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J Biol Chem, Vol. 274, Issue 40, 28071-28074, October 1, 1999

COMMUNICATION
The Talin Head Domain Binds to Integrin  Subunit Cytoplasmic Tails and Regulates Integrin Activation^*

David A. Calderwood^§, Roy Zent^¶, Richard Grant^§§, D. Jasper G. Rees, Richard O. Hynes^**, and Mark H. Ginsberg

From the  Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, ^§§ Cambridge Molecular, Cambridge CB5 8PB, United Kingdom, the  Department of Biochemistry, University of the Western Cape, Bellville 7535, Republic of South Africa, and the ^** Howard Hughes Medical Institute and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The  subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to  integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of ₁A, ₁D and ₃ integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin  tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin _IIb3; activation was dependent on the presence of both the talin head domain and the integrin ₃ cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The connection of integrin adhesion receptors to the actin cytoskeleton regulates cell shape, adhesion, and migration (1). Talin, a cytoplasmic protein composed of ~270-kDa subunits binds to integrin  cytoplasmic tails, vinculin and actin filaments (2-6), and co-localizes with integrins at sites of cell-substratum contact (7). It plays an important role in the establishment and maintenance of integrin-cytoskeleton connections, and loss of talin expression leads to impaired cell adhesion, spreading, and migration (8). Talin consists of an N-terminal ~50-kDa globular head domain and an ~220-kDa C-terminal rod domain (9). The N-terminal talin head domain contains an ~200-residue region similar to a region within the membrane-binding N-terminal ERM association domain (N-ERMAD) in the ezrin, radixin, and moesin (ERM) family of proteins (9). The N-ERMAD domain of ERM proteins binds to the cytoplasmic domain of transmembrane receptors (e.g. CD44), and the C-terminal domain binds to actin, linking the receptor to the cytoskeleton (10). In contrast to ERM proteins, previous studies indicate that the C-terminal rod domain of talin contains the integrin-binding site and the vinculin- and actin-binding sites (5, 6, 11). Thus, talin might differ from other ERM proteins in the manner in which it connects membrane proteins to actin filaments.

Talin binds to recombinant structural mimics of dimerized integrin  cytoplasmic tails (2). Here, we examined the interaction of the talin head domain with three of these integrin  cytoplasmic tails and report that either recombinant or proteolytically derived talin head domains bind specifically to all three  tails. Furthermore, overexpression of the talin fragments containing the head domain "activated" integrin _IIb3 as judged by increased ligand-binding affinity. Consequently, the talin head domain binds to several integrin  tails and can thus mediate the linkage of these integrins to the actin cytoskeleton and modulate integrin function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Antibodies and cDNAs-- Monoclonal antibodies, anti-talin 8d4 (Sigma) and TA205 (Serotec), anti-GST¹ B-14 (Santa Cruz Biotechnology) and anti-Tac 7G7B6 (American Tissue Culture Collection, Manassas, VA) were obtained commercially. The anti-_IIb3 mAb PAC1 and the _IIb3-specific peptide inhibitor Ro43-5054 have been described previously (12). cDNAs encoding Tac-5, GST-chicken talin-(280-435), -(186-435), and -(1-280) and ₁A, ₁A(Y788A), or ₁D integrin cytoplasmic tails have been described previously (2, 13, 14). cDNA encoding the ₃ cytoplasmic tail was amplified by polymerase chain reaction and cloned into a modified pET 15b expression construct (2). Y/A mutations (Fig. 2A) were introduced using the QuikChange^TM site-directed mutagenesis kit (Stratagene). A cDNA encoding amino acids 1-435 of mouse talin (9) was cloned into the bacterial expression vector pGEX-2T (Amersham Pharmacia Biotech, Uppsala, Sweden). cDNAs for mouse talin encoding amino acids 1-1071 and 434-1071 (9) were cloned into pJ6 R mammalian expression vectors.

