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Originally published In Press as doi:10.1074/jbc.M709527200 on December 28, 2007

J. Biol. Chem., Vol. 283, Issue 10, 6118-6125, March 7, 2008
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The N-terminal Domains of Talin Cooperate with the Phosphotyrosine Binding-like Domain to Activate β1 and β3 Integrins*

Mohamed Bouaouina, Yatish Lad, and David A. Calderwood1

From the Department of Pharmacology and Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, Connecticut 06520

Received for publication, November 20, 2007 , and in revised form, December 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of integrin adhesion receptors from low to high affinity in response to intracellular cues controls cell adhesion and signaling. Binding of the cytoskeletal protein talin to the β3 integrin cytoplasmic tail is required for β3 activation, and the integrin-binding PTB-like F3 domain of talin is sufficient to activate β3 integrins. Here we report that, whereas the conserved talin-integrin interaction is also required for β1 activation, and talin F3 binds β1 and β3 integrins with comparable affinity, expression of the talin F3 domain is not sufficient to activate β1 integrins. β1 integrin activation could, however, be detected following expression of larger talin fragments that included the N-terminal and F1 domains, and mutagenesis indicates that these domains cooperate with talin F3 to mediate β1 activation. This effect is not due to increased affinity for the integrin β tail and we hypothesize that the N-terminal domains function by targeting or orienting talin in such a way as to optimize the interaction with the integrin tail. Analysis of β3 integrin activation indicates that inclusion of the N-terminal and F1 domains also enhances F3-mediated β3 activation. Our results therefore reveal a role for the N-terminal and F1 domains of talin during integrin activation and highlight differences in talin-mediated activation of β1 and β3 integrins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are a family of {alpha}β heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. Cells regulate integrin function through tight temporal and spatial control of integrin affinity for extracellular ligands. This is achieved by rapid, reversible changes in the conformation of the integrin extracellular domains; integrin activation (1–3). Activation of the platelet integrin {alpha}IIbβ3 is a pivotal event in thrombus formation (4), and {alpha}IIbβ3 has served as a prototype in studies on integrin activation. However, activation of other integrins, including the widely expressed β1 family, is essential for normal development because it controls cell adhesion, migration, and assembly of an extracellular matrix (5–10), and deregulated β1 integrin activation contributes to neoplasia (11) and impairs cardiac function (12) and the immune response (13).

A large body of evidence points to regulation of integrin activation through interactions of the β subunit tail (3, 14), although {alpha} tail-binding proteins also have a role (15, 16). Using {alpha}IIbβ3 as a model system we and others have shown that binding of talin to the β3 cytoplasmic tail is necessary and sufficient for integrin activation (17–23). Talin, a cytoskeletal actin-binding protein, consists of an N-terminal ~50-kDa globular head and an ~220-kDa C-terminal rod (3, 24, 25). The talin head is composed of an N-terminal 85-amino acid region followed by a FERM (4.1, ezrin, radixin, moesin) domain and a 33-amino acid stretch (18, 24–26). FERM domains are made up of three sub-domains, F1, F2, and F3 (27). The talin F3 domain contains the major integrin-binding site, is structurally very similar to phosphotyrosine binding (PTB)2 domains, engages β3 integrins via a variant of the canonical PTB domain-NPXY ligand interaction, and is sufficient to activate {alpha}IIbβ3 (18, 26). Furthermore, perturbation of the talin F3-β3 integrin interaction by mutagenesis, knockdown, or expression of dominant-negative constructs inhibits β3 integrin activation in vitro and in vivo (19, 21, 28, 29). NMR analysis reveals that talin induces β3 activation through effects on the membrane-proximal region of the integrin tail, which disrupts an inhibitory {alpha}-β tail interaction (20, 22, 26, 30, 31). Thus the major β3 integrin-binding and activating fragment of talin lies within the F3 subdomain and this interaction is necessary and sufficient for β3 activation.

