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At least 19 recordsLinked to original sources
PubMed1999

Identification of a talin-binding site in the integrin beta(3) subunit distinct from the NPLY regulatory motif of post-ligand binding functions. The talin n-terminal head domain interacts with the membrane-proximal region of the beta(3) cytoplasmic tail.

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. In the present study, we performed a double mutation (N744Q/P745A) in the integrin beta(3) subunit 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, we examined the ability of the mutant alpha(IIb)beta(3) to interact with talin in a solid phase binding assay. Both wild type and mutant alpha(IIb)beta(3), purified by RGD affinity chromatography, bound 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) bound 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 was shown to bind through its N-terminal head domain to the membrane-proximal sequence of the beta(3) cytoplasmic tail.

Amino Acid Sequence↗
PubMed1992

A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth rat.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.

Animals↗
PubMed1999

The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH and ionic strength.

We employed quasi-elastic light scattering and electron microscopy to investigate the influence of intact talin and talin tail fragment on actin filament dynamics and network structure. Using these methods, we confirm previous reports that intact talin induces cross-linking as well as filament shortening on actin networks. We now show that 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 showed no effect on actin networks, indicating that the actin binding sites reside probably exclusively within the tail domain.

Actins↗
PubMed1992

Probing actin and liposome interaction of talin and talin-vinculin complexes: a kinetic, thermodynamic and lipid labeling study.

Talin purified from human platelets and chicken gizzard smooth muscle is an actin and lipid binding protein. Here, we have investigated the effect of vinculin on (a) talin-nucleated actin polymerization and (b) insertion of talin into lipid bilayers. Calorimetric data show ternary complex formation between talin, vinculin, and actin. Actin-talin, actin-vinculin and actin-(talin-vinculin) binding and rate constants as well as actin polymerization rates for all three protein species have been determined by steady state titration, stopped-flow, and fluorescence assay. In contrast to an increase of the polymerization rate by a factor of less than 2 for actin-talin and actin-(talin-vinculin) when lowering the temperature, we measured a decrease in rates for actin alone and actin-vinculin. The overall equilibrium constants (Keq) in the van't Hoff plot proved linear and were of one-step reactions. Thermodynamic data exhibited signs of van der Waal's binding forces. Using the photoactivatable lipid analogue [3H]PTPC/11, which selectively labels membrane-embedded hydrophobic domains of proteins, we also show that talin partially inserts into the hydrophobic bilayer of liposomes. This insertion occurs in a similar manner irrespective of preincubation with vinculin.

Actins↗
PubMed1997

Examining F-actin interaction with intact talin and talin head and tail fragment using static and dynamic light scattering.

We examined the binding kinetics of intact talin and talin head and tail fragment with F-actin at pH 7.0 and at low ionic strength. We observed by a transient kinetic method a fast followed by a slower binding process for intact talin and talin tail fragment with filamentous actin. The latter can be attributed to F-actin cross-linking and/or bundling, which was observed in cosedimentation assays as well as by low shear viscometry and electron microscopy [Zhang, J., Robson, R. M., Schmidt, J. M. & Stromer, M. H. (1996) Biochem. Biophys. Res. Commun. 218, 530-537]. This finding is supported by dynamic light scattering measurements, indicating changes in internal actin filament dynamics due to cross-linking/bundling events with intact talin and talin tail fragment. No binding of the talin head fragment with F-actin was detected by either method.

Actins↗
PubMed2000

Phospholipid binding of synthetic talin peptides provides evidence for an intrinsic membrane anchor of talin.

Talin, an actin-binding protein, is assumed to anchor at the membrane via an intrinsic amino acid sequence. Three N-terminal talin fragments, 21-39 (S19), 287-304 (H18), and 385-406 (H17) have been proposed as potential membrane anchors. The interaction of the corresponding synthetic peptides with lipid model systems was investigated with CD spectroscopy, isothermal titration calorimetry, and monolayer expansion measurements. The membrane model systems were neutral or negatively charged small unilamellar vesicles or monolayers with a lateral packing density of bilayers (32 mN/m). S19 partitions into charged monolayers/bilayers with a penetration area A(p) = 140 +/- 30 A(2) and a free energy of binding of DeltaG(0) = -5.7 kcal/mol, thereby forming a partially alpha-helical structure. H18 does not interact with lipid monolayers or bilayers. H17 penetrates into neutral and charged monolayers/bilayers with A(p) = 148 +/- 23 A(2) and A(p) = 160 +/- 15 A(2), respectively, forming an alpha-helix in the membrane-bound state. Membrane partitioning is mainly entropy-driven. Under physiological conditions the free energy of binding to negatively charged membranes is DeltaG(0) = -9. 4 kcal/mol with a hydrophobic contribution of DeltaG(h) = -7.8 kcal/mol, comparable to that of post-translationally attached membrane anchors, and an electrostatic contribution of DeltaG(h) = -1.6 kcal/mol. The latter becomes more negative with decreasing pH. We show that H17 provides the binding energy required for a membrane anchor.

