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Interaction of talin with the cytoplasmic tails of integrin β triggers integrin activation, leading to an increase of integrin affinity/avidity for extracellular ligands. In talin KO mice, loss of talin interaction with platelet integrin αIIbβ3 causes a severe hemostatic defect, and loss of talin interaction with endothelial cell integrin αVβ3 affects angiogenesis. In normal cells, talin is autoinhibited and localized in the cytoplasm. Here, we used an optogenetic platform to assess whether recruitment of full-length talin to the plasma membrane was sufficient to induce integrin activation. A dimerization module (Arabidopsis cryptochrome 2 fused to the N terminus of talin; N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]) responsive to 450 nm (blue) light was inserted into Chinese hamster ovary cells and endothelial cells also expressing αIIbβ3 or αVβ3, respectively. Thus, exposure of the cells to blue light caused a rapid and reversible recruitment of Arabidopsis cryptochrome 2-talin to the N-terminal of cryptochrome-interacting basic helix-loop-helix domain ended with a CAAX box protein [C: cysteine; A: aliphatic amino acid; X: any C-terminal amino acid]-decorated plasma membrane. This resulted in β3 integrin activation in both cell types, as well as increasing migration of the endothelial cells. However, membrane recruitment of talin was not sufficient for integrin activation, as membrane-associated Ras-related protein 1 (Rap1)-GTP was also required. Moreover, talin mutations that interfered with its direct binding to Rap1 abrogated β3 integrin activation. Altogether, these results define a role for the plasma membrane recruitment of talin in β3 integrin activation, and they suggest a nuanced sequence of events thereafter involving Rap1-GTP., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ras-related protein 1 (Rap1) is a major convergence point of the platelet-signaling pathways that result in talin-1 binding to the integrin β cytoplasmic domain and consequent integrin activation, platelet aggregation, and effective hemostasis. The nature of the connection between Rap1 and talin-1 in integrin activation is an important remaining gap in our understanding of this process. Previous work identified a low-affinity Rap1-binding site in the talin-1 F0 domain that makes a small contribution to integrin activation in platelets. We recently identified an additional Rap1-binding site in the talin-1 F1 domain that makes a greater contribution than F0 in model systems. Here we generated mice bearing point mutations, which block Rap1 binding without affecting talin-1 expression, in either the talin-1 F1 domain (R118E) alone, which were viable, or in both the F0 and F1 domains (R35E,R118E), which were embryonic lethal. Loss of the Rap1-talin-1 F1 interaction in platelets markedly decreases talin-1-mediated activation of platelet β1- and β3-integrins. Integrin activation and platelet aggregation in mice whose platelets express only talin-1(R35E, R118E) are even more impaired, resembling the defect seen in platelets lacking both Rap1a and Rap1b. Although Rap1 is important in thrombopoiesis, platelet secretion, and surface exposure of phosphatidylserine, loss of the Rap1-talin-1 interaction in talin-1(R35E, R118E) platelets had little effect on these processes. These findings show that talin-1 is the principal direct effector of Rap1 GTPases that regulates platelet integrin activation in hemostasis., (© 2020 by The American Society of Hematology.)

Rap1 GTPases bind effectors, such as RIAM, to enable talin1 to induce integrin activation. In addition, Rap1 binds directly to the talin1 F0 domain (F0); however, this interaction makes a limited contribution to integrin activation in CHO cells or platelets. Here, we show that talin1 F1 domain (F1) contains a previously undetected Rap1-binding site of similar affinity to that in F0. A structure-guided point mutant (R118E) in F1, which blocks Rap1 binding, abolishes the capacity of Rap1 to potentiate talin1-induced integrin activation. The capacity of F1 to mediate Rap1-dependent integrin activation depends on a unique loop in F1 that has a propensity to form a helix upon binding to membrane lipids. Basic membrane-facing residues of this helix are critical, as charge-reversal mutations led to dramatic suppression of talin1-dependent activation. Thus, a novel Rap1-binding site and a transient lipid-dependent helix in F1 work in tandem to enable a direct Rap1-talin1 interaction to cause integrin activation., (© 2019 Gingras et al.)

Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbβ3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the β3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with β3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1-Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbβ3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1 R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.Tln1 R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1-talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions., (© 2018 by The American Society of Hematology.)

Talin, a cytoskeletal protein essential in mediating integrin activation, has been previously shown to be involved in the regulation of T cell proliferation and function. In this study, we describe a role for talin in maintaining the homeostasis and survival of the regulatory T (Treg) cell pool. T cell-specific deletion of talin in Tln1 mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis.fl/fl Cd4 Cre mice resulted in spontaneous lymphocyte activation, primarily due to numerical and functional deficiencies of Treg cells in the periphery. Peripheral talin-deficient Treg cells were unable to maintain high expression of IL-2Rα, resulting in impaired IL-2 signaling and ultimately leading to increased apoptosis through downregulation of prosurvival proteins Bcl-2 and Mcl-1. The requirement for talin in maintaining high IL-2Rα expression by Treg cells was due, in part, to integrin LFA-1-mediated interactions between Treg cells and dendritic cells. Collectively, our data suggest a critical role for talin in Treg cell-mediated maintenance of immune homeostasis., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Rap1-interacting adaptor molecule (RIAM) is a Rap1 effector that mediates the recruitment of talin to integrins, thereby supporting their activation. In this study, we investigated the role of RIAM in an adoptive transfer model for type I diabetes and report that RIAM expression in T cells is necessary for diabetes development. Loss of RIAM did not prevent lymphocyte recruitment to draining lymph nodes 24 h after transfer, but it was required for Ag-driven proliferation and cytotoxic killing. RIAM is recruited to immune synapses along with talin and LFA-1, and loss of RIAM profoundly suppresses Ag-dependent conjugate formation in primary naive and effector T cells. These data identify the requirement of RIAM for formation of immunological synapses and in resulting T cell functions in autoimmunity. Moreover, because RIAM-null mice are healthy, fertile, and display no bleeding abnormalities, our results identify RIAM and its regulators as potential targets for therapies of T cell-mediated autoimmunity., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Integrin adhesion receptors mediate the adhesion of blood cells, such as leukocytes, to other cells, such as endothelial cells. Integrins also are critical for anchorage of hematopoietic precursors to the extracellular matrix. Blood cells can dynamically regulate the affinities of integrins for their ligands ("activation"), an event central to their functions. Here we review recent progress in understanding the mechanisms of integrin activation with a focus on the functions of blood cells. We discuss how talin binding to the integrin β cytoplasmic domain, in conjunction with the plasma membrane, induces long-range allosteric rearrangements that lead to integrin activation. Second, we review our understanding of how signaling events, particularly those involving Rap1 small guanosine triphosphate (GTP)hydrolases, can regulate the talin-integrin interaction and resulting activation. Third, we review recent findings that highlight the role of the Rap1-GTP-interacting adapter molecule (RIAM), encoded by the APBB1IP gene, in leukocyte integrin activation and consequently in leukocyte trafficking., (© 2016 by The American Society of Hematology.)

The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.

Neutrophil recruitment, mediated by β2 integrins, combats pyogenic infections but also plays a key role in ischemia-reperfusion injury and other inflammatory disorders. Talin induces allosteric rearrangements in integrins that increase affinity for ligands (activation). Talin also links integrins to actin and other proteins that enable formation of adhesions. Structural studies have identified a talin1 mutant (L325R) that perturbs activation without impairing talin's capacity to link integrins to actin and other proteins. Here, we found that mice engineered to express only talin1(L325R) in myeloid cells were protected from renal ischemia-reperfusion injury. Dissection of neutrophil function in vitro and in vivo revealed that talin1(L325R) neutrophils had markedly impaired chemokine-induced, β2 integrin-mediated arrest, spreading, and migration. Surprisingly, talin1(L325R) neutrophils exhibited normal selectin-induced, β2 integrin-mediated slow rolling, in sharp contrast to the defective slow rolling of neutrophils lacking talin1 or expressing a talin1 mutant (W359A) that blocks talin interaction with integrins. These studies reveal the importance of talin-mediated activation of integrins for renal ischemia-reperfusion injury. They further show that neutrophil arrest requires talin recruitment to and activation of integrins. However, although neutrophil slow rolling requires talin recruitment to integrins, talin-mediated integrin activation is dispensable., (© 2015 Yago et al.)