The talin-binding motif is well conserved in integrin β tails and talin F3 binds most integrin β subunits suggesting that talin may be a general integrin activator (3, 26, 32) and function through a conserved mechanism. In support of this contention, overexpressed talin head activates β2 integrins (33), talin knockdown or sequestration inhibits β1 integrin activation (19, 21, 34, 35), and talin knock-out inhibits activation of the Drosophila β1 orthologue βPS (8). However, proof that talin binding is sufficient to activate β1 integrins is lacking. Furthermore, differences between talin-mediated activation of β1 and β3 integrins and the role of regions outside of the talin F3 in integrin activation have not been explored. By investigating activation of β1 integrins we find that the minimal β3-activating F3 fragment of talin is not sufficient for β1 activation but additional N-terminal domains are required.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and DNAs—Monoclonal antibodies, anti-talin 8d4 (Sigma), activating anti-β1 integrin 9EG7 (BD Biosciences), ligand-mimetic anti-{alpha}IIbβ3 PAC1 (BD Biosciences), anti-hamster {alpha}5β1 PB1 (Developmental Studies Hybridoma Bank), anti-HA tag (Covance), and polyclonal anti-GST (Chemicon) were purchased. The anti-{alpha}IIbβ3 monoclonal antibody D57 and the {alpha}IIbβ3-specific antagonist Ro44-9883 have been described previously (36, 37). The {alpha}5β1-specific inhibitor 3F compound was kindly provided by Horst Kessler (38). cDNAs encoding integrins {alpha}IIb, {alpha}IIb{alpha}5, β3β1A, and GST-, GFP- or HA-tagged mouse talin1 F2, F3, or F2F3 (residues 206–305, 309–405, and 206–405, respectively, of NP_006280.2 [GenBank] ), {alpha}IIb and β1A integrin tail model proteins, and GST-fibronectin type III repeats 9–11 (FN9–11) have been described previously (18, 37, 39–41). cDNAs encoding for GFP or GST fused mouse talin1 F0, F0F1, F1F2F3, 1–405, and 1–433 (residues 1–86, 1–205, 86–405, 1–405, and 1–433, respectively, of NP_006280.2 [GenBank] ) were generated by PCR from a talin1 cDNA. Point mutations were introduced with the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by DNA sequencing. Other talin1 fragments were generated by PCR from a talin1 cDNA. HA-tagged human talin2 F3 and F2F3 (residues 312–406 and 208–406, respectively, of NP_055874.1) were generated by PCR from a talin2 cDNA (42), verified by sequencing, and cloned into pcDNA3 (Invitrogen).

Pull-down Assays with Recombinant Integrin Tails—Recombinant integrin tail proteins and GST-talin fragments were produced and purified as described previously (18, 40, 43). Pull-down assays were performed using recombinant integrin tails bound to His-bind resin (Novagen) as described previously (43); bound proteins were fractionated by SDS-PAGE and analyzed by Western blotting or protein staining. To estimate apparent affinity constants the binding of increasing amounts of purified GST-talin proteins to β1A tails was quantified by densitometry, data were plotted as percent maximal binding versus input concentration and fitted to a one site binding model (Y = Bmax·X/(Kd + X)) using GraphPad Prism version 4 for Windows (GraphPad Software).

Analysis of Integrin Activation—The activation state of endogenous {alpha}5β1 was assessed by measuring the binding of a recombinant soluble integrin-binding fragment of fibronectin (FN9–11) in three-color flow cytometric assays as described previously (19, 21, 41). In experiments on CHO cells the {alpha}5β1 integrin expression was assessed in parallel by staining with PB1 (44). Briefly, CHO or HT1080 cells were transfected with the indicated cDNAs using Lipofectamine (Invitrogen) and 24 h later cells were suspended and incubated with biotinylated recombinant GST-FN9–11 in the presence or absence of integrin activators or inhibitors. For each preparation of biotinylated GST-FN9–11 the effective concentration was determined by titration. Cells were washed and bound FN9–11 was detected with R-phycoerythrin-conjugated streptavidin. Five minutes prior to analysis, propidium iodide (2 µg/ml final) was added. FN9–11 binding to live (propidium iodide-negative) transfected (GFP-positive) cells was assessed on a FACSCalibur instrument (BD Biosciences). The activation index was defined either as AI = (F – F0)/(Fmax – F0) or AI = (F – F0)/(Fintegrin), where F is the geometric mean fluorescence intensity (GMFI) of FN9–11 binding, F0 is the GMFI of FN9–11 binding in presence of 0.4 µM 3F inhibitor or 5 mM RGD peptide (Sigma) or 10 mM EDTA, Fmax is the GMFI of FN9–11 binding in the presence of 0.01 mg/ml of 9EG7 or 2 µM Mn2+, Fintegrin is the normalized GMFI of PB1 binding to transfected cells.

