected transiently with vectors encoding Tac-{alpha}5, Tac{beta}3, or Tac{beta}3 mutants, and 24 h later, the binding of PAC-1 and 7G7B6 was assessed by FACS analysis. Results represent the mean percentage inhibition in activation ± S.E. (n ≥ 3). C, the binding of FN9–11 to CHO cells was measured 24 h after transfection with the indicated constructs, and the {alpha}5{beta}1 activation index was calculated as described under "Experimental Procedures." Results represent the mean percentage inhibition in activation ± S.E. (n ≥ 3). *, p < 0.03 when compared with Tac-{beta}3.

 
The experiments described above assessed the activation of ectopically expressed {alpha}IIb{beta}3 or chimeric {alpha}IIb{beta}3 integrins using the mAb PAC-1. As an alternative approach, we used an integrin binding fragment of FN9–11 to assess the activation state of endogenous CHO {alpha}5{beta}1 integrins (42). The binding of FN9–11 is specific, because it can be inhibited by EDTA or function by blocking anti-{alpha}5{beta}1 antibodies and can be stimulated by the {beta}1-activating antibody, 9EG7, and Mn2+ (data not shown). Using this approach, we found that Tac-{beta}3, but not Tac-{beta}3(W739A), or Tac-{beta}3(L746A,K748A) or Tac-{beta}3(L746A) inhibited the activation of endogenous hamster {alpha}5{beta}1A (Fig. 3C). Thus, integrin {beta} tails require an intact talin-binding site to mediate trans-dominant inhibition of a variety of {beta}1 and {beta}3 integrins.

Overexpression of the Integrin-binding Domain of Talin Reverses Tac-{beta}3-mediated Trans-dominant Inhibition—The preceding data suggest that free {beta} tails mediate trans-dominant inhibition of integrin activation by competing with endogenous integrins for talin. We have been unable to reproducibly coimmunoprecipitate talin with integrins expressed in CHO cells (this may be because of the relatively low levels of talin detected in CHO cells), preventing us from directly assessing the effect of Tac-{beta}3 expression on integrin-talin interactions. However, if competition for talin is responsible for trans-dominant inhibition of integrin activation, it should be reversed by the overexpression of the integrin-binding domain of talin. Amino acids 206–405 of mouse talin1 span subdomains F2 and F3 of the talin FERM (Four-point-one, Ezrin, Radixin, Moesin) domain and bind integrin {beta} tails (33, 48). The expression of talin F2F3 along with Tac-{beta}3 reversed trans-dominant suppression of both {alpha}IIb{beta}3 and {alpha}IIb{alpha}5{beta}3 integrins (Fig. 4), supporting the hypothesis that competition for talin mediates trans-dominant inhibition of integrin activation.



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  		FIG. 4.
Overexpression of the integrin-binding domain of talin reverses Tac-{beta}3-mediated trans-dominant suppression. A, CHO cells expressing integrin {alpha}IIb{beta}3 or {alpha}IIb{alpha}5{beta}3 were transfected transiently with vectors encoding Tac-{beta}3 (1 µg) plus hemagglutinin-tagged talin F2F3 or empty vector (2 µg). Twenty-four h later, the binding of PAC-1 and 7G7B6 was assessed by FACS analysis, and the percentage inhibition of activation was calculated. Results represent mean ± S.E. (n ≥ 3). B, the expression of recombinant talin F2F3 in the transfected cells was examined by immunoblotting of SDS-PAGE fractionated cell lysates (10 µg).

 
Expression of Talin-binding Variants of PIPKI{gamma} Inhibits Integrin Activation—The preceding results show that the binding of talin to free {beta}3 tails is necessary for trans-dominant inhibition of integrin activation. To assess whether talin binding was sufficient for trans-dominant inhibition, we examined the effect of cellular expression of PIPKI{gamma}-90, a talin-binding splice variant of PIPKI{gamma} (41, 49), on integrin activation. PIPKI{gamma}-90 binds to talin at a site within the F3 subdomain of talin (41) that overlaps with the integrin-binding site (50, 51). Furthermore, PIPKI{gamma}-90 can compete with integrin {beta} tails for binding to talin (50, 51). To test the hypothesis that competition for talin would lead to trans-dominant inhibition, the effect of GFP fusions of the talin-binding splice variant, PIPKI{gamma}-90, and the non-binding variant, PIPKI{gamma}-87, on integrin activation were assessed by flow cytometry. The activation of integrins {alpha}IIb{beta}3 and {alpha}5{beta}1 (assessed by PAC-1 and FN9–11 binding, respectively) was inhibited by PIPKI{gamma}-90 but not by PIPKI{gamma}-87 (Fig. 5, A and B). Exogenous stimulation of the integrins with activating antibodies (anti-LIBS6 or 9EG7) or Mn2+ promoted ligand binding in all of the cells (data not shown), showing that the inhibition was not the result of a reduction in integrin expression.