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase-associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin-regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3., (© 2014 by The American Society of Hematology.)

Integrin αβ transmembrane heterodimers play central roles in metazoan development and physiology by mediating adhesion and by transmitting forces and biochemical signals across the plasma membrane. In this SnapShot, we present a simplified "modular" view of the integrin adhesome, centered on the talin-integrin interaction, and provide examples of how this view can help to unravel the adhesome's remarkable functional diversity and plasticity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Abstract Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases.

To determine talin1's role in osteoclasts, we mated TLN1(fl/fl) mice with those expressing cathepsin K-Cre (CtsK-TLN1) to delete the gene in mature osteoclasts or with lysozyme M-Cre (LysM-TLN1) mice to delete TLN1 in all osteoclast lineage cells. Absence of TLN1 impairs macrophage colony-stimulating factor (M-CSF)-stimulated inside-out integrin activation and cytoskeleton organization in mature osteoclasts. Talin1-deficient precursors normally express osteoclast differentiation markers when exposed to M-CSF and receptor activator of nuclear factor κB (RANK) ligand but attach to substrate and migrate poorly, arresting their development into mature resorptive cells. In keeping with inhibited resorption, CtsK-TLN1 mice exhibit an ∼5-fold increase in bone mass. Osteoclast-specific deletion of Rap1 (CtsK-Rap1), which promotes talin/β integrin recognition, yields similar osteopetrotic mice. The fact that the osteopetrosis of CtsK-TLN1 and CtsK-Rap1 mice is substantially more severe than that of those lacking αvβ3 is likely due to added failed activation of β1 integrins. In keeping with osteoclast dysfunction, mice in whom talin is deleted late in the course of osteoclastogenesis are substantially protected from ovariectomy-induced osteoporosis and the periarticular osteolysis attending inflammatory arthritis. Thus, talin1 and Rap1 are critical for resorptive function, and their selective inhibition in mature osteoclasts retards pathological bone loss.

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

Talin binding to integrin β tails increases ligand binding affinity (activation). Changes in β transmembrane domain (TMD) topology that disrupt α-β TMD interactions are proposed to mediate integrin activation. In this paper, we used membrane-embedded integrin β3 TMDs bearing environmentally sensitive fluorophores at inner or outer membrane water interfaces to monitor talin-induced β3 TMD motion in model membranes. Talin binding to the β3 cytoplasmic domain increased amino acid side chain embedding at the inner and outer borders of the β3 TMD, indicating altered topology of the β3 TMD. Talin's capacity to effect this change depended on its ability to bind to both the integrin β tail and the membrane. Introduction of a flexible hinge at the midpoint of the β3 TMD decoupled the talin-induced change in intracellular TMD topology from the extracellular side and blocked talin-induced activation of integrin αIIbβ3. Thus, we show that talin binding to the integrin β TMD alters the topology of the TMD, resulting in integrin activation.

In inflammation, neutrophils and other leukocytes roll along the microvascular endothelium before arresting and transmigrating into inflamed tissues. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen-1 (LFA-1). Mutations of the FERMT3 gene encoding kindlin-3 underlie the human immune deficiency known as leukocyte adhesion deficiency-III. Both kindlin-3 and talin-1, another FERM domain-containing cytoskeletal protein, are required for integrin activation, but their individual roles in the induction of specific integrin conformers are unclear. Here, we induce differential LFA-1 activation in neutrophils through engagement of the selectin ligand P-selectin glycoprotein ligand-1 or the chemokine receptor CXCR2. We find that talin-1 is required for inducing LFA-1 extension, which corresponds to intermediate affinity and induces neutrophil slow rolling, whereas both talin-1 and kindlin-3 are required for induction of the high-affinity conformation of LFA-1 with an open headpiece, which results in neutrophil arrest. In vivo, both slow rolling and arrest are defective in talin-1-deficient neutrophils, whereas only arrest is defective in kindlin-3-deficient neutrophils. We conclude that talin-1 and kindlin-3 serve distinct functions in LFA-1 activation.