The activation state of wild-type or chimeric {alpha}IIbβ3 integrins was assessed by measuring the binding of the ligand mimetic anti-{alpha}IIbβ3 monoclonal antibody PAC1 in three-color flow cytometric assays as described previously (17, 19, 21, 39). CHO cell lines stably expressing {alpha}IIbβ3, {alpha}IIb{alpha}5β3β1A, {alpha}IIb{alpha}6Aβ3β1A, or {alpha}IIb{alpha}5β3 (37, 45) were transfected as described above and 24 h later cells were suspended and stained with PAC1. Cells were washed and bound PAC1 was detected with R-phycoerythrin-conjugated goat anti-mouse IgM (Southern Biotech). Five minutes prior to analysis propidium iodide was added. PAC1 binding to live, transfected (GFP-positive) cells was assessed. Activation was quantified and an activation index calculated as defined above, where F is the GMFI of PAC1 binding, F0 is the GMFI of PAC1 binding in presence of EDTA or RGD peptide, and Fmax is the GMFI of PAC1 binding in the presence of 2 µM Mn2+.

To measure the activation of mutant {alpha}IIb{alpha}5β3β1A integrins CHO cells were transfected with vectors encoding {alpha}IIb{alpha}5 and wild-type or mutant β3β1A. Twenty-four hours later cells were suspended and stained with PAC1, washed, and stained with anti-{alpha}IIbβ3 antibody D57 on ice, and the binding of PAC1 and D57 was detected using R-phycoerythrin-conjugated goat anti-mouse IgM and fluorescein isothiocyanate-conjugated donkey anti-mouse IgG. PAC1 binding to live, integrin expressing (D57 positive) cells was measured and an activation index calculated as above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The PTB-like Domain of Talin Is Not Sufficient to Activate β1 Integrins—To investigate activation of β1 integrins by talin we expressed {alpha}IIbβ3-activating fragments of the talin1 FERM domain containing subdomains F3 or F2 and F3 (talin1 F3 and talin1 F2F3) in CHO cells and assessed the activation state of endogenous {alpha}5β1 integrin by measuring the binding of a recombinant soluble integrin-binding fragment of fibronectin (FN9–11), in flow cytometric assays (19, 21, 41). FN9–11 is composed of fibronectin type III repeats 9 to 11 and its binding is specific as it can be inhibited by function blocking anti-{alpha}5β1 antibodies, RGD peptides, 3F, or EDTA and stimulated by the β1-activating antibody 9EG7, or Mn2+ (Fig. 1A and data not shown). However, despite efficient transfection, assessed by measuring expression of co-transfected GFP and good expression of the F3 or F2F3 domains of talin1, assessed by Western blotting, FN9–11 binding was not increased by the talin fragments indicating that {alpha}5β1 was not activated (Fig. 1, A–C). This was confirmed by calculation of an activation index, the ratio of specific (EDTA, 3F, or RGD peptide inhibitable) binding to maximal (9EG7 or Mn2+ stimulated) specific binding. Similar results were obtained with the closely related F3 or F2F3 domains from talin2 (Fig. 1B), talin1 F2F3 constructs fused to GFP, and with HA-tagged full-length talin1 (not shown).


Figure 1
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  		FIGURE 1.
The F3 domain of talin activatesβ3 but notβ1 integrins. A–C, CHO cells were transfected with empty vector or cDNAs encoding HA-tagged F2, F3, or F2F3 domains of talin1 or talin 2, along with GFP as a transfection marker. Cells were harvested and GFP expression and the binding of FN9–11 to live cells in the presence or absence of EDTA or the activating anti-β1 antibody 9EG7 analyzed by three-color FACS. A, dot plots showing GFP expression and FN9–11 binding. Note EDTA inhibits FN9–11 binding resulting in a leftward shift of the cell population and the integrin activating antibody 9EG7 increases FN9–11 binding inducing a rightward shift of the cell population. Expression of talin fragments did not increase FN9–11 binding to transfected cells. B, activation indices were calculated for the transfected (GFP positive) cells. C, the expression of recombinant talin domains in transfected CHO cells was examined by immunoblotting (IB) of SDS-PAGE-fractionated cell lysates. D–F, CHO cells stably expressing chimeric integrin {alpha}IIb{alpha}5β3β1A (D), {alpha}IIb{alpha}6Aβ3β1A (E), or {alpha}IIb{alpha}5β3(F) were transfected with empty vector or cDNAs encoding GFP- or HA-tagged talin domains along with a transfection marker and the activation index of the chimeric integrin assessed by measuring binding of the ligand-mimetic anti-{alpha}IIbβ3 antibody PAC1. G, CHO cells stably expressing integrin {alpha}IIbβ3 were transfected as described above. Twenty-four hours post-transfection, cells were detached, washed, and divided into two sets; one was probed with PAC1 to assess{alpha}IIbβ3 activation, the other with FN9–11 to assess activation of endogenous {alpha}5β1. H, HT1080 cells were transfected with cDNAs encoding GFP or GFP-talin1 F2F3 and the activation index of endogenous {alpha}5β1 integrins assessed as described in A. All bars represent mean activation index ± S.E. (n ≥ 3).

