Talin Binding Research Articles

Involvement of Nectin in Inactivation of Integrin αvβ3 after the Establishment of Cell-Cell Adhesion

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.

A Disease Mechanism Underlying Bleeding in Wiskott-Aldrich Syndrome

The Wiskott-Aldrich Syndrome (WAS) is an × chromosome-linked immunodeficiency disorder. The most common symptom in WAS is bleeding. Several clinical investigations indicate that low platelet counts and defective platelet aggregation are the major causes of bleeding in WAS patients. However, the molecular bases underlying these defects are unclear. This study focuses on the molecular mechanism of defective platelet aggregation of WAS patients. The gene responsible for WAS encodes WAS protein (WASP). The mutations or deletion of WASP causes various functional defects in hematopoietic cells. We previously showed that binding of WASP to calcium- and integrin-binding protein (CIB) is required for activation of platelet integrin, αIIbβ3. I here demonstrate that blocking WASP binding to CIB reduces binding of talin to the β3 cytoplasmic tail, resulting in impaired activation of αIIbβ3. Impaired αIIbβ3 activation causes defective platelet aggregation, resulting in bleeding. This finding suggests a potential disease mechanism underlying bleeding seen in WAS patients.

RGT, a Sythetic Peptide, Selectively Inhibits Integrin αIIbβ3 Outside-In Signaling in Human Platelets through Disassociating SRC Kinase from β3 Subunit.

Integrin αIIbβ3 plays pivotal roles in platelet activation by interacting with ligands and by mediating bidirectional signal transduction as well. The molecular mechanisms underlying the signal transduction pathways by which integrin αIIbβ3 regulates platelet activation have not yet been completely understood. Previous truncational analyses with CHO cell model have established that the C-terminal RGT sequence of integrin β3 subunit is critical to outside-in signaling but not inside-out signaling. To examine whether this mechanism functions in intact human platelets, a synthetic cell-permeable peptide corresponding to C-terminal RGT sequence of integrin β3 was employed in this work in an attempt to compete with native integrin β3 subunit for downstream signaling molecules. Myristoylated RGT peptide (myr-RGT) dose-dependently inhibited adhesion and spreading of normal human platelets on immobilized fibrinogen and prevented fibrin clot retraction. Also, myr-RGT selectively inhibited the second wave of platelet aggregation induced by ADP, ristocetin or thrombin with unaffected first wave of aggregation, while it had no inhibitory effect on ADP-induced soluble fibrinogen binding. These results suggest that the integrin outside-in signaling pathway is impaired by the treatment of RGT peptide and that the inside-out pathway remained undisrupted. Thus we showed for the first time in intact platelets that the outside-in signaling could be selectively regulated by synthetic peptide with sequence corresponding to the last three amino acids of integrin β3 cytoplasmic tail. To further define the molecular basis of the roles of RGT peptide in signal transduction we examined the effect of RGT peptide on tyrosine phosphorylation of integrin β3 cytoplasmic domain. We found that when platelets were stimulated by thrombin the phosphorylation of both cytoplasmic tyrosine residues (Y747 and Y759) of integrin β3 was substantially inhibited by the presence of myr-RGT peptide. This inhibition could be attributed to the findings in immunoprecipitation experiments whereby RGT peptide attenuated the interaction of integrin β3 with Src kinase. On the contrary, the RGT peptide was unable to interfere talin binding to β3 subunit. These results support the conclusion that, in intact human platelets, the RGT peptide selectively blocked outside-in signaling by disrupting the constitutive interaction of Src kinase with integrin β3 and hence causing a decreased phosphorylation level of tyrosine residues at integrin β3 cytoplasmic domain. Thus, in platelets, the interaction of the RGT sequence in integrin β3 cytoplasmic tail with Src kinase is necessary and sufficient to transduce outside-in signals while the disassociation of Src from integrin β3 has no regulatory effect on talin binding to β3 cytoplasmic domain. The application of RGT peptide and derivatives in the practice of platelet studies enables us to regulate outside-in signaling without affecting inside-out pathway and to speculate the better understanding of the molecular basis of mechanisms for integrin signal transdution. These results also underline the potential use of RGT peptide as a new antithrombotic strategy.

