The animal talins are large, modular proteins that link the actin cytoskeleton to the extracellular environment through interactions with β-integrins and actin. Dictyostelium discoideum has two talins, TalA and TalB, which have distinct physiological roles in cell adhesion, cell differentiation, and cytokinesis. A second talin gene has been identified in vertebrates. Thus, talin function in vertebrates is also due to the action of multiple proteins. Using a phylogenomic approach it has been determined that D. discoideum TalA/B and the animal talins are related by descent from a common ancestral talin and that duplication of TLN2 early in the chordate lineage produced TLN1. An additional duplication subsequently produced a second Talin-2 in teleost fishes and a second Talin-1 in Xenopus laevis. Vertebrate Talin-2 mRNA is alternatively processed. In Drosophila melanogaster and in the non-vertebrate chordate Ciona intestinalis, which each have only one talin gene, alternative processing of talin mRNA also produces multiple talin species. Thus, in these organisms, talin function may be due to the action of more than one protein. To identify isoform-specific functions of vertebrate talins it has been shown through proteomic analysis that mammalian Talin-1 and Talin-2 bind to different protein partners. Further characterization of the differences between animal talins, with Talin-1 and Talin-2 in model vertebrates, will provide an experimental system for studying neofunctionalization or subfunctionalization of talin following the vertebrate talin gene duplication, especially the direct comparison of talins in the model urochordate C. intestinalis, which has one talin gene that produces two talins through alternative mRNA splicing (Senetar, 2005).
The amino acid sequence of the rod portion of talin contains strong periodic patterns with a long period of 32 to 34 residues superimposed on short periods of 7 and 7/2 residues. The rod includes 50 to 60 copies of an irregular repeated motif approximately 34 residues long. The motif itself consists of three sections: a short "leader" segment of about six residues, that has a high proportion of the prolines and acidic residues; a relatively well-conserved hydrophobic 'core' pattern of approximately 21 residues; and a highly variable 'linker' region of seven residues which joins onto the next leader. The core section sequence has many of the characteristics of an amphipathic helix. The extensive hydrophobic side of this postulated helix has a characteristic surface pattern of large and small hydrophobic residues (mainly Leu and Ala), with a strong periodicity of seven residues. It also has a narrow hydrophilic edge with a highly variable sequence. The core sequence is unlike either a normal helical coiled coil or a leucine zipper, because it contains several helical ridges and grooves. The helical cores probably form a tightly packed hydrophobic central strand for the fibrous tail. The leader and linker sections are highly variable in length, so that the spacing between the starting points of adjacent cores varies between 20 and 40 residues. The most common spacing is 34, and many spacings are close to this length (McLachlan, 1994).
Thrombin digestion of human platelet talin yields two polypeptide domains of 200 kDa and 47 kDa. These fragments and their functional properties have been analyzed: the 200-kDa fragment is active in nucleating actin filament formation and reduces the viscosity of filamentous actin, comparable to the effects of the intact protein. The 47-kDa fragment is inactive in this respect. However, the 47-kDa polypeptide, but not the 200-kDa fragment, interacts specifically with large liposomes containing acidic phospholipids. The 200-kDa fragment, whether alone or in conjunction with the small fragment, does not co-sediment with liposomes. Thus it is possible to attribute specialized functions to distinct domains on the talin molecule. These enable the protein to interact simultaneously with actin filaments and lipid membranes (Niggli, 1994).
Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, the functional domain structure of intact platelet talin and its proteolytic fragments have been mapped. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide-specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N-terminal 47 kDa domain (i.e., S19, amino acids 21-39; H18, amino acids 287-304; and H17, amino acids 385-406). Antibodies H17 and H18 localize these lipid binding sequences within the N-terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, it is concluded that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell-shaped molecule built of two antiparallel subunits (Isenberg, 1998).
To understand how talin recognizes integrin beta cytoplasmic domains, surface plasmon resonance methodology was configured to measure the interaction of talin with the beta3 integrin cytoplasmic domain. 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 releases talin-H and results 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 is greater than predicted for stoichiometric liberation of free talin-H. This additional increase in binding is 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 (Yan, 2001).
The I/LWEQ module superfamily is a class of actin-binding proteins that contains a conserved C-terminal actin-binding element known as the I/LWEQ module. I/LWEQ module proteins include the metazoan talins, the cellular slime mold talin homologues TalA and TalB, fungal Sla2p, and the metazoan Sla2 homologues Hip1 and Hip12 (Hip1R). These proteins possess a similar modular organization that includes an I/LWEQ module at their C-termini and either a FERM domain or an ENTH domain at their N-termini. As a result of this modular organization, I/LWEQ module proteins may serve as linkers between cellular compartments, such as the plasma membrane and the endocytic machinery, and the actin cytoskeleton. I/LWEQ module proteins bind to F-actin. This report describes a determination of the affinity of the I/LWEQ module proteins Talin1, Talin2, huntingtin interacting protein-1 (Hip1), and the Hip1-related protein (Hip1R/Hip12) for F-actin and identified a conserved structural element that interferes with the actin binding capacity of these proteins. The data support the hypothesis that the actin-binding determinants in native talin and other I/LWEQ module proteins are cryptic and indicate that the actin binding capacities of Talin1, Talin2, Hip1, and Hip12 are regulated by intrasteric occlusion of primary actin-binding determinants within the I/LWEQ module. It was also found that the I/LWEQ module contains a dimerization motif and stabilizes actin filaments against depolymerization. This activity may contribute to the function of talin in cell adhesion and the roles of Hip1, Hip12 (Hip1R), and Sla2p in endocytosis (Senetar, 2004).
Talin is a high-molecular-weight cytoskeletal protein concentrated at regions of cell-substratum contact and, in lymphocytes, at cell-cell contacts. Integrin receptors are involved in the attachment of adherent cells to extracellular matrices and of lymphocytes to other cells. In these situations, talin codistributes with concentrations of integrins in the cell surface membrane. Furthermore, in vitro binding studies suggest that integrins bind to talin, although with low affinity. Talin also binds with high affinity to vinculin, another cytoskeletal protein concentrated at points of cell adhesion. Finally, talin is a substrate for the Ca2(+)-activated protease, calpain II, which is also concentrated at points of cell-substratum contact. To learn more about the structure of talin and its involvement in transmembrane connections between extracellular adhesions and the cytoskeleton, murine talin has been cloned and sequenced. A model is described for the structure of talin based on this sequence and other data. Homologies between talin and other proteins define a novel family of submembranous cytoskeleton-associated proteins all apparently involved in connections to the plasma membrane (Rees, 1990).
The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5' cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mock-transfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both alpha 5 and beta 1 subunits. Surprisingly, beta 1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicates the presence of two forms of beta 1. The processing of beta 1 was analyzed in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature beta 1, while the talin deficient AT22 clone showed that the beta 1 precursor was slowly converted into a very low molecular mass product. These data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of beta 1 integrins (Albiges-Rizo, 1995).