 
We previously showed talin F3-mediated activation of β3 integrins using the {alpha}IIbβ3-specific ligand-mimetic antibody PAC1 as a reporter (18). Therefore, to test whether differences between PAC1 and FN9–11 detection of integrin activation account for the lack of β1 activation, we measured the activation of chimeric {alpha}IIb{alpha}5β3β1A integrins stably expressed in CHO cells. These integrins have the intracellular domains of {alpha}5β1A fused to the {alpha}IIbβ3 extracellular and transmembrane domains, and so can bind PAC1 (45). Talin fragments did not induce PAC1 binding to these integrins (Fig. 1D). To examine whether the lack of talin-mediated activation was due to the presence of the {alpha}5 cytoplasmic tail we also examined activation of chimeric integrins containing the {alpha}6Aβ1A cytoplasmic domains ({alpha}IIb{alpha}6Aβ3β1A) (45). Once again talin fragments containing the F3 domain were unable to activate these integrins (Fig. 1E). However, talin F2F3 could activate chimeric {alpha}IIb{alpha}5β3 integrins (Fig. 1F). This reveals that the inability of talin fragments to activate β1 integrins does not correlate with the presence of the {alpha}5 cytoplasmic tail, but rather with the β1 tail. Furthermore, to exclude the possibility that the differences in β1 and β3 integrin activation were due to other differences between the cell lines used, we expressed talin F2F3 in an {alpha}IIbβ3-expressing CHO cell line. Twenty-four hours post-transfection, cells were detached, washed, and divided into two sets; one was probed with PAC1 to assess {alpha}IIbβ3 activation, the other with FN9–11 to assess {alpha}5β1 activation (to prevent FN9–11 binding to {alpha}IIbβ3, measurements were made in the presence of RO44-9883 a small molecule {alpha}IIbβ3 inhibitor that does not induce trans-dominant inhibition of integrin activation (36)). These experiments clearly show talin F2F3 activation of {alpha}IIbβ3 but not {alpha}5β1 (Fig. 1G). Finally we tested the ability of talin fragments to activate FN9–11 binding to β1 integrins in HT1080 fibrosarcoma cells, in HEK293 human embryonic kidney epithelial cells and in NIH/3T3 murine fibroblastic cells. Once again talin F2F3 failed to induce FN9–11 binding (Fig. 1H and data not shown). Thus using two different reporters of integrin activation, and measuring activation of endogenously and exogenously expressed integrins with a range of basal activation states, in several cell lines, we find that expressed talin fragments are capable of activating β3 but not β1 integrins.