Talin, which is composed of head (THD) and rod domains, plays an important role in cell adhesion events in diverse species including most metazoans and Dictyostelium discoideum. Talin is abundant in the cytosol; however, it mediates adhesion by associating with integrins in the plasma membrane where it forms a primary link between integrins and the actin cytoskeleton. Cells modulate the partitioning of talin between the plasma membrane and the cytosol to control cell adhesion. Here, we combine nuclear magnetic resonance spectroscopy (NMR) with subcellular fractionation to characterize two distinct THD-rod domain interactions that control the interaction of talin with the actin cytoskeleton or its localization to the plasma membrane. An interaction between a discrete vinculin-binding region of the rod (VBS1/2a; Tln1(482-787)), and the THD restrains talin from interacting with the plasma membrane. Furthermore, we show that vinculin binding to VBS1/2a results in talin recruitment to the plasma membrane. Thus, we have structurally defined specific inter-domain interactions between THD and the talin rod domain that regulate the subcellular localization of talin.

Using Tln1(fl/fl);CreER mice, we show that tamoxifen-induced inactivation of the talin1 gene throughout the embryo produces an angiogenesis phenotype that is restricted to newly forming blood vessels. The phenotype has a rapid onset in early embryos, resulting in vessel defects by 48 h and death of the embryo within 72 h. Very similar vascular defects were obtained using a Tie2-Cre endothelial cell-specific Tln1 knockout, a phenotype that was rescued by expression of a Tln1 mini-gene in endothelial cells. We show that endothelial cells, unlike most other cell types, do not express talin2, which can compensate for loss of talin1, and demonstrate for the first time that endothelial cells in vivo lacking talin1 are unable to undergo the cell spreading and flattening required to form vessels., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Cell-directed changes in the ligand-binding affinity ('activation') of integrins regulate cell adhesion and migration, extracellular matrix assembly and mechanotransduction, thereby contributing to embryonic development and diseases such as atherothrombosis and cancer. Integrin activation comprises triggering events, intermediate signalling events and, finally, the interaction of integrins with cytoplasmic regulators, which changes an integrin's affinity for its ligands. The first two events involve diverse interacting signalling pathways, whereas the final steps are immediately proximal to integrins, thus enabling integrin-focused therapeutic strategies. Recent progress provides insight into the structure of integrin transmembrane domains, and reveals how the final steps of integrin activation are mediated by integrin-binding proteins such as talins and kindlins.

Integrins are large membrane-spanning receptors fundamental to cell adhesion and migration. Integrin adhesiveness for the extracellular matrix is activated by the cytoskeletal protein talin via direct binding of its phosphotyrosine-binding-like F3 domain to the cytoplasmic tail of the beta integrin subunit. The phosphotyrosine-binding domain of the signaling protein Dok1, on the other hand, has an inactivating effect on integrins, a phenomenon that is modulated by integrin tyrosine phosphorylation. Using full-length tyrosine-phosphorylated (15)N-labeled beta3, beta1A, and beta7 integrin tails and an NMR-based protein-protein interaction assay, we show that talin1 binds to the NPXY motif and the membrane-proximal portion of beta3, beta1A, and beta7 tails, and that the affinity of this interaction is decreased by integrin tyrosine phosphorylation. Dok1 only interacts weakly with unphosphorylated tails, but its affinity is greatly increased by integrin tyrosine phosphorylation. The Dok1 interaction remains restricted to the integrin NPXY region, thus phosphorylation inhibits integrin activation by increasing the affinity of beta integrin tails for a talin competitor that does not form activating membrane-proximal interactions with the integrin. Key residues governing these specificities were identified by detailed structural analysis, and talin1 was engineered to bind preferentially to phosphorylated integrins by introducing the mutation D372R. As predicted, this mutation affects talin1 localization in live cells in an integrin phosphorylation-specific manner. Together, these results indicate that tyrosine phosphorylation is a common mechanism for regulating integrin activation, despite subtle differences in how these integrins interact with their binding proteins.

Fundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.

Cell migration is a dynamic process that requires temporal and spatial regulation of integrin activation and focal adhesion assembly/disassembly. Talin, an actin and beta-integrin tail-binding protein, is essential for integrin activation and focal adhesion formation. Calpain-mediated cleavage of talin has a key role in focal adhesion turnover; however, the talin head domain, one of the two cleavage products, stimulates integrin activation, localizes to focal adhesions and maintains cell edge protrusions, suggesting that other steps, downstream of talin proteolysis, are required for focal adhesion disassembly. Here we show that talin head binds Smurf1, an E3 ubiquitin ligase involved in cell polarity and migration, more tightly than full-length talin does and that this interaction leads to talin head ubiquitylation and degradation. We found that talin head is a substrate for Cdk5, a cyclin-dependent protein kinase that is essential for cell migration, synaptic transmission and cancer metastasis. Cdk5 phosphorylated talin head at Ser 425, inhibiting its binding to Smurf1, thus preventing talin head ubiquitylation and degradation. Expression of the mutant tal(S425A), which resists Cdk5 phosphorylation thereby increasing its susceptibility to Smurf1-mediated ubiqitylation, resulted in extensive focal adhesion turnover and inhibited cell migration. Thus, talin head produced by calpain-induced cleavage of talin is degraded through Smurf1-mediated ubiquitylation; moreover, phosphorylation by Cdk5 regulates the binding of Smurf1 to talin head, controlling talin head turnover, adhesion stability and ultimately, cell migration.

The adaptor protein talin serves both to activate the integrin family of cell adhesion molecules and to couple integrins to the actin cytoskeleton. Integrin activation has been shown to involve binding of the talin FERM domain to membrane proximal sequences in the cytoplasmic domain of the integrin beta-subunit. However, a second integrin-binding site (IBS2) has been identified near the C-terminal end of the talin rod. Here we report the crystal structure of IBS2 (residues 1974-2293), which comprises two five-helix bundles, "IBS2-A" (1974-2139) and "IBS2-B" (2140-2293), connected by a continuous helix with a distinct kink at its center that is stabilized by side-chain H-bonding. Solution studies using small angle x-ray scattering and NMR point to a fairly flexible quaternary organization. Using pull-down and enzyme-linked immunosorbent assays, we demonstrate that integrin binding requires both IBS2 domains, as does binding to acidic phospholipids and robust targeting to focal adhesions. We have defined the membrane proximal region of the integrin cytoplasmic domain as the major binding region, although more membrane distal regions are also required for strong binding. Alanine-scanning mutagenesis points to an important electrostatic component to binding. Thermal unfolding experiments show that integrin binding induces conformational changes in the IBS2 module, which we speculate are linked to vinculin and membrane binding.

Rap1 small GTPases interact with Rap1-GTP-interacting adaptor molecule (RIAM), a member of the MRL (Mig-10/RIAM/Lamellipodin) protein family, to promote talin-dependent integrin activation. Here, we show that MRL proteins function as scaffolds that connect the membrane targeting sequences in Ras GTPases to talin, thereby recruiting talin to the plasma membrane and activating integrins. The MRL proteins bound directly to talin via short, N-terminal sequences predicted to form amphipathic helices. RIAM-induced integrin activation required both its capacity to bind to Rap1 and to talin. Moreover, we constructed a minimized 50-residue Rap-RIAM module containing the talin binding site of RIAM joined to the membrane-targeting sequence of Rap1A. This minimized Rap-RIAM module was sufficient to target talin to the plasma membrane and to mediate integrin activation, even in the absence of Rap1 activity. We identified a short talin binding sequence in Lamellipodin (Lpd), another MRL protein; talin binding Lpd sequence joined to a Rap1 membrane-targeting sequence is sufficient to recruit talin and activate integrins. These data establish the mechanism whereby MRL proteins interact with both talin and Ras GTPases to activate integrins.