A NPxY-independent β5 integrin activation signal regulates phagocytosis of apoptotic cells

Integrin receptors are heterodimeric transmembrane receptors with critical functions in cell adhesion and migration, cell cycle progression, differentiation, apoptosis, and phagocytosis of apoptotic cells. Integrins are activated by intracellular signaling that alter the binding affinity for extracellular ligands, so-called inside to outside signaling. A common element for integrin activation involves binding of the cytoskeletal protein talin, via its FERM domain, to a highly conserved NPxY motif in the β chain cytoplasmic tails, which is involved in long-range conformation changes to the extracellular domain that impinges on ligand affinity. When the human beta-5 (β5) integrin cDNA was expressed in αv positive, β5 and β3 negative hamster CS-1 cells, it promoted NPxY-dependent adhesion to VTN-coated surfaces, phosphorylation of FAK, and concomitantly, β5 integrin-EGFP protein was recruited into talin and paxillin-containing focal adhesions. Expression of a NPxY destabilizing β5 mutant (Y750A) abrogated adhesion and β5-Y750A-EGFP was excluded from focal adhesions at the tips of stress fibers. Surprisingly, expression of β5 Y750A integrin had a potent gain-of-function effect on apoptotic cell phagocytosis, and further, a β5-Y750A-EGFP fusion integrin readily bound MFG-E8-coated 10μm diameter microspheres developed as apoptotic cell mimetics. The critical sequences in β5 integrin were mapped to a YEMAS motif just proximal to the NPxY motif. Our studies suggest that the phagocytic function of β5 integrin is regulated by an unconventional NPxY-talin-independent activation signal and argue for the existence of molecular switches in the β5 cytoplasmic tail for adhesion and phagocytosis.

The antithrombotic potential of selective blockade of talin-dependent integrin αIIbβ3 (platelet GPIIb–IIIa) activation

In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(3) (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(3)(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(3)(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.

Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions

Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin-integrin complexes into fibronectin-mediated fibrillar adhesions.

Two Synergistic Activation Mechanisms of α2β1 Integrin-mediated Collagen Binding

Activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces ligand-independent aggregation of a cell surface collagen receptor, alpha2beta1 integrin. Concomitantly, TPA increases the avidity of alpha2beta1 for collagen and the number of conformationally activated alpha2beta1 integrins. The structural change was shown using a monoclonal antibody 12F1 that recognizes the "open" (active) conformation of the inserted domain in the alpha2 subunit (alpha2I). Amino acid residue Glu-336 in alpha2 subunit is proposed to mediate the interaction between alpha2I domain and beta1 subunit. Glu-336 seems to regulate a switch between open and "closed" conformations, since the mutation alpha2E336A inhibited the TPA-related increase in the number of 12F1 positive integrins. E336A also reduced cell adhesion to collagen. However, E336A did not prevent the TPA-related increase in adhesion to collagen or alpha2beta1 aggregation. Thus, alpha2beta1 integrin avidity is regulated by two synergistic mechanisms, first an alpha2E336-dependent switch to the open alpha2I conformation, and second an alpha2E336-independent mechanism temporally associated with receptor aggregation.

Stable adhesion and migration of human neutrophils requires phospholipase D-mediated activation of the integrin CD11b/CD18