Talin Binds Integrin β1 and β3 Tails via a Conserved Mechanism—Activation of β3 integrins requires engagement of the β3 tail by the talin F3 domain (19). The β3 tail binds talin via a variant of the canonical PTB domain-NPXY interaction and mutation of β3 residues Trp739, Leu746, or Tyr747 or of Leu746 and Lys748, which contribute to the interface, strongly impairs talin binding and integrin activation (19–21, 26). These residues are conserved in β1A integrins (Trp775, Ile782, Tyr783, and Lys784; numbering according to human β1A NP_596867 [GenBank] ) and mutations at these sites inhibit talin binding (Fig. 2, A–C). Furthermore, as was observed for β3 integrins (19), talin binding was not inhibited by mutagenesis of β1A residue Asp776, which corresponds to β3 residue Asp740 (Fig 2B). In addition, mutation of Arg358, Trp359, or Ala360 in the talin F3 domain, which inhibits binding to β3 tails (19, 26), also inhibits binding to β1A tails (Fig. 2D). Thus sequence conservation and point mutagenesis suggest that the β1-talin F3 interface is very similar to the β3-talin F3 interface. Binding of increasing concentrations of purified GST-F2F3 to β1A tails produces a dose-dependent saturable binding curve (Fig. 2E). Curve fitting using a one-site binding model yields an apparent Kd of 67 ± 14 nM. This is comparable with the reported 91–134 nM affinity of talin head, F3, or F2F3 fragments for similar recombinant β3 tail proteins, measured using pull-down, enzyme-linked immunosorbent, and surface plasmon resonance assays (18, 46–48). Thus the F3 domain of talin binds β1A and β3 tails via a conserved mechanism with comparable affinity and it therefore seems unlikely that a different binding mechanism accounts for the inability of expressed talin fragments to activate β1 integrins.


Figure 2
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  		FIGURE 2.
Talin F3 binds β1A tails via a PTB domain-NPXY-like interaction. A, amino acid sequence of the β1A cytoplasmic tail and the mutants tested in this study. B, pull-down assays using wild-type and mutant recombinant β1A tail proteins were performed with CHO cell lysates. Binding of talin, was assessed by Western blotting. Loading of each tail protein was judged by protein staining. Lysate represents 5% of the starting material in the binding assay. C, talin binding was quantified by densitometry and normalized to the lysate control (mean ± S.E.; n ≥ 3). D, binding of wild-type or mutant GST-talin1 F3 domains to β1A tail proteins was assessed in pull-down assays followed by Western blotting with anti-GST antibodies. E, binding of increasing amounts of GST-talin1 F2F3 domains to β1A tail proteins was assessed in pull-down assays followed by protein staining (left panel). Bound GST-talin1 F2F3 was quantified by scanning densitometry and the amount bound was plotted as percent maximal binding against the input concentration. Non-linear curve fitting was performed with a 1 binding site model (right panel). Results represent mean ± S.E. (n ≥ 4; R2 = 0.86).

 
Talin Binding Is Required for β1 Integrin Activation—Loss of talin expression inhibits activation of β1 integrins (19, 34, 35). To test whether talin binding is required for β1A integrin activation we measured the activation state of integrins containing point mutations that inhibit talin binding. As was observed for β3 integrins (19), point mutations that impair talin binding to β1A tails markedly inhibit integrin activation, whereas mutations within this region that do not inhibit talin binding do not inhibit integrin activation (Fig. 3). Thus, as was observed for β3 integrins, talin binding to the β tail is necessary for β1 integrin activation.

Larger Portions of Talin Are Capable of Activating β1 Integrins—The preceding data indicate that whereas talin binding is necessary for β1 integrin activation, minimal β1A binding fragments of talin are unable to activate β1A integrins. We therefore examined whether the entire talin1 head (residues 1–433) was capable of activating β1 integrins. As shown in Fig. 4A expression of GFP-tagged talin1-(1–433) produced a dose-dependent increase in FN9–11 binding. This was not due to changes in surface expression levels of {alpha}5β1 as these were unchanged in cells expressing the talin head (data not shown). Thus the talin1 head but not the F3 or F2F3 regions alone were capable of activating {alpha}5β1 integrins. Similar results were obtained using HA-tagged talin1 head (data not shown). This difference was not due to higher affinity binding of the integrin tail by talin1 head as binding assays using purified proteins allowed calculation of an apparent Kd of 91 ± 11 nM for the GST-talin1-(1–433) β1A tail interaction (Fig. 4C). This is comparable with, and not tighter than, the 67 ± 14 nM apparent Kd value obtained using the talin1 F2F3 protein (Fig. 2). Very little talin1-(1–433) binding was observed to mutant integrin tails (Fig. 4C).


Figure 3
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  		FIGURE 3.
Talin binding is required for β1 activation. CHO cells were transfected with plasmids encoding {alpha}IIb{alpha}5 and wild-type or mutant β3β1A, as indicated. Integrin expression and PAC1 binding were assessed by flow cytometry and the activation indices of transfected cells were calculated and normalized for integrin expression. Results represent mean ± S.E. (n ≥ 3).