Integrin receptors mediate cell adhesion, signal transduction, and cytoskeletal organization. How a single transmembrane receptor can fulfill multiple functions was clarified by comparing roles of receptor occupancy and aggregation. Integrin occupancy by monovalent ligand induces receptor redistribution, but minimal tyrosine phosphorylation signaling or cytoskeletal protein redistribution. Aggregation of integrins by noninhibitory monoclonal antibodies on beads induces intracellular accumulations of pp125FAK and tensin, as well as phosphorylation, but no accumulation of other cytoskeletal proteins such as talin. Combining antibody-mediated clustering with monovalent ligand occupancy induces accumulation of seven cytoskeletal proteins, including alpha-actinin, talin, and F-actin, thereby mimicking multivalent interactions with fibronectin or polyvalent peptides. Integrins therefore mediate a complex repertoire of functions through the distinct effects of receptor aggregation, receptor occupancy, or both together (Miyamoto, 1995).
A homolog to talin has been identified in Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, it was determined that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci (Moulder, 1996).
Following platelet aggregation, integrin alpha(IIb)beta(3) becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential beta-turn motif in the beta(3) cytoplasmic tail and has been suggested to mediate the interaction of beta(3) integrins with talin. A double mutation (N744Q/P745A) in the integrin beta(3) subunit has been obtained to test the functional significance of this beta-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant beta(3) and wild type alpha(IIb). Cells expressing either wild type (A5) or mutant (D4) alpha(IIb)beta(3) adhered to fibrinogen; however, as opposed to control A5 cells, adherent D4 cells failed to spread, form focal adhesions, or initiate protein tyrosine phosphorylation. To investigate the role of the NPLY motif in talin binding, the ability of the mutant alpha(IIb)beta(3) to interact with talin was examined in a solid phase binding assay. Both wild type and mutant alpha(IIb)beta(3), purified by RGD affinity chromatography, bind to a similar extent to immobilized talin. Additionally, purified talin failed to interact with peptides containing the AKWDTANNPLYK sequence, indicating that the talin binding domain in the integrin beta(3) subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the beta(3) cytoplasmic tail was observed, and this interaction was blocked by a recombinant protein fragment corresponding to the 47-kDa N-terminal head domain of talin (rTalin-N). In addition, RGD affinity purified platelet alpha(IIb)beta(3) binds dose-dependently to immobilized rTalin-N, indicating that an integrin-binding site is present in the talin N-terminal head domain. Collectively, these studies demonstrate that the NPLY beta-turn motif regulates post-ligand binding functions of alpha(IIb)beta(3) in a manner independent of talin interaction. Moreover, talin binds through its N-terminal head domain to the membrane-proximal sequence of the beta(3) cytoplasmic tail (Patil, 1999).
The beta 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 beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. Recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails have been used to characterize integrin-binding sites within talin. An integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta 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 leads to activation of integrin alpha(IIb)beta(3); activation is dependent on the presence of both the talin head domain and the integrin beta(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 (Calderwood, 1999).
Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells) was characterized. A large intracellular pool of beta1 integrins was characterized that is abnormally accumulated in an earlier compartment of the secretory pathway. In talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin is found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies results in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. It is proposed that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains (Martel, 2000).
The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrin biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction the binding of a series of recombinant PTB domains to a panel of short integrin beta tails was examined. In addition to the known integrin-binding proteins, it was found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bind to integrin tails. Furthermore, Dok-1 physically associates with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirm that these interactions are canonical PTB domain-ligand interactions: (1) the interactions are blocked by mutation of an NPXY motif in the integrin tail; (2) integrin class-specific interactions are observed with the PTB domains of Dab, EPS8, and tensin. This specificity, and a molecular model of an integrin beta tail-PTB domain interaction was used to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism (Calderwood, 2003).
The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. The crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail, is reported. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, these studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior (Garcia-Alvarez, 2003).
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). The PIP kinase-binding site was characterized 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 (Barsukov, 2003).
Activation (affinity regulation) of integrin adhesion receptors controls cell migration and extracellular matrix assembly. Talin connects integrins with actin filaments and influences integrin affinity by binding to the integrins' short cytoplasmic beta-tail. The principal beta-tail binding site in talin is a FERM domain, comprised of three subdomains (F1, F2, and F3). Previous studies of integrin alphaIIbbeta3 have shown that both F2 and F3 bind the beta3 tail, but only F3, or the F2-F3 domain pair, induces activation. Talin-induced perturbations of beta3 NMR resonances were examined to explore integrin activation mechanisms. F3 and F2-F3, but not F2, distinctly perturbed the membrane-proximal region of the beta3 tail. All domains also perturbed more distal regions of the beta3 tail that appear to form the major interaction surface, since the beta3(Y747A) mutation suppressed those effects. These results suggest that perturbation of the beta3 tail membrane-proximal region is associated with talin-mediated integrin activation (Ulmer, 2003).
The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. This study reports the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. The in vivo relevance of this interaction was demonstrated by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed mapping out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, a cell biology approach showed that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation (Tremuth, 2004).
The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. This study describes the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and it is suggested to be a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin binding, but not talin binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions (Kiema, 2006).
Migrating cells extend protrusions to establish new adhesion sites at their leading edges. One of the driving forces for cell migration is the directional trafficking of cell-adhesion molecules such as integrins. The endocytic adaptor protein Numb is an important component of the machinery for directional integrin trafficking in migrating cells. In cultured mammalian cells, Numb binds to integrin-βs and localizes to clathrin-coated structures (CCSs) at the substratum-facing surface of the leading edge. Numb inhibition by RNAi impairs both integrin endocytosis and cell migration toward integrin substrates. Numb is regulated by phosphorylation since the protein is released from CCSs and no longer binds integrins when phosphorylated by atypical protein kinase C (aPKC). Because Numb interacts with the aPKC binding partner PAR-3, a model is proposed in which polarized Numb phosphorylation contributes to cell migration by directing integrin endocytosis to the leading edge (Nishimura, 2007).