The pathways regulating integrin-mediated adhesion during neutrophil migration are incompletely defined. Using a flow-based model in which human neutrophils rolling on P-selectin were activated to migrate by the chemoattractant peptide fMLP, we investigated the role of phospholipase D (PLD). fMLP-stimulated PLD generation of phosphatidate (PtdOH); while inhibition of PtdOH production with butan-1-ol had no effect on the initial immobilisation of rolling neutrophils (supported by activation of constitutively surface-expressed β 2-integrin CD11b/CD18) it impaired longer-term stability of adhesion and reduced the rate of migration (supported by activation of de novo-exocytosed CD11b/CD18). PtdOH regulated these processes by controlling activation of exocytosed CD11b/CD18, and appeared to act by directly stimulating phosphatidylinositol 4-phosphate 5-kinase type I to generate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2). Cell-permeable PtdIns(4,5)P 2 recovered migration of neutrophils after PLD inhibition; PtdIns(4,5)P 2 appeared to act by promoting talin binding to CD18 and hence activating CD11b/CD18, as migration was inhibited when neutrophils were loaded with peptides previously shown to block the interaction between PtdIns(4,5)P 2 and talin or talin and CD18. Thus, these data indicate that PLD-synthesised PtdOH stimulates the generation of PtdIns(4,5)P 2, which in turn mediates talin binding to, and activation of, CD11b/CD18 required for neutrophil stable adhesion and migration.

Cytoskeletal protein radixin activates integrin α Mβ 2 by binding to its cytoplasmic tail

Talin binding of integrins, via its band 4.1, ezrin, radixin, and moesin (FERM)-homologous domain, directly activates the integrin receptor. However, it is not known whether other FERM-containing proteins also possess such an integrin activating capability. We report here that radixin, one of the original FERM-domain proteins, binds to the membrane-proximal region of the integrin β 2 but not α M cytoplasmic tail. Importantly, we show that radixin binding significantly enhances the adhesive activity of integrin α Mβ 2. Given the distinct biological activities of radixin and talin, radixin may represent a novel talin-independent pathway for integrin activation under specific settings.

PIP5KIγ Knockout Megakaryocytes Have Defects in Their Cytoskeleton & Demarcation Membrane System, yet Form Proplatlets & Platelets.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical component of intracellular signaling cascades as well as actin dynamics. It is synthesized by the phosphorylation of PI4P by one of three isoforms of PIP5KI (α,β, or γ). We found that the targeted disruption of PIP5KIγ results in early prenatal mortality that prevented studies of bone marrow or liver hematopoietic cells. However, we were able to analyze yolk sac progenitor cells differentiated ex vivo into megakaryocytes. Imaging in the electron microscope showed that PIP5KIγ-null megakaryocytes have normal architectural appearance of their microtubules, and formed proplatelets and platelets. This demonstrates that PIP5KIγ is not essential for the development of megakaryocytes, proplatelets, or platelets. PIP5KIγ knockout megakaryocytes had normal basal levels of PIP2, but decreased synthesis following stimulation with thrombin. Using spinning disk video confocal microscopy, we found that wild type megakaryocytes actively formed and contracted lamellipodia, and rapidly spread upon a fibrinogen matrix. In contrast, PIP5KIγ-null megakaryocytes continuously and slowly extended and retracted membrane blebs, rather than lamellipodia. We analyzed whether PIP5KIγ-null megakaryocytes have a defect in their ability to anchor their membranes to the cytoskeleton accounting for the blebbing phenotype by using laser tweezers to pull the cell membrane apart from the cytoskeleton. Fibrinogen-coated beads bound both wild type and PIP5KIγ-null megakaryocytes. Yet wild type cells had rigid membranes that resisted stretching by trapped beads that were pulled by laser tweezers. In contrast, the majority of PIP5KIγ-null megakaryocytes had flexible membranes that were easily stretched by pulling on the fibrinogen-coated beads, and ultimately allowed membrane tethers to form. We found that the membrane phenotype was completely reverted by adding back wild type PIP5KIγ, but not by a catalytically inactive PIP5KIγ mutant. This shows that the lipid kinase activity of PIP5KIγ regulates the membrane phenotype. Surprisingly, PIP5KIβ-null megakaryocytes have at least as great of a defect in PIP2 synthesis but lack this membrane abnormality. Furthermore, overexpression of PIP5KIβ in PIP5KIγ-null megakaryocytes fails to rescue the cytoskeletal defect. Adding back a splice variant of PIP5KIγ that lacks the binding site for talin, also fails to revert this cytoskeletal defect present in PIP5KIγ-null cells. This suggests that talin binding and PIP2 synthesized specifically by the PIP5KIγ isoform are required for the cytoskeletal organization of megakaryocytes. Given the proposed role of PIP2 on the regulation of the demarcation membrane system (DMS), we expressed GFP-fused to β3-integrin and followed its trafficking from the DMS to the surface membrane. GFP-β3 situated on the DMS externalized to the cellular membrane within minutes of plating a wild type megakaryocyte upon immobilized fibrinogen. In megakaryocytes lacking PIP5KIγ, GFP-β3 situated on the DMS poorly externalized, and was barely detectable on the cell membrane 1 hour after adhering to immobilized fibrinogen. This shows that trafficking of αIIbβ3 from the DMS to the cell membrane requires PIP5KIγ. Together, this demonstrates that spatially restricted production of PIP2 by PIP5KIγ is required to preserve the integrity of the membrane cytoskeleton, and that PIP2 synthesized by PIP5KIβ in megakaryocytes can not contribute to this process.

