Emerging evidence for kindlin oligomerization and its role in regulating kindlin function.

Integrin-mediated cell-extracellular matrix (ECM) interactions play crucial roles in a broad range of physiological and pathological processes. Kindlins are important positive regulators of integrin activation. The FERM-domain-containing kindlin family comprises three members, kindlin-1, kindlin-2 and kindlin-3 (also known as FERMT1, FERMT2 and FERMT3), which share high sequence similarity (identity >50%), as well as domain organization, but exhibit diverse tissue-specific expression patterns and cellular functions. Given the significance of kindlins, analysis of their atomic structures has been an attractive field for decades. Recently, the structures of kindlin and its ß-integrin-bound form have been obtained, which greatly advance our understanding of the molecular functions that involve kindlins. In particular, emerging evidence indicates that oligomerization of kindlins might affect their integrin binding and focal adhesion localization, positively or negatively. In this Review, we presented an update on the recent progress of obtaining kindlin structures, and discuss the implication for integrin activation based on kindlin oligomerization, as well as the possible regulation of this process.

Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state.

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.

Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma.

Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1α) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1α (HIF-1αΔNφ) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1α in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

Ultra-Short Cyclized ß-Boomerang Peptides: Structures, Interactions with Lipopolysaccharide, Antibiotic Potentiator and Wound Healing.

Many antibiotics are ineffective in killing Gram-negative bacteria due to the permeability barrier of the outer-membrane LPS. Infections caused by multi-drug-resistant Gram-negative pathogens require new antibiotics, which are often difficult to develop. Antibiotic potentiators disrupt outer-membrane LPS and can assist the entry of large-scaffold antibiotics to the bacterial targets. In this work, we designed a backbone-cyclized ultra-short, six-amino-acid-long (WKRKRY) peptide, termed cWY6 from LPS binding motif of ß-boomerang bactericidal peptides. The cWY6 peptide does not exhibit any antimicrobial activity; however, it is able to permeabilize the LPS outer membrane. Our results demonstrate the antibiotic potentiator activity in the designed cWY6 peptide for several conventional antibiotics (vancomycin, rifampicin, erythromycin, novobiocin and azithromycin). Remarkably, the short cWY6 peptide exhibits wound-healing activity in in vitro assays. NMR, computational docking and biophysical studies describe the atomic-resolution structure of the peptide in complex with LPS and mode of action in disrupting the outer membrane. The dual activities of cWY6 peptide hold high promise for further translation to therapeutics.

The focal adhesion protein kindlin-2 controls mitotic spindle assembly by inhibiting histone deacetylase 6 and maintaining α-tubulin acetylation.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2-depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3ß (GSK3ß) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

The Systemic Lupus Erythematosus-Associated Single Nucleotide Polymorphism rs1143678 in Integrin αM Cytoplasmic Tail Generates a 14-3-3ζ Binding Site That Is Proinflammatory.

The leukocyte integrin αMß2 (CR3 or Mac-1) has both proinflammatory and immune regulatory functions. Genome-wide association studies have identified several ITGAM (αM subunit) single nucleotide polymorphisms that are associated with systemic lupus erythematosus. The single nucleotide polymorphism rs1143678 substitutes Pro1146 for Ser in the integrin αM cytoplasmic tail. A detailed functional characterization of this substitution is lacking. Using transfected human cell lines, reconstituted mouse bone marrow neutrophils, and bone marrow-derived macrophages (BMDMs), we showed that P1146S (PS) substitution promoted integrin αMß2-mediated adhesion, spreading, and migration of cells on iC3b and fibrinogen. In the presence of LPS together with iC3b or fibrinogen, the expression levels of IL-6 and TNF-α in integrin αM(PS)ß2 BMDMs were significantly higher than those of integrin αM(wild-type)ß2 BMDMs, and they showed faster kinetics of Erk1/2 activation through the src family kinase(s)-Syk signaling pathway. Integrin αM(PS)ß2 BMDMs also exhibited higher levels of active RhoA and phagocytic activity. Mechanistically, P1146S substitution in the αM cytoplasmic tail generates a noncanonical 14-3-3ζ binding site that modulates integrin αM(PS)ß2 outside-in signaling.

Characterization of single amino acid substitutions in the ß2 integrin subunit of patients with leukocyte adhesion deficiency (LAD)-1.

Leukocyte adhesion deficiency 1 (LAD-1) is caused by defects in the ß2 integrin subunit. We studied 18 missense mutations, 14 of which fail to support the surface expression of the ß2 integrins. Integrins with the ß2-G150D mutation fail to bind ligands, possibly due to the failure of the α1 segment of the ßI domain to assume an α-helical structure. Integrins with the ß2-G716A mutation are not maintained in their resting states, and the patient has the severe phenotype of LAD-1. The ß2-S453N and ß2-P648L mutants support the expression of integrins and adhesion functions. They should be re-classified as polymorphic variants.