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  		FIG. 5.
Overexpression of talin-binding variants of PIPKI{gamma} inhibits integrin activation. A, CHO cells expressing integrins {alpha}IIb{beta}3 were transfected transiently with vectors encoding GFP or GFP-PIPKI{gamma} variants and mutants, and 24 h later, the activation state of {alpha}IIb{beta}3 was assessed by measuring PAC-1 binding to GFP-positive cells by FACS analysis. Results represent the mean percentage inhibition in activation ± S.E. (n ≥ 3). B, the binding of FN9–11 to CHO cells was measured 24 h after transfection with the indicated GFP-PIPKI{gamma} variants and mutants, and the {alpha}5{beta}1 activation index was calculated as described under "Experimental Procedures." Results represent the mean ± S.E. (n ≥ 3). C, the expression of the GFP-PIPKI{gamma} variants and mutants in the transfected cells was examined by immunoblotting of SDS-PAGE fractionated cell lysates (10 µg) using an anti-GFP antibody.

 
Suppression of activation is not dependent on the kinase activity of PIPKI{gamma}-90, because PIPKI{gamma}-90 containing a point mutation (K138A) that inhibits kinase activity (49) was still capable of inhibiting integrin activation (Fig. 5, A and B). However, a Trp647 to Phe point mutation within the PIPKI{gamma}-90 talin binding sequence, which inhibits talin binding (41), prevented the inhibition of integrin activation (Fig. 5A). The differing effects of the PIPKI{gamma} variants were not attributed to differing expression levels, because the analyses were performed on cells gated for similar GFP expression and Western blotting of cell lysates revealed that all of the GFP fusions were intact (Fig. 5C). Thus, PIPKI{gamma}-90 can inhibit integrin activation in a manner analogous to the trans-dominant inhibition by free integrin {beta} tails.

Trans-dominant Inhibition of Cell Spreading Requires an Intact Talin-binding Site in the Suppressive Species—The results described above indicate that competition for talin results in the trans-dominant inhibition of integrin activation. Expression of isolated {beta} tails or of PIPKI{gamma}-90 also produces a dose-dependent inhibition of cell spreading (40, 41, 52). In the case of PIPKI{gamma}-90, the inhibition of spreading is dependent a functional talin-binding site within the PIPKI{gamma} protein (41). To test whether talin binding to the isolated integrin {beta} tail is required for trans-dominant inhibition of cell spreading, we expressed Tac-{alpha}5, Tac-{beta}3, Tac-{beta}3(W739A), or Tac-{beta}3(L746A,K748A) in {alpha}IIb{beta}3-expressing CHO cells and assessed the ability of these cells to spread on fibrinogen. Cells expressing Tac-{beta}3 spread significantly less than the cells expressing Tac-{alpha}5, Tac-{beta}3(W739A), or Tac-{beta}3(L746A,K748A) (Student's t two-tailed test p < 10–4) (Fig. 6). Thus, talin binding to the suppressive isolated {beta} tails or to overexpressed PIPKI{gamma}-90 is required for trans-dominant inhibition of integrin activation and cell spreading.