 


Figure 4
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  		FIGURE 4.
Talin1 head activates β1 integrins. A and B, CHO cells were transfected with empty pEGFP vector or cDNAs encoding GFP-tagged talin1 head fragments. Cells were harvested and GFP expression and FN9–11 binding to live cells was analyzed by three-color FACS. A, dot plots showing GFP expression and FN9–11 binding. Expression of talin1-(1–433) but not F2F3 increases FN9–11 binding to transfected cells. B, recombinant talin domains expression in transfected CHO cells was assessed by immunoblotting SDS-PAGE-fractionated cell lysates. C, binding of increasing amounts of purified GST-talin1-(1–433) domain to β1A tail proteins (solid boxes) was assessed in pull-down assays followed by protein staining, then quantified by scanning densitometry and the amount bound (expressed as percent maximal binding) was plotted against the input concentration. Non-linear curve fitting was performed with a 1-binding site model. Talin1-(1–433) binding to the β1A(I782A, K782A) mutant (empty box) is also shown. Results represent mean ± S.E. (n ≥ 4; R2 = 0.84).

 
Talin Residues 1–205 Cooperate with the PTB-like Domain to Activate β1 Integrins—That talin1-(1–433) but not F2F3 (talin1-(206–405)) can activate β1 integrins suggests residues 1–205 and/or 406–433 contribute to the ability of talin1 head to activate β1 integrins. We therefore examined additional fragments of the talin1 head for their ability to activate β1 integrins. As shown in (Fig. 5A) talin1-(1–405) increased FN9–11 binding to a similar extent as talin1-(1–433), implying that residues 406–433 are dispensable for the integrin activation effect.

Sequence analysis indicates that the 3-lobed talin1 FERM domain spans residues 86–405 (25). However, the FERM domain (F1F2F3) is not sufficient to activate β1 integrins (Fig. 5A) indicating a requirement for the N-terminal 1–86 portion. Nonetheless, expression of talin1 F0, tagged with either HA or GFP was insufficient to activate β1 integrins suggesting that the 1–86 portion cooperates with other regions in the talin1 FERM domain to activate β1 integrins. This is confirmed by analysis of talin1-(1–405) constructs containing point mutations in the integrin-binding F3 domain. Thus, the integrin binding-defective A360E mutant of talin1-(1–405) is unable to activate β1 integrins (Fig. 5A).

The data described so far indicate that both the F0 domain and an integrin binding F3 domain are required to induce binding of FN9–11. To test whether the F1 domain is also required we generated a talin1-(1–405) construct lacking F1 (F0F2F3). When expressed in cells this was unable to activate β1 integrins (Fig. 5C). Thus, residues 1–205 and the F3 domain are needed for β1 integrin activation. These regions must be included in the same protein as co-expression of either F0 or F0F1 domains along with F2F3 (206–405) failed to activate β1 integrins. In summary, the N-terminal 405 amino acids, including the N-terminal portion and the entire talin1 FERM domain with an intact integrin β tail binding site are required for detectable activation of β1 integrins.

The N-terminal 205 Amino Acids of Talin Cooperate with the F3 Domain to Activate β3 Integrins—Unlike β1 integrins, β3 integrins are activated by expression of the F3 or F2F3 domains of talin1. Nonetheless, we wished to determine whether the N-terminal portion of the head could enhance this activation. As shown in Fig. 6, {alpha}IIbβ3 activation was further stimulated by larger talin1 fragments indicating that the F1 and N-terminal F0 domain cooperate with the F3 domain to activate β3 integrins. As was observed for β1 integrins this activation depended on an intact β tail binding site within the F3 domain.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have compared the ability of talin fragments to activate β1 and β3 integrins, and find that β1 integrins require larger fragments of talin to produce detectable activation. This observation allowed us to identify a role for the N-terminal 205 amino acids of talin in the activation of both β1 and β3 integrins.