Cells and Cell Lysates-- Human platelets (obtained as described previously (2)) were lysed by sonication on ice in the presence of either Complete^TM protease inhibitor mixture (Roche Molecular Biochemicals), 0.1 mM calpain inhibitor E-64 (Roche Molecular Biochemicals), and 1 mM EDTA, or 1 mM CaCl₂, 1 mM MgCl₂. Following lysis, Complete^TM protease inhibitor mixture, E-64 (0.1 mM final), and EDTA (2 mM final) were added to the inhibitor-free sample. CHO cells stably expressing human integrin _IIb3 or _IIb3728 (15) were transfected using lipofectAMINE (Life Technologies, Inc.), and activation of _IIb3 was assayed by PAC1 binding as described previously (12).

Purification of Recombinant Proteins-- Recombinant integrin cytoplasmic tails were expressed, purified and characterized as described previously (2). GST-talin fusion proteins were expressed and purified using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech), according to the manufacturer's instructions.

Affinity Chromatography with Recombinant Integrin Cytoplasmic Tails-- Affinity chromatography was performed using recombinant integrin cytoplasmic tails bound to His-Bind^® Resin (Novagen). Binding of 200 µl of cell lysate, or 20 µl of purified GST-talin fragments, to 50 µl of coated resin was performed in a total volume of 800 µl as described previously (2). Bound proteins were fractionated by SDS-PAGE and analyzed by immunoblotting. Binding of recombinant integrin tails to the resin was verified by Coomassie Blue staining.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Binding of Talin Domains to the Integrin ₁D Cytoplasmic Tail-- Talin, a large cytoskeletal actin-binding protein, binds to the muscle-specific integrin ₁D cytoplasmic tail (2). To determine which domains of talin bind, we permitted endogenous platelet calpain to cleave talin into an ~50-kDa head domain and an ~220-kDa rod domain (Ref. 9; Fig. 1A). In the presence of calpain inhibitors, intact talin resulted in a band with an apparent molecular mass of 240 kDa that reacted with antibodies specific for the talin head (TA205) and rod (8d4) domains. Platelet lysis in the absence of calpain inhibitors resulted in the complete disappearance of the intact talin and generation of a 50-kDa, TA205-binding, head domain and an apparent 190-kDa, 8d4-reactive, rod domain (Fig. 1B). The discrepancy between the predicted and apparent molecular masses of both intact talin and the talin rod domain is consistent with previous reports (9). ₁D affinity chromatography revealed binding of both head and rod domains (Fig. 1C). Binding of both domains was specific in that it was abrogated by a tyrosine to alanine (Y/A) substitution in the first NPXY motif of ₁D (Figs. 1C and 2A). Mutation of this residue in ₁A or ₁D inhibited binding of full-length talin (data not shown) (2). Thus both domains of talin bind to a ₁D affinity matrix.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   The talin head and rod domains both bind to integrin 1D cytoplasmic tails. A, a schematic representation of talin. Cleavage by calpain II (after amino acid 433) results in a 50-kDa head domain and a 220-kDa rod domain. The location of the epitopes for the head domain-specific mAb TA205 (amino acids 139-433 in chicken talin (17) and the rod domain-specific mAb 8d4 (amino acids 433-1071 in mouse talin) are indicated. B, talin head and rod domains were generated by lysing platelets (109/ml) in Tris-buffered saline, 0.5% Triton X-100, 0.1% deoxycholate in the presence of protease inhibitors (+), or in 1 mM CaCl2, (). Proteins were fractionated by SDS-PAGE under reducing conditions and were transferred to Immobilon-P membranes and probed with monoclonal antibodies TA205 or 8d4. C, lysate prepared in the absence of protease inhibitors was mixed with beads coated with recombinant 1D or 1D(Y/A) cytoplasmic domains. Bound proteins were fractionated by SDS-PAGE, and binding of talin fragments was analyzed by Western blotting with TA205 or 8d4 mAbs. Amounts of the recombinant integrin  cytoplasmic tails bound to the beads were assayed by Coomassie Blue staining of eluted proteins (bottom panels).