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  		FIG. 6.
Trans-dominant inhibition of cell spreading requires an intact talin-binding site in the suppressive {beta} tail. CHO cells expressing integrin {alpha}IIb{beta}3 were transfected transiently with vectors encoding GFP and Tac-{alpha}5, Tac-{beta}3 or Tac-{beta}3 mutants. Twenty-four h later, cells were plated on fibrinogen-coated dishes and allowed to spread for 1 h. GFP-positive cells were examined by epifluorescence microscopy. The mean area of GFP-positive cells from at least four separate fields was calculated. Results are expressed as the mean ± S.E. *, p < 10–4 when compared with Tac-{beta}3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cross-talk among different integrins and between integrins and other receptors impacts many essential cellular processes including adhesion, migration, proliferation, and matrix assembly (19). Here we report that competition for talin underlies one form of integrin cross-talk, the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i) free integrin {beta} tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii) trans-dominant inhibition can be reversed by the overexpression of integrin binding and activating fragments of talin, and (iii) expression of another non-integrin talin-binding protein, PIPKI{gamma}-90, also inhibits integrin activation. The latter finding further indicates that cross-talk between integrins and other signaling molecules also can be mediated via competition for talin. Thus, talin binding to the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.

Talin binds to the integrin {beta}3 tail via a variant of the canonical phosphotyrosine-binding domain-NPXY motif interaction, and this binding leads to integrin activation (33, 48). The structural and functional analysis of the talin-integrin complex permitted the identification of integrin point mutations that selectively impair talin binding (35) such as alanine substitutions at Trp739 or Leu746. The introduction of either of these mutations into integrin {beta}3 tails impairs their ability to mediate trans-dominant inhibition. Furthermore, {beta}1A tails containing mutations at the site corresponding to the {beta}3 Trp739, {beta}1A(W775A), also are impaired in their ability to mediate trans-dominant inhibition of integrin activation (52). Additional support for the importance of talin binding in transdominant inhibition of integrin activation comes from the correlation between talin binding and the potency of different {beta} tails in the inhibition of integrin activation. When expressed as free {beta} tails, {beta}1D exhibits a greater inhibition of integrin activation than {beta}1A, which in turn is more potent than {beta}7 (53). The {beta}1D tails bind talin more tightly than the {beta}1A tail, whereas the {beta}7 tails bind only low levels of talin (43, 54). Finally, the expression of talin-binding variants of PIPKI{gamma} also inhibits integrin activation. Thus, the competition for integrin {beta} tail binding to talin is the basis for trans-dominant inhibition of integrin activation.

Trans-dominant inhibition of integrin function occurs when occupancy of one integrin can suppress the function of other integrins (9). In addition to the inhibition of integrin activation, trans-dominant inhibition of integrin-mediated cell adhesion, signaling, migration, phagocytosis, focal adhesion formation, and gene expression has been observed (9–13, 40, 52, 55–57). Integrin activation is an important component of cell adhesion, migration, phagocytosis, and integrin-mediated signaling; therefore, the titration of talin may contribute to the trans-dominant inhibition of these processes by blocking integrin activation. However, because the exogenous activation of integrins does not reverse this inhibition fully (11–13, 56), either sequential activation and inactivation or additional activation-independent processes also are involved in these processes. Talin binds to most integrin {beta} tails and connects integrins to the actin cytoskeleton. It is probable that these connections are central to many integrin functions (3, 35, 58–62). Therefore, titration of talin may inhibit activation and disrupt the formation and reinforcement of links from integrins to the actin cytoskeleton (61, 62). A requirement for talin binding in trans-dominant inhibition of cell spreading is evidenced by our observation that isolated {beta}3 tails containing mutations that impair talin binding are ineffective in inhibiting cell spreading. Furthermore, isolated {beta}1A(W775A) tails (containing a mutation predicted to disrupt talin binding) are unable to inhibit cell spreading (52), and the expression of talin binding but not non-binding variants of PIPKI{gamma} inhibits cell spreading, attachment, and focal adhesion formation (41, 49). However, mutagenesis of the {beta}1A tail reveals that some mutants, which retain the ability to inhibit activation, are unable to inhibit cell spreading (52), suggesting that titration of additional factors may be required for this activity. Furthermore, at least in K562 cells and monocyte-derived macrophages, when integrins are activated exogenously with Mn2+, an {alpha}V{beta}3-mediated suppression of calcium/calmodulin-dependent protein kinase is required for the trans-dominant inhibition of {alpha}5{beta}1-mediated migration and phagocytosis (13). Thus, the binding of talin to the suppressive tail is necessary and sufficient for trans-dominant inhibition of integrin activation, but other pathways can contribute to the inhibition of additional integrin functions.