Amino Acid Sequence↗
PubMed2001

Localization of an integrin binding site to the C terminus of talin.

Talin, consisting of a 47-kDa N-terminal head domain (residues 1-433) and a 190-kDa C-terminal rod domain (residues 434-2541), links integrins to the actin cytoskeleton. We previously reported that the binding stoichiometry of integrin alpha(IIb)beta(3):talin is approximately 2:1. More recently, an integrin binding site has been localized to the talin head domain. In the present study, we identified another integrin binding site at the C-terminal region of the talin rod domain. In a solid phase binding assay, RGD affinity-purified alpha(IIb)beta(3) bound in a dose-dependent manner to microtiter wells coated with the isolated 190-kDa proteolytic fragment of the talin rod domain. Additionally, alpha(IIb)beta(3) also bound to the talin rod domain captured by 8d4, an anti-talin monoclonal antibody. Polyclonal antibodies raised against a recombinant protein fragment corresponding to the entire talin rod domain (anti-talin-R) inhibited alpha(IIb)beta(3) binding to intact talin by approximately 50% but completely blocked alpha(IIb)beta(3) binding to the talin rod domain. To localize the integrin binding site, we examined alpha(IIb)beta(3) binding to recombinant polypeptide fragments corresponding to partial sequences of the talin rod domain. Whereas alpha(IIb)beta(3) bound effectively to talin-(1075-2541) and talin-(1984-2541), it failed to bind to talin-(434-1076) and talin-(434-1975). Furthermore, the binding of alpha(IIb)beta(3) to talin-(1984-2541) was inhibited by anti-talin-R. These results indicate that an integrin binding site is located within residues 1984-2541 of the talin rod domain. Thus, talin contains two integrin binding sites, one in the homologous FERM (band four-point-one, ezrin, radixin, moesin) domain and another near its C terminus. Because talin exists as an anti-parallel homodimer in focal adhesions, the two integrin binding sites in the adjacent talin molecules would be in close proximity with each other.

Actins↗
PubMed2001

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.

Amino Acid Sequence↗
PubMed1993

Talin distribution and phosphorylation in thrombin-activated platelets.

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

Biological Transport↗
PubMed1999

Interaction of talin with actin: sensitive modulation of filament crosslinking activity.

Talin is an adhesion plaque protein believed important in linking actin filaments to the plasma membrane. The nature of a direct talin-actin interaction, however, is complex and has remained unclear. We have systematically characterized the effects of pH, ionic strength, temperature, and protein molar ratio on the interaction between highly purified talin and actin. The ability of talin to increase viscosity of F-actin at 25 degrees C and low ionic strength increased with decreasing pH from 7.3 to 6.4 and increasing molar ratio of talin to actin. At pH 6.4 and low ionic strength, talin could extensively crosslink actin filaments into ordered bundles as shown by negative staining and could cosediment with F-actin at molar ratios as high as one talin to two actin monomers. Talin crosslinked prepolymerized actin filaments to a similar extent as actin filaments polymerized in its presence. The 190-kDa calpain-generated proteolytic fragment of talin bound poorly to actin under conditions favorable for intact talin, but was able to crosslink actin filaments at a lower pH. Increasing the ionic strength within a relatively narrow range significantly decreased ability of talin to bind to actin, regardless of pH. The effects of pH and ionic strength on the talin-actin interaction were rapid and reversible. Low-shear-viscosity studies revealed a strong temperature dependence in the talin-actin interaction with significant crosslinking activity at physiological-like ionic conditions and temperature (37 degrees C). Our results consistently demonstrated that talin crosslinks actin filaments and that this direct interaction is highly sensitive to, and dependent upon, ionic conditions and temperature.

Actins↗
PubMed1996

Direct binding of the platelet integrin alphaIIbbeta3 (GPIIb-IIIa) to talin. Evidence that interaction is mediated through the cytoplasmic domains of both alphaIIb and beta3.

As a consequence of platelet activation and fibrinogen binding, glycoprotein (GP)IIb-IIIa (integrin alphaIIbbeta3) becomes associated with the cytoskeleton. Although talin has been suggested to act as a linkage protein mediating the attachment of GPIIb-IIIa to actin filaments, direct binding of GPIIb-IIIa to this cytoskeletal protein has not been demonstrated. In the present study, we examined the interaction of GPIIb-IIIa with purified talin using a solid-phase binding assay. Soluble GPIIb-IIIa bound in a time- and dose-dependent manner to microtiter wells coated with talin but not with BSA. Time course studies demonstrated that steady-state binding was achieved after 4-5 h incubation at 37 degrees C. Binding isotherms with varying concentrations of GPIIb-IIIa showed that half-saturation binding was achieved at approximately 15 nM GPIIb-IIIa. At saturation, there was 211 +/- 8 fmol of GPIIb-IIIa bound per well containing 117 +/- 10 fmol of immobilized talin. Besides binding to immobilized talin, GPIIb-IIIa also bound to talin captured by the anti-talin monoclonal antibody 8d4. Moreover, the interaction of GPIIb-IIIa to 8d4-captured talin was blocked by mAb10B2, a monoclonal antibody raised against a synthetic peptide encompassing the entire cytoplasmic sequence of GPIIb. The interaction of talin with the cytoplasmic domain of GPIIb-IIIa was further investigated using peptide-coated wells. Purified talin was found to bind to both synthetic peptides corresponding to the cytoplasmic sequences of GPIIb (P2b) and GPIIIa (P3a). As expected, the binding of talin to P2b-coated wells was specifically blocked by mAb10B2. Thus, these results demonstrate direct binding of GPIIb-IIIa to talin and suggest a role of the cytoplasmic sequences of both GPIIb and GPIIIa in mediating this interaction.