Figure 5
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  		FIGURE 5.
Talin1 F0 and F1 domains are needed for F3-mediated activation of β1 integrins. A, CHO cells were transfected with empty pEGFP vector or cDNAs encoding GFP-tagged talin1 head domain fragments and {alpha}5β1 integrin activation assessed. Activation indices of transfected cells were calculated and normalized for {alpha}5β1 integrin expression. Results represent mean ± S.E. (n ≥ 3). B, expression of recombinant talin domains in transfected CHO cells was assessed by immunoblotting SDS-PAGE-fractionated cell lysates. C, CHO cells were transfected with GFP-tagged talin1 fragments or GFP-tagged talin1 fragments and HA-tagged talin1 F2F3, and {alpha}5β1 integrin activation in transfected (GFP-positive) cells was assessed in fluorescence-activated cell sorter assays. Activation indices were calculated and normalized for integrin expression. Results represent mean ± S.E. (n ≥ 3). D, expression of recombinant talin fragments was assessed by immunoblotting (IB) SDS-PAGE-fractionated cell lysates using the correspondent antibodies.

 
We previously demonstrated that an interaction between the talin PTB-like F3 domain and an NPLY motif in the β3 integrin tail triggered {alpha}IIbβ3 integrin activation (17, 18). We have since established a more general role for talin in activation of β1 and β3 integrins (19, 21). Others have confirmed this (23, 34), and shown that talin functions during β2 and Drosophila βPS integrin activation (8, 33). A requirement for talin therefore seems to be a general feature of integrin activation.

The β1 integrins are the most widely expressed β integrin subunits. In mammals they can heterodimerize with 11 different {alpha} subunits, and they play essential roles in organisms as diverse as nematodes, flies, fish, and mammals (10, 49–52). Here we have investigated the regulation of β1 activation by talin and report that whereas interactions between the talin PTB-like domain, F3, and the WXXXXNPIY motif in the β1 integrin tail are required for activation, expression of the talin F3 domain alone is not sufficient to activate β1 integrins.

Our conclusion that talin binding is required for β1 activation is based on the observation that point mutations in the β1A tail that inhibit talin binding also inhibit integrin activation, whereas adjacent mutations that do not inhibit talin binding do no inhibit activation. This is consistent with findings that β1 activation is inhibited by loss of talin expression (19, 34, 35), or by talin sequestration (21). Furthermore, when we use larger fragments of talin capable of activating β1 integrins, activation is blocked by point mutations in the talin F3 domain that inhibit β1 tail binding. Taken together this clearly points to a requirement for talin binding for β1 integrin activation.

Despite the requirement for a talin F3-β1A tail interaction, short talin fragments containing the β1A tail-binding F3 domain are unable to induce β1 activation even in situations where they clearly activate β3 integrins. We do not believe that this difference can be accounted for by any differences in the sensitivity of the β1 and β3 activation assays, differences in the basal activation state of β1 and β3 integrins, or differences in the affinity of F3 for β1 and β3 tails. These conclusions are based on the observations that: 1) when assessed using the same PAC1 binding assay used for β3 integrins short talin fragments were unable to activate chimeric β3β1A integrins composed of the extracellular and transmembrane domains of β3 and the β1A cytoplasmic tail. 2) Integrin activation states vary between integrins and between cell types (37) and we have assessed {alpha}5β1 activation in a variety of cell types with a range of basal activation levels, however, neither talin F3 nor talin F2F3 was able to increase {alpha}5β1 activation in any of these cells. 3) Our data suggest that talin binds β1A and β3 integrins via a conserved PTB-domain WXXXXNP(I/L)Y motif interaction with comparable in vitro affinities so differences in the mode of binding are unlikely to account for the failure of talin F3 to activate β1A integrins.

Given the requirement of talin binding for activation but the inability of short PTB-like domain containing fragments of talin to activate β1 integrins we investigated whether larger pieces of talin could activate β1 integrins. Because full-length talin may be autoinhibited and hence poorly activating we tested activation by the talin head. This produced a consistent dose-dependent activation of {alpha}5β1 integrins. Analysis of various portions of the talin head revealed that the N-terminal 205 amino acids cooperated with F3 during β1 activation. This region contains the F1 portion of the talin FERM domain and F0, an N-terminal 85-amino acid region, both of which are required for β1 activation. A similar situation exists for β3 integrins, thus although F3 or F2F3 domains are capable of activating β3 integrins activation is significantly enhanced by addition of the F1 and F0 portions.