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.

Platelet aggregation requires agonist-induced alphaIIbbeta3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered alphaIIbbeta3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates alphaIIbbeta3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to alphaIIbbeta3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or beta3 that disrupt their mutual interaction block both talin recruitment and alphaIIbbeta3 activation. However, one talin mutant (L325R) is recruited to alphaIIbbeta3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated alphaIIbbeta3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and alphaIIbbeta3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to alphaIIbbeta3 by RIAM mediates agonist-induced alphaIIbbeta3 activation, with implications for hemostasis and thrombosis.

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin alphaIIbbeta3-mediated platelet aggregation and beta1 integrin-mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet beta1 and beta3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian alpha2beta1 and alphaIIbbeta3 integrins in vivo.

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.

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 beta subunit cytoplasmic domains (tails) causes activation, whereas numerous other PTB-domain-containing proteins bind integrins without activating them. Here we define the structure of a complex between talin and the membrane-proximal integrin beta3 cytoplasmic domain and identify specific contacts between talin and the integrin tail required for activation. We used structure-based mutagenesis to engineer talin and beta3 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.

The cytoskeletal protein talin binds to a short C-terminal sequence in phosphatidylinositol phosphate kinase type Igamma (PIPKIgamma), activating the enzyme and promoting the local production of phosphatidylinositol 4,5 bisphosphate, which regulates focal adhesion dynamics as well as clathrin-mediated endocytosis in neuronal cells. Here we show by crystallographic, NMR, and calorimetric analysis that the phosphotyrosine binding (PTB)-like domain of talin engages the PIPKIgamma C terminus in a mode very similar to that of integrin binding. However, PIPKIgamma binds in the canonical PTB-peptide mode with an SPLH motif replacing the classic NPXY motif. The tighter packing of the SPLH motif against the hydrophobic core of talin may explain the stronger binding of PIPKIgamma. Two tyrosine residues flanking the SPLH motif (Tyr-644 and Tyr-649) have been implicated in the regulation of talin binding. We show that phosphorylation at Tyr-644, a Src phosphorylation site in vivo, has little effect on the binding mode or strength, which is consistent with modeling studies in which the phosphotyrosine makes surface-exposed salt bridges, and we suggest that its strong activating effect arises from the release of autoinhibitory restraints in the full-length PIPKIgamma. Modeling studies suggest that phosphorylation of Tyr-649 will likewise have little effect on talin binding, whereas phosphorylation of the SPLH serine is predicted to be strongly disruptive. Our data are consistent with the proposal that Src activity promotes a switch from integrin binding to PIPKIgamma binding that regulates focal adhesion turnover.

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

The ability of integrin adhesion receptors to undergo rapid changes in affinity for their extracellular ligands (integrin activation) is essential for the development and function of multicellular animals and is dependent on interactions between the integrin beta subunit-cytoplasmic tail and the cytoskeletal protein talin. Cross-talk among different integrins and between integrins and other receptors impacts many cellular processes including adhesion, spreading, migration, clot retraction, proliferation, and differentiation. One form of integrin cross-talk, transdominant inhibition of integrin activation, occurs when ligand binding to one integrin inhibits the activation of a second integrin. This may be relevant clinically in a number of settings such as during platelet adhesion, leukocyte trans-migration, and angiogenesis. Here we report that competition for talin underlies the trans-dominant inhibition of integrin activation. This conclusion is based on our observations that (i). beta tails selectively defective in talin binding are unable to mediate trans-dominant inhibition, (ii). trans-dominant inhibition can be reversed by overexpression of integrin binding and activating fragments of talin, and (iii). expression of another non-integrin talin-binding protein, phosphatidylinositol phosphate kinase type Igamma-90, also inhibits integrin activation. Thus, the sequestration of talin by the suppressive species is both necessary and sufficient for trans-dominant inhibition of integrin activation.