Binding of Talin Domains to Other Talin-binding Integrin  Cytoplasmic Tails-- Talin also binds to ₁A and ₃ cytoplasmic tails, (2, 3). However, binding to ₁A is weaker than to ₁D and ₃ (Ref. 2 and data not shown). We therefore tested whether the same talin domains bound to ₁A and ₃ cytoplasmic tails. Both head and rod domains bound to ₁A and ₃ (Fig. 2B). Furthermore, the relative binding of each domain was similar to that of intact talin (data not shown). Substitution of Ala for Tyr in the membrane-proximal NPXY motif inhibited binding of both full-length talin (data not shown), and head and rod domains (Fig. 2B).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Integrin 1A and 3 cytoplasmic tails bind talin head and rod domains. A shows an alignment of the amino acid sequences of human 1A, 1D, and 3 integrin cytoplasmic tails. The tyrosine residues mutated to alanine in the Y/A mutants are shown as bold underlined and correspond to amino acid number 788 in chicken 1A (22) and 747 in human 3 (23). B, the binding experiments described in the legend to Fig. 1 were repeated using 1A, 1A(Y/A), 3, and 3(Y/A) coated beads. Bound talin head and rod domain were detected by Western blotting with mAbs TA205 or 8d4, respectively. The lysate shown corresponds to 10% of the starting material in the binding assay.

Talin forms antiparallel homodimers (6), and previous work (11) implicated the rod domain as the integrin-binding site. Consequently, the head domain could have bound through an association with intact integrin-bound talin. However, the absence of intact talin in the lysates (Fig. 1B) suggests that binding of the isolated domains is not mediated through their dimerization with full-length talin. Furthermore, localization of the dimerization domains to the central region of the rod domain (6) suggests that the head and rod domains do not associate following calpain cleavage. In support of this hypothesis, head and rod domains did not co-immunoprecipitate from calpain-cleaved lysates using either 8d4 or TA205 as precipitating antibodies (data not shown). Consequently, the binding of both head and rod domains to integrins probably represents independent interactions. Independent binding of the head and tail domains would explain the observation that integrin _IIb3 binds to talin with a stoichiometry of 2:1 (3).

Talin Head Domain Binds Directly to Integrin  Cytoplasmic Tails-- The experiments reported above suggested an interaction between the talin head domain and integrin  cytoplasmic tails. However, binding of the head domain could occur via a talin-binding intermediary protein present in the platelet lysate. We therefore examined the binding of purified recombinant talin head domain (talin 1-435) to ₁A, ₁D, and ₃ integrin cytoplasmic tails. Talin head domain bound to all three tails. Furthermore, Tyr to Ala mutations in the first NPXY motif inhibited binding (Fig. 3B). In addition, GST failed to bind to any of the cytoplasmic tails, and the head domain failed to bind to the _IIb cytoplasmic tail (data not shown). These results demonstrate that the isolated talin head domain contains an integrin-binding site.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   The talin head domain binds directly to integrin  cytoplasmic tails. A, schematic representation of recombinant talin head domain fragments. The region of ERM homology (9) is indicated. Beads coated with recombinant 1A, 1A(Y/A), 1D, 1D(Y/A), 3, or 3(Y/A) cytoplasmic tails were incubated with 30 µg of purified GST-talin head domain (talin-(1-435)) (B) or 7 µg of purified GST-talin-(280-435), GST-talin-(186-435), and GST-talin-(1-280) (C). Bound protein was eluted by heating in SDS-sample buffer and samples separated on 4-20% SDS-polyacrylamide gels under reducing conditions. Binding was detected by Western blotting with an anti-GST mAb. Starting material used for the assays is shown.

To further localize the integrin binding site within the talin head domain, we investigated the binding of three overlapping talin head domain fragments to ₁D cytoplasmic tails (Fig. 3, A and C). Western blotting revealed that fusion proteins of the predicted molecular mass were present (Fig. 3C). Only talin-(186-435) bound to the ₁D tails, and this binding was sensitive to Tyr to Ala mutations in the ₁D tail (Fig. 3C). A similar binding pattern was seen for ₁A and ₃ tails (data not shown). The failure of talin-(280-435) and -(1-280) to bind integrin tails indicates that amino acids within the 186-280 region are necessary, but not sufficient, for tail binding. Further localization of the binding site within amino acids 186-435 was not possible due to the insolubility of all smaller fragments tested.