Dissociation of the Extracellular α-Tail/β-Tail Interface Interaction Activates αIIbβ3 Integrin.

It is postulated that integrins undergo structural rearrangement from the low affinity bent conformer to the high affinity extended conformer upon activation. In inside-out signaling, the binding of talin to β cytoplasmic tail has been shown to activate integrin by disrupting the endogenous α/β cytoplasmic tail interaction that constrains integrin in low affinity state. How this intracellular event triggers structural rearrangement in the extracellular part of the integrin has not been elucidated. In integrin bent conformer, β-tail not only makes interface with β-head, but also makes large interface with α-tail. We previously reported that the membrane-proximal α-tail/β-tail interface interaction regulates integrin activation, and that stabilization of this interface completely blocks the propagation of inside-out signaling. From these results, we hypothesized that dissociation of the α-tail/β-tail following the dissociation of the α/β cytoplasmic tails triggers disruption of the β-head/β-tail interaction that constrains integrin in the bent state, thus activates integrin in inside-out signaling. Here we report that dissociation of the extracellular α-tail/β-tail actually activates αIIbβ3 integrin. To induce dissociation of the tails, we introduced N-linked glycosylation site (N-X-T/S) in αIIb amino acid residue Met-660 (M660NRT) which is located at the calf-1/EGF-3 interface that compose part of the α-tail/β-tail interface. When expressed in CHO cells, the mutant αIIbβ3 constitutively bound fibrinogen. Introduction of any sequences other than N-X-T/S in this site failed to activate αIIbβ3, suggesting this activation depends on the binding of a bulky N-glycan to the site. This activation was completely abolished when the β-head/β-tail interface was stabilized. Notably, stabilization of the membrane-proximal calf-2/EGF4-βTD interface with a disulfide bridge significantly inhibited activation induced by calf-1/EGF-3 dissociation, while it did not have any effect on fully extended αIIbβ3. These results suggest that disruption of the membrane-proximal α-tail/β-tail interface rather than the dissociation of the membrane-distal calf-1/EGF-3 interface is critical for initiating the structural rearrangement. Next, we examined whether disruption of the β-head/β-tail interface activates αIIbβ3 by introducing N-linked glycosylation site in β3 amino acid residue Gly-382 or in Thr-564 that are located at the β-head/β-tail interface. The resulting G382NLT and T564N mutants constitutively bound fibrinogen. This activation was completely abolished when αIIbβ3 was constrained in the bent conformer. Different from the M660NRT, this activation was not inhibited by stabilizing the calf-2/EGF4-βTD interface. In summary, these results suggest, 1) that disruption of the β-head/β-tail interface is required for the α-tail/β-tail dissociation to activate αIIbβ3, 2) that αIIbβ3 has to be able to extend for the disruption of the β-head/β-tail interface to activate αIIbβ3, 3) dissociation of the α-tail/β-tail is not required for activation, once β-head/β-tail interface is disrupted or αIIbβ3 is fully extended. Taken together, these results are consistent with our hypothesis and indicate that the α-tail/β-tail interface interaction is the key regulator of integrin activation in inside-out signaling.