Control of integrin affinity for ligands (integrin activation) is essential for normal cell adhesion, migration, and assembly of an extracellular matrix. Integrin activation is usually mediated through the integrin beta subunit cytoplasmic tail and can be regulated by many different biochemical signaling pathways. We report that specific binding of the cytoskeletal protein talin to integrin beta subunit cytoplasmic tails leads to the conformational rearrangements of integrin extracellular domains that increase their affinity. Thus, regulated binding of talin to integrin beta tails is a final common element of cellular signaling cascades that control integrin activation.

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. Here, 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.

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report 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. 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, our 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.

Cellular regulation of the ligand binding affinity of integrin adhesion receptors (integrin activation) depends on the integrin beta cytoplasmic domains (tails). The head domain of talin binds to several integrin beta tails and activates integrins. This head domain contains a predicted FERM domain composed of three subdomains (F1, F2, and F3). An integrin-activating talin fragment was predicted to contain the F2 and F3 subdomains. Both isolated subdomains bound specifically to the integrin beta3 tail. However, talin F3 bound the beta3 tail with a 4-fold higher affinity than talin F2. Furthermore, expression of talin F3 (but not F2) in cells led to activation of integrin alpha(IIb)beta3. A molecular model of talin F3 indicated that it resembles a phosphotyrosine-binding (PTB) domain. PTB domains recognize peptide ligands containing beta turns, often formed by NPXY motifs. NPX(Y/F) motifs are highly conserved in integrin beta tails, and mutations that disrupt this motif interfere with both integrin activation and talin binding. Thus, integrin binding to talin resembles the interactions of PTB domains with peptide ligands. These resemblances suggest that the activation of integrins requires the presence of a beta turn at NPX(Y/F) motifs conserved in integrin beta cytoplasmic domains.

Rap1 GTPase drives assembly of the Mig-10/RIAM/Lamellipodin (MRL protein)-integrin-talin (MIT) complex that enables integrin-dependent lymphocyte functions. Here we used tandem affinity tag-based proteomics to isolate and analyze the MIT complex and reveal that Phostensin (Ptsn), a regulatory subunit of protein phosphatase 1, is a component of the complex. Ptsn mediates dephosphorylation of Rap1, thereby preserving the activity and membrane localization of Rap1 to stabilize the MIT complex. CRISPR/Cas9-induced deletion of PPP1R18, which encodes Ptsn, markedly suppresses integrin activation in Jurkat human T cells. We generated apparently healthy Ppp1r18-/- mice that manifest lymphocytosis and reduced population of peripheral lymphoid tissues ascribable, in part, to defective activation of integrins αLβ2 and α4β7. Ppp1r18-/- T cells exhibit reduced capacity to induce colitis in a murine adoptive transfer model. Thus, Ptsn enables lymphocyte integrin-mediated functions by dephosphorylating Rap1 to stabilize the MIT complex. As a consequence, loss of Ptsn ameliorates T cell-mediated colitis.

β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2 reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3 min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.

Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-to-cell and cell-to-extracellular matrix interactions, play an important role in facilitating Treg cell contact-mediated suppression. In this article, we show that integrin activation plays an essential, previously unappreciated role in maintaining murine Treg cell function. Treg cell-specific loss of talin, a β integrin-binding protein, or expression of talin(L325R), a mutant that selectively abrogates integrin activation, resulted in lethal systemic autoimmunity. This dysfunction could be attributed, in part, to a global dysregulation of the Treg cell transcriptome. Activation of integrin α4β1 led to increased suppressive capacity of the Treg cell pool, suggesting that modulating integrin activation on Treg cells may be a useful therapeutic strategy for autoimmune and inflammatory disorders. Taken together, these results reveal a critical role for integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell function.

Integrin activation regulates adhesion, extracellular matrix assembly, and cell migration, thereby playing an indispensable role in development and in many pathological processes. A proline mutation in the central integrin β3 transmembrane domain (TMD) creates a flexible kink that uncouples the topology of the inner half of the TMD from the outer half. In this study, using leukocyte integrin α4β7, which enables development of gut-associated lymphoid tissue (GALT), we examined the biological effect of such a proline mutation and report that it impairs agonist-induced talin-mediated activation of integrin α4β7, thereby inhibiting rolling lymphocyte arrest, a key step in transmigration. Furthermore, the α4β7(L721P) mutation blocks lymphocyte homing to and development of the GALT. These studies show that impairing the ability of an integrin β TMD to transmit talin-induced TMD topology inhibits agonist-induced physiological integrin activation and biological function in development.