Sequences within the talin head domain suggest that this region of talin is structurally related to the N-ERMAD of the ERM family of proteins (Ref. 9; Fig. 3B). ERM proteins generally function as membrane-cytoskeleton linker molecules, in which the N-ERMAD domain binds to the cytoplasmic domains of transmembrane receptors, while the C-terminal, C-ERMAD binds filamentous actin (10). Talin resembles ERM family members in the interaction of its head domain with membranes by binding to phospholipids (6) and to the cytoplasmic domain of layilin, a transmembrane protein (14). Furthermore, the talin rod domain contains binding sites for filamentous actin (5, 6). Nevertheless, talin is concentrated at focal adhesions, sites devoid of layilin (14). Our finding that talin can bind to integrin  cytoplasmic tails via its head domain, and that the integrin- binding site maps to a fragment containing most of the ERM domain, suggests that this interaction is involved in the linkage of actin filaments to integrins in a manner analogous to other ERM proteins. In addition, the head domain localizes to focal adhesions (16), and microinjection of the anti-head domain mAb TA205, or of a recombinant talin fragment containing the TA205 epitope (residues 102-497), disrupts stress fibers (5, 17). Thus, the talin head domain is implicated in linkage of integrins to the actin cytoskeleton.

Integrin Activation by N-terminal Fragments of Talin Requires the Head Domain-- Increases in integrin affinity for ligands ("activation") can be influenced by cytoskeletal linkages (4, 18-20). We reasoned that if the talin head domain bound to integrin cytoplasmic tails, then overexpression of talin fragments containing this domain might alter integrin affinity. We, therefore, expressed N-terminal fragments of talin (Fig. 4A) and assessed the activation state of _IIb3 by measuring binding of the activation-specific mAb PAC1 (12). CHO cells expressing recombinant human integrin _IIb3 were co-transfected with talin-(1-1071) cDNA along with Tac 5 as a marker of transfection. Cells expressing high levels of the transfection marker (FL-2) exhibited increased PAC1 binding (FL-1), resulting in a rightward tilt in the density plot (Fig. 4B). This effect was not seen if the expressed talin fragment lacked the head domain (talin-(434-1071)) or with vector DNA (Fig. 4, A and B). Talin-(1-1071)-induced PAC1 binding was inhibited by the _IIb3 antagonist, Ro43-5054, demonstrating the specificity of binding (data not shown). Expression of talin-(1-1071) resulted in a 2.7-fold increase in mean activation index (from 0.21 to 0.56), while a fragment lacking the head domain, talin-(434-1071), had little effect (activation index: 0.18) (Fig. 4D). These fragments had no obvious effect on cell shape (data not shown), consistent with previous results obtained by microinjection of recombinant talin-(102-497) (5). Expression of the talin fragments was confirmed by Western blotting with mAb 8d4, which recognizes endogenous hamster talin and both recombinant mouse talin fragments (Fig. 4C). Densitrometry of the blot shown in Fig. 4C revealed that the intensities of signal from talin-(1-1071) and talin-(434-1071) were 0.66 and 1.36 times, respectively, the intensity of the endogenous talin band. However, these fragments are only expressed in the transfected cells. Thus, we calculated that on a per cell basis talin-(1-1071) was 2-fold and talin-(434-1071) was 6-fold overexpressed with respect to endogenous talin, based on transfection efficiencies of 29 and 22%, respectively. We were unable to test whether overexpression of intact talin or isolated head domain results in similar integrin activation as we detected no increase in their expression following transient transfection with cDNAs encoding them in either pJ6 R or pcDNA3.1 expression vectors.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Expression of a talin fragment containing the head domain activates integrin IIb3. A, a schematic representation of mouse talin. The 8d4 epitope is shown, and bars indicate the sizes of recombinant talin fragments (1-1071 and 434-1071) used in this experiment. B, CHO cells expressing integrin IIb3 were co-transfected with cDNAs encoding Tac-5 (1 µg) and talin-(1-1071), talin-(434-1071), or vector control (4 µg), as indicated. Two days after transfection cells were harvested and analyzed by two-color flow cytometry for binding of the activation-specific anti-IIb3 ligand-mimetic mAb PAC1 and the anti-Tac mAb 7G7B6 (12). Density plots showing 7G7B6 staining (FL2-H) and PAC1 staining (FL1-H) allow comparison of integrin activity in transfected and untransfected cells. C, anti-talin (8d4) Western blot of lysates of cells used in B to show expression of talin-(1-1071), and -(434-1071) fragments. D, CHO cells expressing integrin IIb3 or integrin IIb3728 were transfected and stained as in B, and the activation index of transfected cells was calculated as (F  Fo)/(Fm  Fo), in which F is the median fluorescence intensity (MFI) of PAC1 binding; Fo is the MFI of PAC1 binding in the presence of competitive inhibitor (Ro43-5054, 1 µM); and Fm is the MFI of PAC1 binding in the presence of the integrin-activating antibody anti-LIBS6 (2 µM). Results represent means ± S.E. (n = 5). Expression of talin fragments was confirmed by Western blotting and was equal (data not shown).