Computational Design of a Model for the Platelet Integrin αIIbβ3.

Integrins, the principal receptors linking the cytoskeleton to extracellular ligands, are responsible, in large part, for platelet adhesion and aggregation. Like many membrane spanning proteins, the molecular structure of integrins has been difficult to determine, although crystal and NMR structures are available for water soluble portions of αVβ3 and αIIbβ3. In addition, models for the αIIb and β3 transmembrane (TM) heterodimer have been obtained using computational methods, but with conflicting results. Accordingly, we employed two different modeling strategies to distinguish between existing models. First, we used a Monte Carlo-simulated annealing (MCSA) protocol that features a selective advantage for structures that are consistent with previously characterized αIIb and β3 TM domain point mutations. During each step of the protocol, we calculated the effects of selected mutations and assigned a penalty to the step if it produced a structure that was inconsistent with experimental results. Second, we threaded the αIIb and β3 TM sequences onto a database of membrane helix dimers and rank-ordered the structures based on their calculated energies and their agreement with the cysteine crosslinking experiments reported by Luo et al (PLoS Biol 2:776–86, 2004). The MCSA and threaded TM models were similar with a root mean squared deviation (RMSD) of 1.5 Å. Moreover, they differed from a model reported by Kay Gottschalk (Structure 13:703–12, 2005) most notably by an ~50° rotation of the αIIb TM helix about its helical axis. This positions αIIb residue Leu980 in the TM interface and in van der Waals contact with β3 residue Gly708, a result consistent with mutagenesis experiments. Further, the αIIb and β3 TM helices pack along conserved glycine residues (Gly972 and Gly976) in the GXXXG motif of αIIb with a right-handed crossing angle of −18°. To create a model for the full-length integrin, the N-termini of the TM model were connected to the crystal structure of the αVβ3 extracellular domain and its C-termini were connected to the corresponding NMR structures of the αIIb and β3 cytosolic domains. The model predicts an αIIb Arg995-β3 Asp723 salt bridge in the membrane proximal cytosolic domains and a change in the secondary structure of the extracellular domains from an extended to a helical conformation that occurs at Pro965 in αIIb and Pro693 in β3, just proximal to the TM domains. Finally, the full length model provides insight into distal interactions since its extracellular portion is held close to the membrane and is therefore sterically inaccessible to large ligands such as fibrinogen, whereas cytosolic regulatory sites such as the talin binding site in β3 are accessible and not sequestered by α/β interactions. In conclusion, we have modeled the αIIbβ3 TM heterodimer using two fundamentally different computational strategies, both of which converge upon similar structures that were used to construct a model for the full-length integrin.

Tyrosine Phosphorylation of the Integrin β3 Subunit Regulates β3 Cleavage by Calpain

Outside-in signaling of beta(3) integrins induces and requires phosphorylation at tyrosine 747 (Tyr(747)) and tyrosine 759 (Tyr(759)) of the beta(3) subunit, but the mechanism for this requirement is unclear. On the other hand, a key consequence of integrin signaling, cell spreading, is inhibited by calpain cleavage of beta(3) cytoplasmic domain. Here we show that beta(3) tyrosine phosphorylation inhibits calpain cleavage. Mutating both tyrosines to phenylalanine sensitizes beta(3) to calpain cleavage. Furthermore, phosphorylation at Tyr(747) and Tyr(759) of beta(3) in the focal adhesion sites and the leading edge of spreading platelets was differentially regulated. Selective dephosphorylation of Tyr(759) is associated with calpain cleavage at Tyr(759). Thus, one mechanism by which tyrosine phosphorylation promotes integrin signaling and cell spreading is its inhibition of calpain cleavage of the beta(3) cytoplasmic domain.

