The talin FERM domain is not so FERM

The structure of the head domain of talin, an intracellular activator of integrin membrane adhesion receptors, has been solved by Elliott et?al. (2010). A FERM domain can be identified in the head from sequence comparisons but, rather than having a compact structure of three subdomains, it has linear arrangement of four subdomains.     

The talin-tail interaction places integrin activation on FERM ground

Integrins are essential receptors for the development and functioning of multicellular animals because they mediate cell migration and cell adhesion, and regulate cell proliferation and apoptosis. Cellular regulation of the affinity of integrins for ligands - so-called 'integrin activation' - is a central property of these receptors. Integrin activation controls cell adhesion, migration and extracellular matrix assembly, thereby contributing to processes such as angiogenesis, tumor cell metastasis, inflammation, the immune response and hemostasis. Recent studies indicate that a crucial, final step in integrin activation is the binding of talin, a cytoskeletal protein, to the cytoplasmic domain of the integrin beta subunit. These results provide a focus for unraveling the many biochemical pathways implicated in integrin activation and suggest a general structural model for the connections between integrins and diverse cellular signal transduction pathways.     

Characterization of an actin-binding site within the talin FERM domain

Talin is a large cytoskeletal protein that couples integrins to F-actin. Three actin-binding sites (ABS1-3) have been reported: one in the N-terminal head, and two in the C-terminal rod domain. Although the C-terminal ABS3 has been partially characterized, the presence and properties of ABS1 within the talin head are less well defined. We show here that the talin head binds F-actin in vitro and in vivo at a specific site within the actin filament. Thus, purified talin head liberated from gizzard talin by calpain cleavage cosediments with F-actin in a low salt buffer at pH 6.4 (conditions that are optimal for binding intact talin), and using recombinant polypeptides, we have mapped ABS1 to the FERM domain within the talin head. Both the F2 and F3 FERM subdomains contribute to binding, and EGFP-tagged FERM subdomains colocalize with actin stress fibers when expressed in COS cells. High-resolution electron microscopy of actin filaments decorated with F2F3 localizes binding to a site that is distinct from that recognized by members of the calponin-homology superfamily. Finally, we show that the FERM domain can couple F-actin to PIPkin, and by inference to integrins, since they bind to the same pocket in the F3 subdomain. This suggests that the talin FERM domain functions as a linker between PIPkin or integrins and F-actin at sites of cell-matrix adhesions.     

The integrin binding site 2 (IBS2) in the talin rod domain is essential for linking integrin beta subunits to the cytoskeleton

Talin1 is a large cytoskeletal protein that links integrins to actin filaments through two distinct integrin binding sites, one present in the talin head domain (IBS1) necessary for integrin activation and a second (IBS2) that we have previously mapped to talin residues 1984-2113 (fragment J) of the talin rod domain (1 Tremuth, L., Kreis, S., Melchior, C., Hoebeke, J., Ronde, P., Plancon, S., Takeda, K., and Kieffer, N. (2004) J. Biol. Chem. 279, 22258-22266), but whose functional role is still elusive. Using a bioinformatics and cell biology approach, we have determined the minimal structure of IBS2 and show that this integrin binding site corresponds to 23 residues located in alpha helix 50 of the talin rod domain (residues 2077-2099). Alanine mutation of 2 highly conserved residues (L2094A/I2095A) within this alpha helix, which disrupted the alpha-helical structure of IBS2 as demonstrated by infrared spectroscopy and limited trypsin proteolysis, was sufficient to prevent in vivo talin fragment J targeting to alphaIIbbeta3 integrin in focal adhesions and to inhibit in vitro this association as shown by an alphaIIbbeta3 pulldown assay. Moreover, expression of a full-length mouse green fluorescent protein-talin LI/AA mutant in mouse talin1(-/-) cells was unable to rescue the inability of these cells to assemble focal adhesions (in contrast to green fluorescent protein-talin wild type) despite the presence of IBS1. Our data provide the first direct evidence that IBS2 in the talin rod is essential to link integrins to the cytoskeleton.     