Epigallocatechin gallate (EGCG) is the principal bioactive ingredient in green tea and has been reported to have many health benefits. EGCG influences multiple signal transduction pathways related to human diseases, including redox, inflammation, cell cycle, and cell adhesion pathways. However, the molecular mechanisms of these varying effects are unclear, limiting further development and utilization of EGCG as a pharmaceutical compound. Here, we examined the effect of EGCG on two representative transmembrane signaling receptors, integrinαIIbβ3 and epidermal growth factor receptor (EGFR). We report that EGCG inhibits talin-induced integrin αIIbβ3 activation, but it activates αIIbβ3 in the absence of talin both in a purified system and in cells. This apparent paradox was explained by the fact that the activation state of αIIbβ3 is tightly regulated by the topology of β3 transmembrane domain (TMD); increases or decreases in TMD embedding can activate integrins. Talin increases the embedding of integrin β3 TMD, resulting in integrin activation, whereas we observed here that EGCG decreases the embedding, thus opposing talin-induced integrin activation. In the absence of talin, EGCG decreases the TMD embedding, which can also disrupt the integrin α-β TMD interaction, leading to integrin activation. EGCG exhibited similar paradoxical behavior in EGFR signaling. EGCG alters the topology of EGFR TMD and activates the receptor in the absence of EGF, but inhibits EGF-induced EGFR activation. Thus, this widely ingested polyphenol exhibits pleiotropic effects on transmembrane signaling by modifying the topology of TMDs.

Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of "inside-out" signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

The capacity of integrins to mediate adhesiveness is modulated by their cytoplasmic associations. In this study, we describe a novel mechanism by which alpha4-integrin adhesiveness is regulated by the cytoskeletal adaptor paxillin. A mutation of the alpha4 tail that disrupts paxillin binding, alpha4(Y991A), reduced talin association to the alpha4beta1 heterodimer, impaired integrin anchorage to the cytoskeleton, and suppressed alpha4beta1-dependent capture and adhesion strengthening of Jurkat T cells to VCAM-1 under shear stress. The mutant retained intrinsic avidity to soluble or bead-immobilized VCAM-1, supported normal cell spreading at short-lived contacts, had normal alpha4-microvillar distribution, and responded to inside-out signals. This is the first demonstration that cytoskeletal anchorage of an integrin enhances the mechanical stability of its adhesive bonds under strain and, thereby, promotes its ability to mediate leukocyte adhesion under physiological shear stress conditions.

Integrins are adhesion receptors important for hematopoiesis, leukocyte trafficking, and formation of immunological synapses; hence, they may provide targets for therapeutic intervention in leukocyte-driven pathologies. Blocking integrin–ligand binding is one strategy for inhibiting integrins; however, a complete loss of integrin function can lead to mechanism-based toxicities. Because integrin α and β subunits interact with a variety of other proteins to receive and transmit cellular signals, targeting these integrin-associated proteins may utilize alternative sites for intervention that lead to therapies with fewer side effects. This review summarizes integrin-associated proteins in leukocytes and focuses on four of these proteins with perceived therapeutic potential. Specific mutations in the α4 integrin cytoplasmic tail block or enforce binding to paxillin and thus modulate integrin signaling required for efficient cell migration. Similarly, the association of RAPL(NORE1B) with β2 integrins may participate in adhesive and migratory events in leukocytes. The β integrin cytoplasmic tail-binding protein talin is critical for increasing the affinity of integrins (activation), and blockade of talin binding can prevent leukocyte arrest on the endothelium. Finally, the membrane protein CD98 mediates β1 and β3 integrin signaling and may be involved in leukocyte functions. Identification of biologically important interactions of integrins and signaling proteins can thus pave the way to new strategies for manipulating leukocyte functions. [ABSTRACT FROM AUTHOR]