Genetic analysis of β1 integrin “activation motifs” in mice

Akey feature of integrins is their ability to regulate the affinity for ligands, a process termed integrin activation. The final step in integrin activation is talin binding to the NPXY motif of the integrin β cytoplasmic domains. Talin binding disrupts the salt bridge between the α/β tails, leading to tail separation and integrin activation. We analyzed mice in which we mutated the tyrosines of the β1 tail and the membrane-proximal aspartic acid required for the salt bridge. Tyrosine-to-alanine substitutions abolished β1 integrin functions and led to a β1 integrin–null phenotype in vivo. Surprisingly, neither the substitution of the tyrosines with phenylalanine nor the aspartic acid with alanine resulted in an obvious defect. These data suggest that the NPXY motifs of the β1 integrin tail are essential for β1 integrin function, whereas tyrosine phosphorylation and the membrane-proximal salt bridge between α and β1 tails have no apparent function under physiological conditions in vivo.

Reconstructing and Deconstructing Agonist-Induced Activation of Integrin αIIbβ3

Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.

Insights into Allosteric Control of Vinculin Function from Its Large Scale Conformational Dynamics

Vinculin is an important constituent of both cell-cell and cell-matrix junctions, where it plays crucial roles in the regulation of cell adhesion and migration. When activated, it mediates the linkage between cadherins (cell-cell) or integrins (cell-matrix) and the actin cytoskeleton through interactions with various proteins. The activation of vinculin requires structural conversions from an autoinhibited conformation to the "open" conformations in which the occluded binding sites of its different ligands become exposed, while the structural dynamics underlying the vinculin activation remains largely unknown. Here we report the first computational study of large scale conformational dynamics of full-length vinculin. We find that the "holding" and "releasing" motions between vinculin tail and pincer-like structure formed by first three domains of vinculin are the dominant motions near the native state of vinculin, indicating that an inherent flexibility of vinculin has a large influence on its allostery. We also find a cooperative dissociation between the head and tail domains of vinculin with increasing temperature in both thermodynamic and kinetic simulations, implying that vinculin may function as an allosteric switch in response to external signals. We show that the kinetics of vinculin unfolding exhibits specific sequential patterns, suggesting that a sophisticated interplay between domains may synergistically contribute to vinculin activation. We further find that the interaction between vinculin-binding site peptide from talin and vinculin significantly destabilizes the intramolecular head-tail interactions, suggesting a direct role of talin binding in vinculin activation.

A Conformational Switch in Vinculin Drives Formation and Dynamics of a Talin-Vinculin Complex at Focal Adhesions

Dynamic interactions between the cytoskeleton and integrins control cell adhesion, but regulatory mechanisms remain largely undefined. Here, we 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 drove assembly of talin-vinculin complexes, whereas ectopic complexes did not form between talin and wild-type vinculin. Moreover, reduction of HTI altered the dynamic assembly of vinculin and talin in focal adhesions. Using fluorescence recovery after photobleaching, we show that the focal adhesion residency time of vinculin was enhanced up to 3-fold by HTI mutations. The slow dynamics of vinculin correlated with exposure of its cryptic talin-binding site, and a talin-binding site mutation rescued the dynamics of activated vinculin. Significantly, HTI-deficient vinculin inhibited 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. We therefore conclude that vinculin conformation, as modulated by the strength of HTI, directly regulates the formation and lifetime of talin-vinculin complexes in cells.

Integrin activation—the importance of a positive feedback

Integrins mediate cell adhesion and are essential receptors for the development and functioning of multicellular organisms. Integrin activation is known to require both ligand and talin binding and to correlate with cluster formation but the activation mechanism and precise roles of these processes are not yet resolved. Here mathematical modeling, with known experimental parameters, is used to show that the binding of a stabilizing factor, such as talin, is alone insufficient to enable ligand-dependent integrin activation for all observed conditions; an additional positive feedback is required.