To determine whether the _IIb3 activation by talin-(1-1071) required the talin-binding ₃ cytoplasmic tail, we tested CHO cells expressing _IIb3728, which lacks the C-terminal 35 amino acids of the ₃ subunit (15). Loss of the ₃ cytoplasmic tail results in an integrin that is deficient in cytoskeletal interactions, will not support cell spreading or initiation of focal adhesions, and which is not recruited to existing focal adhesions (15). As shown in Fig. 4D expression of talin-(1-1071) did not activate _IIb3728 in CHO cells; however, _IIb3728 could be activated exogenously by addition of the activating mAb anti-LIBS6 (data not shown (12)). Consequently, expression of fragments of talin containing the head domain can increase the ligand-binding affinity of integrin _IIb3 when the integrin contains an intact ₃ cytoplasmic tail.

Following platelet activation talin is redistributed from the cytoplasm to the cytoplasmic face of the plasma membrane (21), raising the possibility that it plays a role in the regulation of adhesive functions of platelets. The data presented above implicate talin in the regulation of _IIb3 ligand binding affinity; however, the molecular mechanism of this regulation remains unclear. Intact talin binding to integrin  tails via its head domain may retain the integrin in an inactive state (4), and the isolated fragment may break these tethers and so activate the integrin (19, 20). Alternatively, talin binding may alter integrin affinity through direct conformational changes or clustering the integrin. Additional studies are under way to distinguish among these potential mechanisms.

In conclusion, we have found (i) that the talin head domain contains a binding site for the cytoplasmic tails of integrins ₁A, ₁D, and ₃ and (ii) that expression of a talin fragment containing the head domain activates integrin _IIb3 in a manner dependent on the ₃ cytoplasmic tail. Identification of an integrin-binding site in the talin head domain is consistent with talin's similarity to the ERM family of proteins. Thus, the talin head domain can form an integrin-binding element in a physical link between integrins and the actin cytoskeleton.

	Note Added in Proof

The paper by Patil et al. (Patil, S., Jedsadayanmata, A., Wencel-Drake, J. D., Wang, W., Knezevic, I., and Lam, S. C.-T. (1999) J. Biol. Chem. 274, 28575-28583) describes the interaction of the talin head domain with integrin _IIb3.

	FOOTNOTES

^* This work was supported by Grants HL48728, HL59007, CA17007, and AR27214 from the National Institutes of Health. This is publication number 12590-VB.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Wellcome Trust International Prize Traveling Fellow.

^¶ Fellow of the National Kidney Foundation.

To whom correspondence should be addressed. Tel.: 858-784-7143; Fax: 858-784-7343; E-mail: ginsberg@scripps.edu.

	ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; CHO, Chinese hamster ovary MFI, median fluorescence intensity.

	REFERENCES

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