Numb localizes at a part of CCSs and functions in integrin endocytosis as a cargo-selective adaptor. Integrin is thought to be recycled from the tail to the front of migrating cells by endocytosis. However, many focal adhesions or focal complexes formed at the cell front disassemble behind the F-actin-rich lamellipodia. Numb mainly accumulated behind lamellipodia, although a certain population of Numb still remained and colocalized with integrin at the trailing edge. In addition, localization of Numb among CCSs correlated with the position of integrin adhesions, supporting the role of Numb in integrin endocytosis. Talin is a key molecule that tethers integrin to components of focal adhesions and actin stress fibers and is critical for focal-adhesion disassembly. Mutation of a conserved tyrosine residue within the integrin-β3 intracellular domain abolished the binding of both talin and Numb, suggesting that talin and Numb cannot bind to integrin simultaneously. Consistent with these observations, interaction of Numb with talin could not be detected. In addition, the binding of Numb to integrin does not activate the integrin extracellular domain, whereas the binding of talin does. The overexpression or knockdown of Numb does not directly affect cell adhesion. Thus, it appears that Numb does not actively promote focal-adhesion disassembly, but rather recruits free integrins without the components of focal adhesions to the AP-2 complex for internalization. Preferential localization of Numb around focal adhesions at the substratum-facing surface would facilitate recruitment of integrin during focal adhesion disassembly (Nishimura, 2007).
Recent genetic screening isolated Numb as a mutant defective for peripheral glia migration along axons in Drosophila (Edenfeld, 2007). Migration defects of postmitotic neurons have been described in Numb-knockout mice, indicating that Numb regulates particular cell migration in vivo. However, the defects of Numb knockdown on integrin endocytosis and cell migration are less marked than those of AP-2 and clathrin knockdown, suggesting that another adaptor molecule(s) may function in integrin endocytosis. A good candidate is disabled-2 (Dab2), which has a similar domain structure as Numb and binds to both components of clathrin-mediated endocytosis and to integrin-β. Dab2 is expressed in HeLa cells and positively controls cell adhesion and spreading. In contrast to Numb, Dab2 appears to preferentially localize to the apical surface. Thus, Numb and Dab2 may coordinately function in integrin endocytosis in different subcellular compartments for cell motility (Nishimura, 2007).
How does Numb localize at the substratum-facing surface and polarize toward the leading edge? Integrin adhesions could activate several intracellular signaling events and promote protein transport to adhesion sites by targeting microtubules and linking actin stress fibers. The actin cytoskeleton and/or adhesion itself are important for the preferential localization of Numb around adhesions. However, Numb still localized at the substratum-facing surface in the presence of cytochalasin-D, indicating that an additional mechanism may exist. Observations indicate that direct phosphorylation by aPKC may be a part of the regulatory mechanism underlying Numb localization at the substratum-facing surface. In addition, polarized localization of Numb toward the leading edge was lost upon aPKC knockdown. In support of these observations, asymmetric localization of Numb in Drosophila has been shown to be dependent on cortical actomyosin and the polarized localization/function of aPKC and PAR-3. Conclusive evidence will require isolation of the responsible motor(s) and anchor protein(s) for specific Numb localization (Nishimura, 2007).
Numb-full-3A, mutated at three phosphorylation sites, did not function as a constitutively active form that promotes integrin endocytosis and cell migration, but rather inhibited these processes. Similarly, both the phospho-mimic and nonphosphorylated form of μ2-adaptin, which is phosphorylated by AAK1, inhibit transferrin endocytosis, suggesting that clathrin-mediated endocytosis is tightly controlled by cycles of phosphorylation and dephosphorylation. Additional phosphorylation during endocytosis may be required for the dissociation of Numb from the binding proteins, integrin-β, and α-adaptin. Numb is indeed phosphorylated by several PKCs and CaMKs, and phosphatase inhibitor dramatically increases the phosphorylation level of Numb. Thus, local phosphorylation and dephosphorylation seems to allow Numb to localize defined CCSs around adhesion sites (Nishimura, 2007).
Trafficking of internalized integrin is regulated by growth factors and the extracellular matrix through several adaptors/kinases, including PI3-kinase, PKB/Akt, GSK3β, and PKCs. Several growth factors and adhesions indeed promote the recycling of integrins, leading to the upregulation of cell-surface expression, whereas treatment of cells with PDGF does not affect the internalization rate of integrin. The degree of colocalization and interaction of Numb with integrin-β1 was not significantly altered in HeLa cells before and after wounding. These data suggest that Numb functions constitutively in integrin endocytosis, although it is possibile that polarized migration promotes the internalization rate and amount of integrin endocytosis. It might be difficult to detect the changes in the interaction of Numb and integrin during migration due to the nature of rapid cycling of endocytosis and exocytosis and possibly due to the transient interaction. It has been reported that the inhibition of directional membrane trafficking causes membrane extension in all directions. Membrane trafficking controls the directionality of migrating cells. Taking into account the fact that Numb localization becomes polarized coincidently with directional migration, the subcellular region at which integrin is internalized and the subsequent coupling with the recycling processes could be important for efficient cell migration suitable for the particular environment (Nishimura, 2007).
Density-Enhanced Phosphatase-1 (DEP-1) (see Drosophila Ptp4E) de-phosphorylates various growth factor receptors and adhesion proteins to regulate cell proliferation, adhesion and migration. Moreover, dep-1/scc1 mutations have been detected in various types of human cancers, indicating a broad tumor suppressor activity. During C. elegans development, DEP-1 mediates binary cell fate decisions by negatively regulating EGFR signaling (see Drosophila EGFR signaling). Using a substrate-trapping DEP-1 mutant in a proteomics approach, this study identified the C. elegans β-integrin subunit PAT-3 (see Drosophila mys) as a specific DEP-1 substrate. DEP-1 selectively de-phosphorylates tyrosine 792 in the membrane-proximal NPXY motif to promote integrin activation via talin (see Drosophila rhea) recruitment. The non-phosphorylatable β-integrin mutant pat-3(Y792F) partially suppresses the hyperactive EGFR signaling phenotype caused by loss of dep-1 function. Thus, DEP-1 attenuates EGFR signaling in part by de-phosphorylating Y792 in the β-integrin cytoplasmic tail, besides the direct de-phosphorylation of the EGFR. Furthermore, in vivo FRAP analysis indicates that the αβ-integrin/talin complex attenuates EGFR signaling by restricting receptor mobility on the basolateral plasma membrane. The study proposes that DEP-1 regulates EGFR signaling via two parallel mechanisms, by direct receptor de-phosphorylation and by restricting receptor mobility through αβ-integrin activation (Walser, 2017).
Regulation of integrin affinity (activation) is essential for metazoan development and for many pathological processes. Binding of the talin phosphotyrosine-binding (PTB) domain to integrin β subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. This study defined the structure of a complex between talin and the membrane-proximal integrin β3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. Structure-based mutagenesis was used to engineer talin and β3 variants that interact with comparable affinity to the wild-type proteins but inhibit integrin activation by competing with endogenous talin. These results reveal the structural basis of talin's unique ability to activate integrins, identify an interaction that could aid in the design of therapeutics to block integrin activation, and enable engineering of cells with defects in the activation of multiple classes of integrins (Wegener, 2007).