Amino Acid Sequence↗
PubMed1986

Demonstration of a relationship between talin and P235, a major substrate of the calcium-dependent protease in platelets.

Talin is a 225,000-Dalton protein we have purified from smooth muscle. In chick embryo fibroblasts talin is found in adhesion plaques (focal contacts), areas where the cell is closely apposed to the substratum. In comparison with other cytoskeletal proteins, we found talin to be unusually susceptible to proteolysis and have identified a 190,000-Dalton proteolytic fragment of talin in the immunoblots of many tissues. These observations raised the possibility that the cleavage of talin to this fragment has physiological relevance. One system that we have investigated in which significant proteolysis occurs is platelets. During platelet activation several high-molecular-weight proteins are cleaved to lower-molecular-weight forms. Here we demonstrate that talin is closely related to one of these platelet high-molecular-weight proteins, P235. The purification of talin is comparable to that developed for P235, and the two proteins have similar biophysical properties. In addition, antibodies raised against chicken gizzard talin recognize P235 in purified form as well as in crude platelet extracts. The platelet protein also resembles smooth-muscle talin in its susceptibility to endogenous proteolysis: P235 is rapidly cleaved to a 190-200 kD polypeptide by a calcium-activated protease found in platelet extracts. Moreover, partial proteolysis of P235 and talin with chymotrypsin, elastase, or trypsin also generates remarkably similar one-dimensional peptide maps. Because of their similar biophysical properties, immunological crossreactivity, and similar one-dimensional partial peptide maps, we conclude that P235 is the platelet form of talin.

Animals↗
PubMed2001

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.

3T3 Cells↗
PubMed1990

Functional studies of the domains of talin.

The protein talin has two domains of approximately 200 and 47 kD, which can be cleaved apart by a variety of proteases. To examine the function of these two structural domains of talin, we have digested purified talin with a calcium-dependent protease and separated the resulting fragments chromatographically. Both fragments were radioiodinated and used to probe Western blots of whole fibroblasts and chicken gizzard extracts. The large talin fragment bound to vinculin and metavinculin. The small fragment did not demonstrate any binding in this assay. The fragments were labeled fluorescently and microinjected into fibroblasts in tissue culture. The large talin fragment incorporated quickly into focal adhesions where it remained stable for at least 14 h. The small fragment associated with focal adhesions of fibroblasts but was also distributed diffusely in the cytoplasm and the nucleus. These experiments suggest that talin has at least two sites that contribute to its localization in focal adhesions. Intact talin microinjected into Madin-Darby bovine kidney epithelial cells localized to the focal adhesions but was excluded from the zonulae adherentes, despite the localization of vinculin to both of these sites. In contrast, the large talin fragment, when microinjected into these epithelial cells, incorporated into both focal adhesions and zonulae adherentes. The difference in localization between the large talin fragment and intact talin seems to be due to the removal of the small domain. This difference in localization suggests that talin binding sites in zonulae adherentes have limited accessibility.

Actins↗
PubMed1990

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.

Animals↗
PubMed1992

Vinculin binding site mapped on talin with an anti-idiotypic antibody.

Vinculin and talin are major adhesion plaque components which interact in vitro and presumably in vivo. The amino acid sequence of talin is now known so details of its domain structure can be mapped. We localized vinculin binding sites in the talin sequence by overlaying peptide maps of talin with an anti-idiotypic vinculin antibody that recognizes talin and with 125I-vinculin. A rabbit injected only twice with vinculin and producing anti-vinculin antibodies spontaneously generated a second antibody that recognizes talin. Vinculin and anti-vinculin antibodies specifically compete with this second antibody for binding to talin as determined by solid-phase binding and overlay assays. The antibody is thus most likely an anti-idiotypic antibody which mimics a region of vinculin that interacts with talin. The binding site of the anti-idiotypic antibody on talin was mapped to the 196 amino acids spanning residues 1653 to 1848. A second vinculin binding site identified with an 125I-vinculin blot overlay technique was located between residues 483 and 1652. The observation that talin has two immunologically distinct vinculin binding sites suggests that vinculin may have two different talin binding sites or one "complex" site with two interacting regions.

Amino Acid Sequence↗