Calpain cleavage promotes talin binding to the beta 3 integrin cytoplasmic domain

Talin links integrin beta cytoplasmic domains to the actin cytoskeleton and is involved in the clustering and activation of these receptors. To understand how talin recognizes integrin beta cytoplasmic domains, we configured surface plasmon resonance methodology to measure the interaction of talin with the beta3 integrin cytoplasmic domain. Here we report that the N-terminal approximately 47-kDa talin head domain (talin-H) has a 6-fold higher binding affinity than intact talin for the beta3 tail. The affinity difference is mainly due to a difference in k(on). Calpain cleavage of intact talin released talin-H and resulted in a 16-fold increase in apparent K(a) and a 100-fold increase in apparent k(on). The increase in talin binding after cleavage was greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding was due to cooperative binding of talin-H and talin rod domain to the beta3 tail. Talin resembles ERM (ezrin, radixin, moesin) proteins in possessing an N-terminal FERM (band four-point-one, ezrin, radixin, moesin) domain. These data show that the talin FERM domain, like that in the ERM proteins, is masked in the intact molecule. Furthermore, they suggest that talin cleavage by calpain may contribute to the effects of the protease on the clustering and activation of integrins.     

The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.     

Phosphatidylinositol phosphate kinase type 1gamma and beta1-integrin cytoplasmic domain bind to the same region in the talin FERM domain

Talin is an essential component of focal adhesions that couples beta-integrin cytodomains to F-actin and provides a scaffold for signaling proteins. Recently, the integrin beta3 cytodomain and phosphatidylinositol phosphate (PIP) kinase type 1gamma (a phosphatidylinositol 4,5-bisphosphate-synthesizing enzyme) were shown to bind to the talin FERM domain (subdomain F3). We have characterized the PIP kinase-binding site by NMR using a 15N-labeled talin F2F3 polypeptide. A PIP kinase peptide containing the minimal talin-binding site formed a 1:1 complex with F2F3, causing a substantial number of chemical shift changes. In particular, two of the three Arg residues (Arg339 and Arg358), four of eight Ile residues, and one of seven Val residues in F3 were affected. Although a R339A mutation did not affect the exchange kinetics, R358A or R358K mutations markedly weakened binding. The Kd for the interaction determined by Trp fluorescence was 6 microm, and the R358A mutation increased the Kd to 35 microm. Comparison of these results with those of the crystal structure of a beta3-integrin cytodomain talin F2F3 chimera shows that both PIP kinase and integrins bind to the same surface of the talin F3 subdomain. Indeed, binding of talin present in rat brain extracts to a glutathione S-transferase integrin beta1-cytodomain polypeptide was inhibited by the PIP kinase peptide. The results suggest that ternary complex formation with a single talin FERM domain is unlikely, although both integrins and PIP kinase may bind simultaneously to the talin anti-parallel dimer.     

The Structure of the talin head reveals a novel extended conformation of the FERM domain

FERM domains are found in a diverse superfamily of signaling and adaptor proteins at membrane interfaces. They typically consist of three separately folded domains (F1, F2, F3) in a compact cloverleaf structure. The crystal structure of the N-terminal head of the integrin-associated cytoskeletal protein talin reported here reveals a novel FERM domain with a linear domain arrangement, plus an additional domain F0 packed against F1. While F3 binds β-integrin tails, basic residues in F1 and F2 are required for membrane association and for integrin activation. We show that these same residues are also required for cell spreading and focal adhesion assembly in cells. We suggest that the extended conformation of the talin head allows simultaneous binding to integrins via F3 and to PtdIns(4,5)P2-enriched microdomains via basic residues distributed along one surface of the talin head, and that these multiple interactions are required to stabilize integrins in the activated state.     