Development requires activation but not phosphorylation of β1 integrins

Named after the ability of many of them to “integrate” the extracellular matrix with the intracellular cytoskeleton, integrins are well-characterized mediators of cellular adhesion and signaling (Giancotti and Ruoslahti 1999; Hynes 2002). Each integrin consists of an and a subunit and binds to a distinct, but often overlapping, spectrum of ligands. Whereas 1 integrins mediate cell adhesion to extracellular matrix components, 2 integrins play key roles in immune cell recognition and activation by interacting with counterreceptors of the Ig superfamily, and IIb 3 orchestrates platelet aggregation by binding to the polyvalent blood protein fibrinogen. Genetic studies using mouse models have demonstrated that 1 integrins have unique roles in embryonic development, hematopoiesis, wound healing, and cancer (Brakebusch et al. 1997; Grose et al. 2002; White et al. 2004). Successful execution of a number of physiological processes requires precisely choreographed changes in the activity of several integrins. The regulation of integrin functions is complex, and recent studies suggest that it hinges on long-range structural changes, which are propagated across the plasma membrane in both directions (Hynes 2002). In spite of its importance, genetic analysis of this aspect of integrin function has lagged behind. A new paper by Chen et al. (2006), published in the April 15 issue of Genes and Development, intends to remedy this imbalance. Many integrins are maintained in a default low-affinity state and, hence, need to be activated to exert their function. The past decade has seen the emergence and consolidation of two major paradigms for integrin activation and signaling. The first describes the process through which cytoplasmic signals trigger integrin activation (“inside-to-outside” integrin signaling) (Ginsberg et al. 1992). Most, perhaps all, activating signals promote the binding of talin to the cytoplasmic portion of the integrin subunit (Liddington and Ginsberg 2002). Talin binding promotes a separation of the integrin cytoplasmic tails and triggers a series of long-range conformation changes, including a “switch-blade” movement of the integrin head domains, resulting in increased availability of the extracellular ligand-binding site (Shimaoka et al. 2002). The immediate outcome of this form of regulation is an increase in the affinity of integrins for their matrix ligands. The second process, “outside-to-inside” integrin signaling, flows in the opposite direction. Upon binding to extracellular matrix components, integrins multimerize and recruit complexes of cytoskeletal and signaling molecules. Using this latter mechanism, integrins cooperate with receptors for cytokines and growth factors to control a multitude of cellular functions, including cell adhesion, migration, survival, differentiation, and proliferation (Assoian and Schwartz 2001; Miranti and Brugge 2002; Giancotti and Tarone 2003). Both inside-to-outside and outside-to-inside integrin signaling rely on a combination of regulatory controls that involves alterations in the structure of the receptors’ cytoplasmic domains, long-range conformational changes in integrin structure, a steep increase in ligand-binding affinity, and finally, clustering of integrins and downstream signal transduction. Mutagenesis and structural studies have painted a particularly vivid picture of integrin activation. Integrins are maintained inactive by interactions between the transmembrane domains and the membrane-proximal segments of the cytoplasmic tails of their constituent subunits (Fig. 1; Liddington and Ginsberg 2002). Substitution with Ala of an Asp residue in the membraneproximal segment of the subunit or an Arg residue in the corresponding region of the subunit causes constitutive integrin activation, suggesting that a salt bridge between the side chains of the two amino acids stabilizes the inactive conformation (Hughes et al. 1996). In agreement with this notion, forced dimerization of the and tails prevents integrin activation (Lu et al. 2001). What is the mechanism through which cytoplasmic signals disrupt the – tail “clasp”? The C-terminal portion of all subunits contains two tandem NPxY motifs, and compelling evidence indicates that the N-terminal motif functions to “unclasp” the integrin tails (Fig. 1). NMR structural analysis indicates that this motif forms a turn, creating a binding interface for a phospho-tyroCorrespondence. E-MAIL f-giancotti@ski.mskcc.org; FAX (212) 794-6236. E-MAIL yvp2001@med.cornell.edu; FAX (212) 794-6236. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1432006.

The Vinculin Binding Sites of Talin and α-Actinin Are Sufficient to Activate Vinculin

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. We tested the hypothesis that talin and alpha-actinin activate vinculin through their vinculin binding sites. Indeed, we show that 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.