This study has revealed how the talin F3 domain is uniquely designed to activate integrins. The talin F3 domain forms a well-defined complex with the helix-forming MP region of the β-integrin tail, and this interaction holds the key to the molecular recognition required for activation. Mutations in integrin or talin that inhibit this interaction in vitro also prevent integrin activation in cells, and mutants with intermediate functional effects in cells retain a partial ability to form the F3-MP interaction. These findings are consistent with studies that identified a variety of activating mutations within the MP region of integrins, thus establishing that this region is critical for stabilizing the low-affinity conformation (Wegener, 2007).
The unique feature of talin F3 that promotes interaction with the MP region of β-integrin appears to be the flexible loop between strands S1 and S2 that forms a hydrophobic pocket that accepts the side chains of F727 and F730 in the complex. The talin mutation L325R within this pocket abolishes binding to the MP region and thus hinders activation. Other PTB-domain-containing proteins (DOK [“IRS-like”], Shc [”Shc-like”], and NUMB [”Dab-like”] lack such a mobile loop and so are unlikely to interact with the MP region. Indeed, DOK binds and inhibits integrin activation and binding occurs to the MD site but not the MP site. Several point mutants in talin also transform the F3 domain from an activator into an inhibitor. This is consistent with competition between endogenous talin and various PTB domains, including mutated talin F3, for the MD site. The concept of inhibition of talin activation by competitive binding to the β tails by a variety of proteins, especially those with PTB domains, may be an important feature in the regulation of integrin activity. These conclusions also apply to PTB (F3) subdomains in other FERM-domain proteins. The FERM-domain structures of band 4.1, ezrin, radixin, and moesin all have very short loops between strands 1 and 2 with no hydrophobic residues that could bury the two integrin phenylalanines (Wegener, 2007).
Activation of the ligand binding function of integrin heterodimers requires transmission of an 'inside-out' signal from the integrin small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alphaIIß3 has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the ß cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin, both of which are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the ß cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation (Vinogradova, 2002).
As the trigger point of inside-out signaling, the integrin cytoplasmic face has been the focus of intense investigations. These studies have revealed that (1) while intact integrin can remain latent both in unstimulated cells and in a purified state, deletion of the cytoplasmic and transmembrane region activates the receptor; (2) point mutations in the membrane-proximal regions of the cytoplasmic tails or deletion of either can result in constitutive activation of the receptor; (3) replacement of the cytoplasmic-transmembrane regions by heterodimeric coiled-coil peptides or an artificial linkage of the tails inactivates the receptor, and breakage of the coiled-coil or clasp activates the receptor, and (4) overexpression of certain intracellular proteins that bind to the cytoplasmic tails, including the cytoskeletal protein talin, which binds to the ß cytoplasmic tail, can result in integrin activation. These data suggest that a direct interaction between the alpha/ß cytoplasmic tails might maintain the receptor in a latent state. However, vigorous biochemical/biophysical studies aimed at examining such interaction have yielded contradictory results. While certain studies have suggested such interactions, recent NMR studies have failed to detect a complex. The most recent NMR study reported interaction between truncated versions of the alphaIIb and ß3 cytoplasmic tail peptides, but structural analyses revealed two different complexes of unknown physiological relevance. Thus, the current view of the cytoplasmic face and its regulation on the integrin inside-out signaling remains unclear; this is a major impediment to an understanding of integrin structure-function relationships (Vinogradova, 2002 and references therein).
In this study, the successful structure determination of the intact integrin alphaIIbß3 cytoplasmic face using NMR spectroscopy is reported. The structure reveals that the alphaIIb and ß3 cytoplasmic tails do, indeed, interact; they engage in a weak handshake within their membrane-proximal regions. This handshake is unclasped by 'activating' mutations in the binding interface and by a known integrin activator, the talin head domain. The results provide a structural basis for how an integrin cytoplasmic face regulates inside-out activation (Vinogradova, 2002).
The cytoplasmic protein talin is an essential part of the integrin-cytoskeleton link. This study has characterized the interaction between integrin and two conserved regions of talin, the N-terminal 'head' domain and the C-terminus, which includes the I/LWEQ domain, within the living organism. Green-fluorescent-protein-tagged head and C-terminal domains are recruited to integrin adhesion sites. Both required integrins for recruitment, but the C-terminal domain also requires endogenous talin, showing it was not recruited directly by integrins. Chimeric transmembrane proteins containing the cytoplasmic domain of the integrin β subunit were used to examine the integrin-talin head interaction. Monomeric chimeric proteins did not recruit talin head, whereas dimeric chimeras efficiently recruited it and caused a strong inhibition of integrin-mediated adhesion. These chimeras recruited surprisingly few integrin-associated proteins, indicating that recruitment of talin does not initiate a cascade of recruitment. Mutagenesis of the integrin cytoplasmic domain, within the chimera, showed the dominant-negative inhibition is not due to talin sequestration alone and that additional interactions are required (Tanentzapf, 2006a).
For most integrin heterodimers, the majority of interactions with cytoplasmic proteins are made by the 47-residue cytoplasmic domain of the ß subunit. This short peptide has been subjected to extensive study, but there is no clear model of how it functions. Recent work by a number of groups has highlighted the importance of the large cytoskeletal linker talin, which was the first protein to be identified that binds to the ß tail. This study focused on the interaction between two domains of talin and the cytoplasmic domain of the ß subunit, using the Drosophila embryo. This has provided new insights into the early steps in the linkage between integrins and the cytoskeleton (Tanentzapf, 2006a).
Using live imaging within the intact animal, in vivo evidence is provided to support a direct interaction between the head domain of talin and the cytoplasmic tail of the ß subunit. This interaction requires an 'alteration' to the cytoplasmic domain of integrin because only one of three chimeric transmembrane proteins containing this domain was able to recruit the talin head. Since the active chimera (integrin ß tail is placed on a heterologous transmembrane protein) is derived from a constitutively active receptor tyrosine kinase, it seems likely that its ability to constitutively dimerize or oligomerize accounts for its special activities, leading to it being referred to as diß. Previous work showed that diß was constitutively active in sending integrin signals that regulate gene expression, and this study shows that it also acts as a dominant negative protein on integrin-mediated adhesion at muscle attachment sites. The dominant negative phenotype of diß consists of a detachment between integrins and the cytoskeleton, similar to the phenotype seen in the absence of talin. This suggests an explanation for the dominant negative activity of diß: it sequesters talin away from the endogenous integrins. Consistent with this, diß recruited talin and TalinH-GFP, but not other proteins required for integrin adhesion, such as PINCH, ILK and tensin. In addition, this latter finding further supports the direct interaction between integrins and talin, and shows that recruitment of talin is not sufficient to trigger the assembly of the whole complex of proteins that contribute to the link between integrins and the cytoskeleton (Tanentzapf, 2006a).