Structural basis for the autoinhibition of talin in regulating integrin activation

Activation of heterodimeric (alpha/beta) integrin transmembrane receptors by the 270 kDa cytoskeletal protein talin is essential for many important cell adhesive and physiological responses. A key step in this process involves interaction of phosphotyrosine-binding (PTB) domain in the N-terminal head of talin (talin-H) with integrin beta membrane-proximal cytoplasmic tails (beta-MP-CTs). Compared to talin-H, intact talin exhibits low potency in inducing integrin activation. Using NMR spectroscopy, we show that the large C-terminal rod domain of talin (talin-R) interacts with talin-H and allosterically restrains talin in a closed conformation. We further demonstrate that talin-R specifically masks a region in talin-PTB where integrin beta-MP-CT binds and competes with it for binding to talin-PTB. The inhibitory interaction is disrupted by a constitutively activating mutation (M319A) or by phosphatidylinositol 4,5-bisphosphate, a known talin activator. These data define a distinct autoinhibition mechanism for talin and suggest how it controls integrin activation and cell adhesion.     

The talin wags the dog: new insights into integrin activation

Integrin transmembrane receptors have a unique property that distinguishes them from other signaling receptors. Their affinity for ligands can be modulated from the inside out in response to intracellular signals generated by non-integrin receptors. Recent findings provide novel mechanistic insights into this process by demonstrating that talin, a protein that links integrins to actin, is necessary for the inside-out activation of integrins.     

Structure of a double ubiquitin‐like domain in the talin head: a role in integrin activation

Talin is a 270‐kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N‐terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β‐integrin tails, F0 and F1 are also required for activation of β1‐integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin‐like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin‐mediated activation of β1‐integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.     

Dynamic interaction between Src and C-terminal Src kinase in integrin alphaIIbbeta3-mediated signaling to the cytoskeleton

Integrin-bound Src tyrosine kinase mediates alpha(IIb)beta(3) out-side-in signaling to the cytoskeleton required for platelet adhesion and thrombus formation. Src activation (signal initiation) by phosphorylation of Tyr-418 occurs at lamellipodia leading edges. However, little is known about Src inactivation mediated by C-terminal Src kinase (Csk) Tyr-529 phosphorylation. In an established platelet model cell line (A5-Chinese hamster ovary), we studied the inactivation of Src during alpha(IIb)beta(3)-mediated adhesion to fibrinogen with live cell fluorescence resonance energy transfer (FRET) microscopy. Imaging revealed highly dynamic Src-Csk interactions at the leading edges of active lamellipodia. The Src-Csk interaction followed a highly dynamic pattern. Every 2-3 min, Src-Csk complexes moved inward in the cell, reorganized, and formed stable focal adhesions. These accumulations were primarily seen during retraction of lamellipodia, whereas no interaction was observed during protrusions. Western blot analysis during the run time of FRET signaling revealed an increase in Csk-mediated SrcTyr-529 phosphorylation with a parallel decline of tyrosine 418 phosphorylation. Mutation analysis provided additional insights into the role of Src. Although inactivation of Csk (CskK222R) had no effect on cell adhesion and spreading efficiency, cells with constitutively active expressed Src (SrcY529F) exhibited hardly any adhesion and no spreading. The few adherent cells showed weak focal adhesions that were disorganized and oversized. The data clearly demonstrate the important role of tight Src control by Csk for functional cell adhesion and spreading. The novel experimental FRET approach reported here for the inactivation of Src can be readily applied to other integrin and signaling pathways, including closely related Src family kinase members.     