Although there is evidence for the physiological role of trans-dominant integrin inhibition, the in vivo significance of competition between integrins and PIPKI{gamma}-90 for binding to talin is less certain. However, there is probably a complex relationship between integrins, talin, and PIPKI{gamma}-90. PIPKI{gamma}-90 localizes to sites of integrin-mediated adhesion, and talin binding activates PIPKI{gamma}-90, which catalyzes the production of phosphatidylinositol 4,5-bisphosphate (41, 49). Phosphatidylinositol 4,5-bisphosphate binds to talin and induces a conformational change that enhances talin association with integrin {beta} tails (63). Therefore, talin can stimulate phosphatidylinositol 4,5-bisphosphate production, which in turn enhances talin-integrin interactions, suggesting that PIPKI{gamma}-90 may stimulate integrin activation. However, as shown here, by competing with integrin {beta} tails for binding to talin, PIPKI{gamma}-90 can inhibit integrin activation. Hence, PIPKI{gamma}-90 probably plays both positive and negative roles in integrin activation and thus may contribute to dynamic processes such as adhesion turnover and cell migration. This activity probably is controlled by Src, acting downstream of focal adhesion kinase, because Src-mediated tyrosine phosphorylation of PIPKI{gamma}-90 greatly increases its affinity for talin resulting in a more effective displacement of integrin tails from talin (51).

Src family kinases also may be involved in modulating competition for talin among integrins and thus influence trans-dominant inhibition. Integrin-cytoplasmic tails can be tyrosinephosphorylated by Src leading to reduced talin binding (51, 64). Therefore, differential phosphorylation of integrin tails will influence their ability to compete for talin. In this regard, it is noteworthy that the integrin {beta} tails show differential specificities for interaction with Src family kinases (65).

The vital role of integrin-talin interactions in integrin function suggested that the regulation of these interactions provides a mechanism to control integrin function (1, 3). The results presented here indicate that one mechanism by which such regulation can be achieved is through competition for talin among different integrins or between integrins and other talin-binding proteins. Competition for talin among integrins, leading to trans-dominant inhibition of activation, may have a role in normal physiology during leukocyte trans-migration, platelet adhesion, and control of angiogenesis (10, 14, 21, 22), processes that require temporal and spatial control of integrin activation. Furthermore trans-dominant inhibition may have implications for the mechanisms of action of drugs that target integrins (14, 19, 21). Our identification of {beta}3 mutants incapable of mediating trans-dominant inhibition should facilitate the investigation of the role of trans-dominant inhibition in the anti-angiogenic effects of agents targeting {alpha}V{beta}3 and {alpha}V{beta}5 integrins.


   FOOTNOTES
 
* This work was supported by Grants GM68600, HL31950, AR27214, NS36251, and CA46128 from the National Institutes of Health and a Scientist Development Award from the American Heart Association (to D. A. C.). This is publication number 16429-CB from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** Present address: Dept. of Medicine, University of California, San Diego, La Jolla, CA 92037. Back

To whom correspondence should be addressed. E-mail: david.calderwood{at}yale.edu.

1 The abbreviations used are: PIPK{gamma}, phosphatidylinositol phosphate kinase type I{gamma}; GFP, green fluorescent protein; GST, glutathione S-transferase; FN9–11, fibronectin, type III repeats 9–11; CHO, Chinese hamster ovary; PI, propidium iodide; AI, activation index; MFI, mean fluorescence intensity; mAb, monoclonal antibody; FACS, fluorescence-activated cell sorter. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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D. M. Cohen, H. Chen, R. P. Johnson, B. Choudhury, and S. W. Craig
Two Distinct Head-Tail Interfaces Cooperate to Suppress Activation of Vinculin by Talin
J. Biol. Chem., April 29, 2005; 280(17): 17109 - 17117.
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S. Y. Lee, S. Voronov, K. Letinic, A. C. Nairn, G. Di Paolo, and P. De Camilli
Regulation of the interaction between PIPKI{gamma} and talin by proline-directed protein kinases
J. Cell Biol., February 28, 2005; 168(5): 789 - 799.
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J. R. Morgan, G. Di Paolo, H. Werner, V. A. Shchedrina, M. Pypaert, V. A. Pieribone, and P. De Camilli
A role for talin in presynaptic function
J. Cell Biol., October 11, 2004; 167(1): 43 - 50.
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