Based on these observations, it is possible to propose a simple model where talin is the only protein recruited directly by integrins and the dominant negative activity of diß is solely due to talin sequestration. This was tested by generating point mutations in the ß subunit of the cytoplasmic domain within the diß chimera and by assaying their ability to cause muscle detachment and recruit TalinH-GFP, surmising that, if this model was correct there should be a clear correlation between the two. However, this proved not to be the case. Mutations throughout the length of the integrin tail caused a loss of dominant negative activity, but only those in the half closest to the membrane could not recruit TalinH-GFP. This leads a to hypothesis that a factor X binds to the second half of the integrin cytoplasmic domain and contributes to the dominant negative effect (Tanentzapf, 2006a).
Based on previous in vitro studies it has been proposed that the head domain of talin acts as the preferred site for integrin binding. The talin head domain, which in vertebrates can be found endogenously due to cleavage by the calcium dependent protease calpain, has also been shown to mediate integrin activation. This study has shown that the talin head can localize to the muscle attachments in an integrin-dependent manner and that expression of dimeric integrin cytoplasmic tail chimeras is sufficient to recruit it to the cell cortex. Experiments in myoblasts and the direct correlation between levels of integrin and talinH-GFP recruitment strongly suggest that, in vivo recruitment of talinH-GFP by the integrin cytoplasmic tail occurs independently of any accessory factors and, therefore, is most probably direct. The finding that talinH-GFP is the only protein tested that was found around the entire cortex of muscles containing diß or excess integrin also supports a direct interaction between them. There might be a difference between the membrane domains on the lateral sides of the muscles versus the ends, which blocks recruitment of additional integrin associated proteins to the lateral sides (Tanentzapf, 2006a).
Further support for a direct interaction between the ßPS cytoplasmic domain and the talin head domain in vivo comes from mutational analysis. Those residues essential to recruit talinH-GFP in vivo are within the same regions where the cytoplasmic domain binds to talin in vitro. Thus, the first NPxY motif of the ß integrin cytoplasmic tail is crucial for binding to the talin head domain both in vivo and in vitro. Both approaches show that talin binding requires more than just this region. For example, peptides that correspond to a small region covering the NPLY motif of ß3 integrin and the preceding eight amino acid residues are unable to bind to talin by themselves in comparison to peptides covering the membrane proximal region of the tail. This leads to the suggestion that the NPLY domain by itself can mediate talin binding in the context of the full-length cytoplasmic tail but that the membrane proximal region is sufficient by itself. NMR studies showed that, the region of the integrin cytoplasmic tail that is perturbed upon binding of the talin head includes residues in the membrane proximal region and this is abolished when NPxY is mutated. Peptides only containing the region that includes the NPxY region showed a surprisingly low binding affinity for the talin head, about 100 times less than the affinity for PIP4, 5-kinase. In vivo results support the idea that talin binding requires interaction with multiple regions of the integrin cytoplasmic tail either simultaneously or sequentially (Tanentzapf, 2006a).
In contrast to the talin head, the C-terminal domain of talin behaves quite differently. It localizes to sites of adhesion and is also specifically and strongly recruited to the developing Z-lines. Like the talinH-GFP, the C-terminal region requires integrins for its localization but, by contrast, it is not recruited by overexpression of integrins or diß, nor is it recruited more efficiently when the levels of endogenous talin are reduced, instead it requires talin for its localisation. This is not too surprising because GFP-talinC does not include the recently defined C-terminal integrin-binding domain (Tanentzapf, 2006a).
Since GFP-talinC contains the I/LWEQ domain, which is thought to have actin-binding activity, its failure to colocalise with actin filaments is surprising. In contrast to other fusion proteins of GFP to actin-binding domains tested, the talin C-term does not decorate actin filaments in the developing muscle. Instead, it decorates the Z-lines and muscle ends, with a distribution that is very similar to that of the N-terminal domain of tensin, which also binds actin in vitro. Recently it was reported that the actin-binding site is cryptic, supporting the current findings. Some possible candidates for the recruitment of this domain seem unlikely, such as vinculin and talin itself. GFP-talinC does not include any of the identified vinculin-binding sites. The talinC domain does contain a recently identified homodimerization site, but if it is recruited solely by dimerizing with endogenous talin, such strong Z-line relative to muscle-end staining would not be expected, because talin is much more enriched at the muscle ends. In addition, it would be expected to be recruited by talin associated with diß, which it is not. Therefore, the identity of the molecules that mediate the localization of GFP-talinC remains a topic for further study (Tanentzapf, 2006a).
Different chimeric proteins containing the ßPS cytoplasmic domain did not have an equal ability to recruit talinH-GFP to the cell membrane and, thus, the extracellular domain of the chimera influences intracellular interactions. One clear difference is that, using extracellular domains from a receptor tyrosine kinase that has been mutated so that it signals independently of ligand-binding resulted in recruitment of talinH-GFP. The strength of the constitutive signalling produced by these mutant receptors correlated with their ability to recruit talinH-GFP as chimeric proteins. These findings point to dimerisation or oligomerisation as a key trigger for talin recruitment. This fits with the recent discovery of homotypic interactions between the transmembrane domains of integrin subunits, leading to a model where ligand-bound integrin heterodimers exist in a complex-cluster on the cell surface. There, the transmembrane and cytoplasmic domains of the ß subunit form trimers and those of the alpha subunits form dimers in the membrane, whereas outside the cell alphaß heterodimers bind to ligand. The increased recruitment of talinH-GFP to the dimers or trimers of the ß cytoplasmic domain could occur by cooperative binding, which would require some interaction between different talin head domains, or the oligomers could stabilise a conformation of the ß cytoplasmic domain that binds tightly to the talin head. This latter model gives an alternate explanation: the chimeras with the mutant forms of the receptor tyrosine kinase could induce the ßPS cytoplasmic domain to adopt a different conformation compared with the CD2 chimera, which bind to talin more tightly. At present, tools are not available to unambiguously distinguish between these different possibilities, but oligomerization is favored as the working model (Tanentzapf, 2006a).
The formation of ß subunit oligomers might also explain why overexpression of the ßPS integrin subunit is dominant negative on integrin adhesion – similar to that produced by diß. Formation of oligomers can be suppressed by co-overexpressing the alphaPS2 subunit, indicating that it is not overexpression of integrin heterodimers that is the problem, but free ß subunits. One explanation is that free ßPS subunits form – just like diß – homodimers or homotrimers at low levels, which can be transported to the plasma membrane where they recruit cytoplasmic proteins but cannot mediate extracellular adhesion. This might also explain why the dependence on dimerization for dominant negative activity that was observed has not been reported in other systems, because even a low level of dimerization may induce dominant negative activity (Tanentzapf, 2006a).