The copper binding domain of SPARC mediates cell survival in vitro via interaction with integrin beta1 and activation of integrin-linked kinase

Secreted protein acidic and rich in cysteine (SPARC) is important for the normal growth and maintenance of the murine lens. SPARC-null animals develop cataracts associated with a derangement of the lens capsule basement membrane and alterations in lens fiber morphology. Cellular stress and disregulation of apoptotic pathways within lens epithelial cells (LEC) are linked to cataract formation. To identify molecular targets of SPARC that are linked to this disorder, we stressed wild-type (WT) and SPARC-null LEC by serum deprivation or exposure to tunicamycin. SPARC enhanced signaling by integrin-linked kinase (ILK), a serine/threonine kinase known to enhance cell survival in vitro. In response to stress, an ILK-dependent decrease in apoptosis was observed in WT relative to SPARCg-null LEC. Co-immunoprecipitation and cross-linking of cell lysates revealed enhanced levels of a SPARC-integrin beta1 complex during stress. Competition with monoclonal antibodies and peptides indicated that the copper binding domain of SPARC is required for SPARC-mediated response to stress. Inhibiting the binding and/or activity of ILK, integrin beta1, or SPARC resulted in increased apoptosis of stressed LEC. We conclude that SPARC protects cells from stress-induced apoptosis in vitro via an interaction with integrin beta1 heterodimers that enhances ILK activation and pro-survival activity.     

Cooperativity between integrin activation and mechanical stress leads to integrin clustering

Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays.     

Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain

Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P₂ by type 1 phosphatidyl inositol phosphate kinase type 1γ (PIPK1γ) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin β-subunit cytoplasmic domain and PIPK1γ, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1γ and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.     

A truncated FAK lacking the FERM domain displays high catalytic activity but retains responsiveness to adhesion-mediated signals

In order to determine the role of the FERM domain in the regulation of FAK phosphorylation at Tyr-397, the major autophosphorylation site, we generated a truncated FAK lacking a region of the N-terminus corresponding to amino acids 1-384 (FAKDelta384). FAKDelta384 showed a striking increase in phosphorylation, as compared with wild type FAK, in lysates of either HEK 293 or FAK-/- cells. Interestingly, the truncated form of FAK lacking the N-terminal domain retains responsiveness to integrin-mediated signals, as judged by its dephosphorylation by holding cells in suspension and by the recovery of the phosphorylation when replating the cells on fibronectin. We propose a model in which removal of FERM-mediated auto-inhibition is important to increase FAK catalytic activity but the translocation and clustering of this enzyme at the focal adhesions is required for maximal phosphorylation at Tyr-397.     

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.     

An interaction between alpha-actinin and the beta 1 integrin subunit in vitro

A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely alpha-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect the additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of beta 1 integrin and affinity chromatography to identify additional integrin-binding proteins. Here we report our finding of an interaction between the cytoplasmic domain of beta 1 integrin and the actin-binding protein alpha-actinin. Beta 1-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at approximately 100 kD was identified by immunoblot analysis as alpha-actinin. Solid phase binding assays indicated that alpha-actinin bound specifically and directly to the beta 1 peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a beta 1 integrin) or the platelet integrin glycoprotein IIb/IIIa complex (a beta 3 integrin), binding of alpha-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the beta 1 peptide. alpha-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-alpha-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.     

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.     

An integrin phosphorylation switch: the effect of beta3 integrin tail phosphorylation on Dok1 and talin binding

Integrins play a fundamental role in cell migration and adhesion; knowledge of how they are regulated and controlled is vital for understanding these processes. Recent work showed that Dok1 negatively regulates integrin activation, presumably by competition with talin. To understand how this occurs, we used NMR spectroscopy and x-ray crystallography to investigate the molecular details of interactions with integrins. The binding affinities of beta3 integrin tails for the Dok1 and talin phosphotyrosine binding domains were quantified using 15N-1H hetero-nuclear single quantum correlation titrations, revealing that the unphosphorylated integrin tail binds more strongly to talin than Dok1. Chemical shift mapping showed that unlike talin, Dok1 exclusively interacts with the canonical NPXY motif of the beta3 integrin tail. Upon phosphorylation of Tyr 747 in the beta3 integrin tail, however, Dok1 then binds much more strongly than talin. Thus, we show that phosphorylation of Tyr 747 provides a switch for integrin ligand binding. This switch may represent an in vivo mechanism for control of integrin receptor activation. These results have implications for the control of integrin signaling by proteins containing phosphotyrosine binding domains.