Figure 6
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  		FIGURE 6.
Talin1 F0F1 domains help F3 domain activating β3 integrins. CHO cells stably expressing integrin {alpha}IIbβ3 were transfected with empty pEGFP vector or cDNAs encoding GFP-tagged talin1 domains. Cells were harvested, then GFP expression and the binding of PAC1 to live cells in the presence or absence of inhibitors or activators analyzed by three-color FACS. A, representative dot plots showing GFP expression and PAC1 binding. Expression of talin1-(1–433) domain increases PAC1 binding more than expression of talin1 F2F3 domains. B, {alpha}IIbβ3 integrin activation by different domains of talin1 head. Activation indices were calculated for the transfected (GFP positive) cells and normalized for integrin expression. Results represent mean ± S.E. (n ≥ 3). C, expression of recombinant talin domains was assessed by immunoblotting SDS-PAGE-fractionated cell lysates. IB, immunoblot.

 
The mechanisms by which these portions of the talin head contribute to integrin activation remain unclear and will be the subject of future studies. However, we find that activation requires an intact integrin binding site in the F3 domain and that the PTB domain needs to be attached to the 1–205 portion as neither expression of F0, F0F1, talin 1–405(A360E), nor coexpression of F0 or F0F1 along with F2F3 was sufficient to activate β1 integrins or to enhance the activation of β3 integrins (Fig. 7). This is consistent with our data showing that an F3-β tail interaction is required for activation. Thus the N-terminal half of the talin head cooperates with the PTB domain to support integrin activation. Talin F0- or F1-binding proteins have not been reported and we could not detect direct binding of either F0 or F0F1 domains to integrin β tails (data not shown). The basis of the cooperation between F0F1 and talin F3 is apparently not an increase in affinity for the β tails as we have previously reported that the β3 tail binds the talin head and F3 with comparable affinities (18, 46) and we show here that the talin head does not bind β1 tails with higher affinity than the isolated F3 domain does. Instead, we speculate that residues in F0F1 may play a role in targeting and or recruiting talin to integrins allowing the F3 domain to bind to and activate integrins. In this regard we note that Wegener et al. (30) have reported data indicating that interactions between F3 and the membrane are also important for talin-mediated integrin activation and point out that when the complete FERM domain is modeled, further basic residues from the F1 and F2 domains are brought into apposition with the membrane. Thus membrane binding sites in F1 and/or F0 may contribute to talin targeting and differences in the composition of the membrane in the vicinity of β1 or β3 integrins could contribute to the different requirements for β1 versus β3 integrin activation.


Figure 7
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  		FIGURE 7.
Summary of β1 and β3 integrin activation by talin domains.

 
We note that even the larger more active fragments of talin produce a relatively small increase in activation of {alpha}5β1 integrins. The effect on β3 integrins is more pronounced, although even there activating fragments do not achieve maximal activation. This is consistent with the suggestion of Ma et al. (22) that other co-regulators may be involved in integrin activation and that talin may induce a partial or intermediate state of integrin activation. Additional structural and functional studies will be required to determine whether the ability of F3 to activate β3 but not β1A integrins represents a difference in the mechanism of activation of the two integrins or instead represents a difference in susceptibility of the integrins to activation by talin. For example, β1 integrins may have a higher threshold for activation perhaps due to a tighter association between the {alpha} and β membrane proximal regions or due to a difference in the availability of co-activating factors or inhibitors, and so require the more potent larger fragments of talin.

In summary, whereas many questions remain to be answered, our findings that there is a difference in the talin-mediated activation of β1 and β3 and that the N-terminal domains of the talin head cooperate with the PTB-like domain to mediate integrin activation should impact future thinking about how talin-mediated integrin activation is regulated in cells and provides a starting point for studies that address the mechanisms by which cells differentially regulate their integrins.


   FOOTNOTES
 
* This work was supported by Grants GM068600 and HL089433 from the National Institutes of Health (to D. A. C.) and by a fellowship from the Fondation pour la Recherche Médicale (to M. B.). 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

1 To whom correspondence should be addressed: 333 Cedar St., P. O. Box 208066, New Haven, CT 06520-8066. Tel.: 203-737-2311; Fax: 203-785-7670; E-mail: david.calderwood{at}yale.edu.

2 The abbreviations used are: PTB domain, phosphotyrosine binding domain; HA, hemagglutinin; GST, glutathione S-transferase; GFP, green fluorescent protein; FN, fibronectin; CHO, Chinese hamster ovary; GMFI, geometric mean fluorescence intensity. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Horst Kessler and Dominik Heckmann (Department of Chemistry, Technical University München, Garching, Germany) for providing the {alpha}5β1 specific inhibitor 3F.



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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