Two models have been proposed that explain the dominant negative effect of chimeric proteins containing the integrin ß subunit cytoplasmic tail: a feedback model, involving excess signalling, and a competition model, involving sequestration of key cytoplasmic proteins required for endogenous integrin function. The results suggest that sequestration of talin accounts for some but not all of the dominant negative effect. Talin is one of the few integrin-associated proteins tested that is recruited by diß and its overexpression partially suppressed the dominant negative activity. Mutants of diß that have lost talinH-GFP binding also have lost dominant negative activity. However, mutations in the C-terminal part of the ß tail still recruited talin yet also lost dominant activity, suggesting involvement of another protein. It is therefore hypothesized that, binding of a factor X to the C-terminus is also required for the dominant negative effect. The requirement for two factors received some support from the finding that coexpression of certain pairs of diß mutants partially restored dominant negative activity; the thinking is that the heterodimers formed can now recruit both talin and factor X (Tanentzapf, 2006a).
It hard to explain how diß can produce a dominant negative effect only when sequestering two proteins, because it would be expected that recruiting a single protein has some effect, and recruiting both has an additive effect. Instead very strong synergy is seen, that is easier to explain if factor X has a signalling role. For example, one speculative model is that factor X is a kinase that phosphorylates and inactivates talin when it is bound at the adjacent site on the ß cytoplasmic tail. Exchange of the inactivated talin would then lead to a gradual inactivation of the cytoplasmic pool of talin, which could be partially alleviated by increasing the amount of talin by overexpression. In the case of the endogenous integrins, there would have to be a mechanism to inactivate this inhibition. This could be achieved by one of the integrin-associated proteins that is recruited by the endogenous integrins but not by the diß-inactivating factor X or by removing the inhibitory phosphate from talin. The inactivation of the inhibition by endogenous integrins must not be efficient enough to counter the negative effect of diß. Thus, in the end the best model combines both sequestration and excessive signalling (Tanentzapf, 2006a).
A number of candidates for factor X that interact with the relevant region of the ß subunit cytoplasmic domain have already been identified, including filamin, non-muscle myosin, and Src. Non-muscle myosin can be recruited by the CD2ßPS chimera. Src is activated by binding to the C-terminal region the of the ß3 integrin cytoplasmic tail, and this is enhanced by clustering and homo-oligomerization of the integrins. The role of Src in integrin function in Drosophila has yet to be elucidated. Phosphorylated FAK was recruited by diß, and overexpression of FAK causes muscle detachment. By contrast, sequestration of FAK is unlikely to cause a defect because the absence of FAK did not cause defects in integrin-mediated adhesion in Drosophila. Thus, there are candidate kinases that interact with this region of the ß tail and could send inhibitory signals (Tanentzapf, 2006a).
This study found that when the integrin ß tail is placed on a heterologous transmembrane protein it recruited only some integrin associated proteins. In particular, talin and FAK were recruited, but not ILK, PINCH or tensin. This suggests additional input, required to assemble the full complement of proteins that contribute to the integrin-cytoskeleton link. Either of the two domains missing from these chimeras could provide the input: the extracellular ligand-binding domain, composed of both α and ß subunits, or the α subunit cytoplasmic domain. Since an integrin heterodimer lacking the α subunit cytoplasmic domain can mediate muscle attachment, the extracellular ligand-binding domain is favored. This could either provide unique conformational changes to the ß tail, or it could allow tension to be placed on the integrin-cytoskeletal linkage (Tanentzapf, 2006a).
Transmembrane adhesion receptors, such as integrins, mediate cell adhesion by interacting with intracellular proteins that connect to the cytoskeleton. Talin, one such linker protein, is thought to have two roles: mediating inside-out activation of integrins, and connecting extracellular matrix (ECM)-bound integrins to the cytoskeleton1. Talin's amino-terminal head, which consists of a FERM domain, binds an NPxY motif within the cytoplasmic tail of most integrin β subunits. This is consistent with the role of FERM domains in recruiting other proteins to the plasma membrane. The role of the talin-head-NPxY interaction in integrin function was tested in Drosophila. Introduction of a mutation that perturbs this binding in vitro into the isolated talin head disrupts its recruitment by integrins in vivo. Surprisingly, when engineered into the full-length talin, this mutation does not disrupt talin recruitment by integrins nor its ability to connect integrins to the cytoskeleton. However, it reduces the ability of talin to strengthen integrin adhesion to the ECM, indicating that the function of the talin-head-NPxY interaction is solely to regulate integrin adhesion (Tanentzapf, 2006b).
A model to explain these findings is as follows. It starts with the natural equilibrium between low- and high-affinity integrin conformations. For talin to activate integrins, it follows that the talin head must bind low-affinity integrins, but additional integrin-binding sites in talin may only interact with high-affinity, ligand-bound integrins. It is envisioned that integrin adhesion starts with transient high-affinity integrins binding the adjacent ECM and recruiting talin via the predicted novel integrin binding site. The bound talin clusters other high-affinity integrins by an interaction that does not involve the head (R367), but could involve IBS2. This leaves the head domain of the bound talin free to bind new low-affinity integrin heterodimers that diffuse into proximity, activating them so that they bind the ECM and further strengthen the adhesive cluster. This model, therefore, provides a mechanism for integrins to be preferentially activated close to existing adhesive sites (Tanentzapf, 2006b).
This work has two major implications: first, when the ability of the talin head to interact with the integrin cytoplasmic tail is compromised, talin can still localize to sites of adhesion and carry out its function as a cytoplasmic linker. This implies that talin forms different kinds of links to integrin to mediate its different functions. Second, disrupting the talin-head-integrin interaction reduces the ability of talin to function, resulting in defective attachment of integrin to the ECM. The results support the view that this is due to reduced conversion of integrins into a high-affinity state, and therefore that regulation of integrin affinity has an important role during development, as it generates strong adhesion of cells to an insoluble ECM (Tanentzapf, 2006b).
Electron microscopy of glycerol-sprayed and rotary metal-shadowed talin from human platelets reveals a dumbbell-shaped molecule with an average length of approximately 51 nm. Analytical ultracentrifugation of native talin yields a single molecular species with an apparent molecular mass of 412 kDa and a sedimentation coefficient of S20w = 11.2. Chemical cross-linking with glutaraldehyde (GA) and corresponding SDS-PAGE analysis shows that the monomer band of talin can be quantitatively converted to a dimer band at GA concentrations 0.45%, indicating that there is no significant amount of monomer present in solution. These structural and biophysical data are compatible with native talin being an antiparallel homodimer. Actin filaments polymerized in the presence of native talin show increased nucleation and polymerization rates and an overall reduction of actin filament length. Hence, it is concluded that talin in its native biological state is a dimer when promoting nucleation of actin filaments (Goldmann, 1994).
Talin is an actin-binding protein involved in integrin-mediated cell adhesion and spreading. The C-terminal 197 amino acids of vertebrate talin are 45% similar to the C-terminal residues of Sla2, a yeast protein implicated in polarized assembly of the yeast actin cytoskeleton. Talin is also homologous in this region to nematode talin, cellular slime mold filopodin, and an Sla2 homolog from nematode. Analysis of the conserved C-terminal sequences of these five proteins with BLOCK MAKER reveals a series of four blocks, which have been named the I/LWEQ module after the conserved initial residues in each block. The conserved protein domain represented by the I/LWEQ module competes quantitatively with native talin for binding to F-actin in vitro. Furthermore, the corresponding domain of Sla2 binds to both yeast and vertebrate F-actin in vitro. Mutation of one of the conserved residues in the fourth conserved block abolishes the interaction of the Sla2 I/LWEQ module with F-actin. These results establish the location of an F-actin binding domain in native talin, demonstrate that direct interaction of Sla2 with actin is a possible basis for its effect on the actin cytoskeleton in vivo, and define the I/LWEQ consensus as a new actin-binding motif (McCann, 1997).
Light scattering and electron microscopy have been used to investigate the influence of intact talin and talin tail fragment on actin filament dynamics and network structure. Intact talin induces cross-linking as well as filament shortening on actin networks. The effect of intact talin as well as talin tail fragment on actin networks is controlled by pH and ionic strength. At pH 7.5, actin filament dynamics in the presence of intact talin and talin tail fragment are characterized by a rapid decay of the dynamic structure factor and by a square root power law for the stretched exponential decay which is in contrast with the theory for pure actin solutions. At pH 6 and low ionic strength, intact talin cross-links actin filaments more tightly than talin tail fragment. Talin head fragment shows no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain (Goldmann, 1999).
The sequence of chicken talin [2,541 amino acids, M(r) 271,881] is very similar (89% identity) to that of the mouse protein. Alignments with the Caenorhabditis elegans and Dictyostelium discoideum talin sequences show that the N- and C-terminal regions of the protein are conserved, whereas the central part of the molecule is more divergent. By expressing overlapping talin polypeptides as fusion proteins, at least three regions of the protein have been identified that can bind F-actin: residues 102-497, 951-1,327 and 2,269-2,541. The N-terminal binding site contains a region with homology to the ERM family of actin-binding proteins, and the C-terminal site is homologous to the yeast actin-binding protein Sla2p. Each of the actin-binding sites is close to, but distinct from, a binding site for vinculin, a protein that also binds actin. The Pro1176 to Thr substitution found in talin from Wistar-Furth rats does not destroy the capacity of this region of the protein to bind actin or vinculin. Microinjection studies showed that a fusion protein containing the N-terminal actin-binding site localized weakly to stress fibers, whereas one containing the C-terminal site initially localized predominantly to focal adhesions. The former was readily solubilized, and the latter was resistant to Triton extraction. The N-terminal talin polypeptide eventually disrupts actin stress fibers whereas the C-terminal polypeptide is without effect. However, a larger C-terminal fusion protein also containing a vinculin-binding site does disrupt stress fibers and focal adhesions. The results suggest that, although both the N- and C-terminal regions of talin bind actin, the properties of these two regions of the protein are distinct (Hemmings, 1999).
Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, three regions of talin have been identified that are involved in binding to vinculin. These observations have been confirmed by using a yeast two-hybrid assay; talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. The three vinculin-binding sites in talin have been further localized to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic alpha-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites. To test the possibility that this might constitute a leucine zipper involved in vinculin binding, each leucine residue was mutated to an alanine. The results show that this leucine repeat is not essential to the interaction between talin and vinculin. The yeast two-hybrid system was used to define further the talin-binding site within vinculin residues 1-258. C-terminal deletions made in accordance with exon boundaries show that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin. However, all N-terminal deletions abolish binding. The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain (Bass, 1999).
The cytoskeletal protein talin, which is thought to couple integrins to F-actin, contains three binding sites (VBS1-VBS3) for vinculin, a protein implicated in the negative regulation of cell motility and whose activity is modulated by an intramolecular interaction between the vinculin head (Vh) and vinculin tail (Vt) domains. Recombinant talin polypeptides containing the three VBSs (VBS1, residues 498-636; VBS2, residues 727-965; and VBS3, residues 1943-2157) each bind tightly to the same or overlapping sites within vinculin(1-258). A short synthetic talin VBS3 peptide (residues 1944-1969) is sufficient to inhibit binding of a (125)I-labelled talin VBS3 polypeptide to vinculin(1-258), and NMR spectroscopy confirmed that this peptide forms a 1:1 complex in slow exchange with vinculin(1-258). Binding of the (125)I-labelled VBS3 polypeptide is markedly temperature dependent, but is not inhibited by 1 M salt or 10% (v/v) 2-methyl-2-propanol. Attempts to further define the talin-binding site within vinculin(1-258) using a gel-blot assay were unsuccessful, but near maximal talin-binding activity was retained by a construct spanning vinculin residues 1-131 in a yeast two-hybrid assay. Interestingly, the talin VBS3 polypeptide is a potent inhibitor of the Vh-Vt interaction, and the VBS3 synthetic peptide is able to expose the actin-binding site in intact vinculin, which is otherwise masked by the Vh-Vt interaction. The results suggest that under certain conditions, talin may be an effective activator of vinculin (Bass, 2002).
Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. This study tested the extent to which the autoinhibitory head-tail interaction (HTI) in vinculin regulates formation and lifetime of the talin-vinculin complex, a proposed mediator of integrin-cytoskeleton bonds. In an ectopic recruitment assay, mutational reduction of HTI drives assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI alters the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, it was shown that the focal adhesion residency time of vinculin is enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlates with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescues the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibits the focal adhesion dynamics of talin, but not paxillin or alpha-actinin. These data show that talin conformation in cells permits vinculin binding, whereas the autoinhibited conformation of vinculin constitutes the barrier to complex formation. Down-regulation of HTI in vinculin to Kd approximately 10-7 is sufficient to induce talin binding, and HTI is essential to the dynamics of vinculin and talin at focal adhesions. It is therefore concluded that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells (Cohen, 2006).
Vinculin regulates cell adhesion by strengthening contacts between extracellular matrix and the cytoskeleton. Binding of the integrin ligand, talin, to vinculin's head domain and F-actin to its tail domain is a potential mechanism for this function, but vinculin is autoinhibited by intramolecular interaction between head and tail domain and must be activated to bind talin and actin. Since autoinhibition of vinculin occurs by synergism between two head and tail interfaces, one hypothesis is that activation could occur by two ligands that coordinately disrupt both interfaces. To test this idea, a FRET probe was used that reports directly on activation of vinculin. Neither talin rod, VBS3 (a talin peptide that mimics a postulated activated state of talin), nor F-actin can activate vinculin. But in the presence of F-actin, either talin rod or VBS3 induces dose-dependent activation of vinculin. The activation data are supported by solution phase binding studies which show that talin rod or VBS3 fail to bind vinculin whereas the same two ligands bind tightly to vinculin head domain (Kd ~ 100nM). These data strongly support a combinatorial mechanism of vinculin activation; moreover, they are inconsistent with a model in which talin or activated talin is sufficient to activate vinculin. Combinatorial activation implies that at cell adhesion sites, vinculin is a coincidence detector, awaiting simultaneous signals from talin and actin polymerization to unleash its scaffolding activity (Chen, 2006).
The talin rod contains approximately 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin fails to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin does bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K(d) of approximately 9 mum. Interestingly, Vd1 binding exposes a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K(d) approximately 0.14 mum), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localizes efficiently to focal adhesions, whereas 482-655 does not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold binds Vd1 much less well than wild type. It is concluded that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs (Patel, 2006).
Vinculin regulates both cell-cell and cell-matrix junctions and anchors adhesion complexes to the actin cytoskeleton through its interactions with the vinculin binding sites of alpha-actinin or talin. Activation of vinculin requires a severing of the intramolecular interactions between its N- and C-terminal domains, which is necessary for vinculin to bind to F-actin; yet how this occurs in cells is not resolved. The hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites was tested. Indeed, these vinculin binding sites have a high affinity for full-length vinculin, are sufficient to sever the head-tail interactions of vinculin, and they induce conformational changes that allow vinculin to bind to F-actin. Finally, microinjection of these vinculin binding sites specifically targets vinculin in cells, disrupting its interactions with talin and alpha-actinin and disassembling focal adhesions. In their native (inactive) states the vinculin binding sites of talin and alpha-actinin are buried within helical bundles present in their central rod domains. Collectively, these results support a model where the engagement of adhesion receptors first activates talin or alpha-actinin, by provoking structural changes that allow their vinculin binding sites to swing out, which are then sufficient to bind to and activate vinculin (Bois, 2006).
Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. A new binding partner for talin is reported, that has been named layilin. Layilin contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten-amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. A short region has been identified within talin's amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase (Borowsky, 1998).
Membrane phosphoinositides control a variety of cellular processes through the recruitment and/or regulation of cytosolic proteins. One mechanism ensuring spatial specificity in phosphoinositide signalling is the targeting of enzymes that mediate their metabolism to specific subcellular sites. Phosphatidylinositol phosphate kinase type 1 gamma (PtdInsPKI gamma) is a phosphatidylinositol-4-phosphate 5-kinase that is expressed at high levels in brain, and is concentrated at synapses. The predominant brain splice variant of PtdInsPKI gamma (PtdInsPKI gamma-90) binds, by means of a short carboxy-terminal peptide, to the FERM domain of talin, and is strongly activated by this interaction. Talin, a principal component of focal adhesion plaques, is also present at synapses. PtdInsPKI gamma-90 is expressed in non-neuronal cells, albeit at much lower levels than in neurons, and is concentrated at focal adhesion plaques, where phosphatidylinositol-4,5-bisphosphate has an important regulatory role. Overexpression of PtdInsPKI gamma-90, or expression of its C-terminal domain, disrupts focal adhesion plaques, probably by local disruption of normal phosphoinositide balance. These findings define an interaction that has a regulatory role in cell adhesion and suggest new similarities between molecular interactions underlying synaptic junctions and general mechanisms of cell adhesion (Di Paolo, 2002).
The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate is essential for development, wound healing, metastasis, cell survival and the immune response. These events depend on the binding of integrin to the extracellular matrix, and assembly of focal adhesions, which are complexes comprising scaffolding and signalling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to beta-integrin is strengthened by PtdIns(4,5)P(2), suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. The type I phosphatidylinositol phosphate kinase isoform-gamma 661 (PIPKI gamma 661), an enzyme that makes PtdIns(4,5)P(2), is targeted to focal adhesions by an association with talin. PIPKI gamma 661 is tyrosine phosphorylated by focal adhesion associated kinase signalling, increasing both the activity of phosphatidylinositol phosphate kinase and its association with talin. This defines a mechanism for spatial generation of PtdIns(4,5)P(2) at focal adhesions (Ling, 2002).
Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing β3-GFP-integrins with enhanced talin-binding affinity, integrin activation, clustering, and substrate binding was experimentally uncoupled from its function in cell spreading. Mutational analysis revealed that Tyr747, located in the first cytoplasmic NPLY(747) motif, induced spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, is affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins form adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell-matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions (Pinon, 2014).
Gene disruption has been used to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin; (b) showed reduced adhesion to laminin, but not fibronectin; (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type; (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant and (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. It is concluded that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics (Priddle, 1998).
Mice have been generated with a targeted disruption of the talin gene. Heterozygotes are normal, but no surviving homozygous mutant animals were obtained, proving that talin is required for embryogenesis. Mutant embryos develop normally to the blastocyst stage and implant, but there is a gross disorganization of the embryos at gastrulation (6.5-7.5 days post coitum), and they die around 8.5-9.5 days post coitum. The embryonic ectoderm is reduced in size, with fewer cells, and is incompletely organized compared with wild-type embryos. The mutant embryos show disorganized extraembryonic tissues, and the ectoplacental and exocoelomic cavities are not formed. This seems to be because embryonic mesoderm accumulates as a mass on the posterior side of the embryos and fails to migrate to extraembryonic regions, although mesodermal cells are evident in the embryo proper. Spreading of trophoblast cells derived from cultured mutant blastocysts on fibronectin and laminin is also considerably reduced. Therefore, the fundamental deficit in these embryos seems to be a failure of cell migration at gastrulation (Monkley, 2000)
The function of talin has been investigated by inactivating talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. These experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum (Nuckolls, 1992).
Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. This study shows that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. It is concluded that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs (Conti, 2009).
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Cell sensing of externally applied mechanical strain through integrin-mediated adhesions is critical in development and physiology of muscle, lung, tendon, and arteries, among others. This study examined the effects of strain on force transmission through the essential cytoskeletal linker talin (Rhea in Drosophila). Using a fluorescence-based talin tension sensor (TS), it was found that uniaxial stretch of cells on elastic substrates increased tension on talin, which was unexpectedly independent of the orientation of the focal adhesions relative to the direction of strain. High-resolution electron microscopy of the actin cytoskeleton revealed that stress fibers (SFs) are integrated into an isotropic network of cortical actin filaments in which filamin A (FlnA; Cheerio in Drosophila) localizes preferentially to points of intersection between SFs and cortical actin. Knockdown (KD) of FlnA resulted in more isolated, less integrated SFs. After FlnA KD, tension on talin was polarized in the direction of stretch, while FlnA reexpression restored tensional symmetry. These data demonstrate that a FlnA-dependent cortical actin network distributes applied forces over the entire cytoskeleton-matrix interface (Kumar, 2019).
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