TWEAK/Fn14 signalling driven super-enhancer reprogramming promotes pro-metastatic metabolic rewiring in triple-negative breast cancer.

Triple Negative Breast Cancer (TNBC) is the most aggressive breast cancer subtype suffering from limited targeted treatment options. Following recent reports correlating Fibroblast growth factor-inducible 14 (Fn14) receptor overexpression in Estrogen Receptor (ER)-negative breast cancers with metastatic events, we show that Fn14 is specifically overexpressed in TNBC patients and associated with poor survival. We demonstrate that constitutive Fn14 signalling rewires the transcriptomic and epigenomic landscape of TNBC, leading to enhanced tumour growth and metastasis. We further illustrate that such mechanisms activate TNBC-specific super enhancers (SE) to drive the transcriptional activation of cancer dependency genes via chromatin looping. In particular, we uncover the SE-driven upregulation of Nicotinamide phosphoribosyltransferase (NAMPT), which promotes NAD+ and ATP metabolic reprogramming critical for filopodia formation and metastasis. Collectively, our study details the complex mechanistic link between TWEAK/Fn14 signalling and TNBC metastasis, which reveals several vulnerabilities which could be pursued for the targeted treatment of TNBC patients.

Kindlin-3 mediates integrin αLß2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1).

Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLß2 outside-in signaling. It also promotes micro-clustering of integrin αLß2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLß2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLß2 outside-in signaling.

Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells.

Cyclic di-GMP (c-di-GMP) has emerged as a prominent intracellular messenger that coordinates biofilm formation and pathogenicity in many bacterial species. Developing genetically encoded biosensors for c-di-GMP will help us understand how bacterial cells respond to environmental changes via the modulation of cellular c-di-GMP levels. Here we report the design of two genetically encoded c-di-GMP fluorescent biosensors with complementary dynamic ranges. By using the biosensors, we found that several compounds known to promote biofilm dispersal trigger a decline in c-di-GMP levels in Escherichia coli cells. In contrast, cellular c-di-GMP levels were elevated when the bacterial cells were treated with subinhibitory concentrations of biofilm-promoting antibiotics. The biosensors also revealed that E. coli cells engulfed by macrophages exhibit lower c-di-GMP levels, most likely as a response to the enormous pressures of survival during phagocytosis.

ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters.

Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5ß1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5ß1-mediated Rac1/PAK signaling to weaken cell-cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

Molecular condensation and mechanoregulation of plant class I formin, an integrin-like actin nucleator.

The actin cytoskeleton (AC) undergoes rapid remodelling to coordinate cellular processes during signal transduction, including changes in actin nucleation, crosslinking, and depolymerization in a time- and space-dependent manner. Switching the initial actin nucleation often provides timely control of the entire actin network formation. Located at the cell surface, the plant class I formin family is a major class of actin nucleators that rapidly respond to exterior chemical and environmental cues. Plant class I formins are structurally integrated within the plant cell wall-plasma membrane-actin cytoskeleton (CW-PM-AC) continuum, sharing similar biophysical properties to mammalian integrins that are embedded within the extracellular matrix-PM-AC continuum. In plants, perturbation of structural components of the CW-PM-AC continuum changes the biophysical properties of two dimensional-scaffolding structures, which results in uncontrolled molecular diffusion and interactions of class I formins, as well as their clustering and activities in the nucleation of the AC. Emerging studies have shown that the PM-integrated formins are highly responsive to the mechanical perturbation of CW and AC integrity changes that tune the oligomerization and condensation of formin on the cell surface. However, during diverse signalling transductions, the molecular mechanisms that spatiotemporally underlie the mechanosensing and mechanoregulation of formin for remodelling actin remain unclear. Here, the emphasis will be placed on recent developments in understanding how the molecular condensation of class I formin regulates the biochemical activities in tuning actin polymerization during plant immune signalling, as well as how the plant structural components of the CW-PM-AC continuum control formin condensation at a nanometre scale.

Sustained activation of non-canonical NF-κB signalling drives glycolytic reprogramming in doxorubicin-resistant DLBCL.

DLBCL is the most common lymphoma with high tumor heterogeneity. Treatment refractoriness and relapse from R-CHOP therapy in patients remain a clinical problem. Activation of the non-canonical NF-κB pathway is associated with R-CHOP resistance. However, downstream targets of non-canonical NF-κB mediating R-CHOP-induced resistance remains uncharacterized. Here, we identify the common mechanisms underlying both intrinsic and acquired resistance that are induced by doxorubicin, the main cytotoxic component of R-CHOP. We performed global transcriptomic analysis of (1) a panel of resistant versus sensitive and (2) isogenic acquired doxorubicin-resistant DLBCL cell lines following short and chronic exposure to doxorubicin respectively. Doxorubicin-induced stress in resistant cells activates a distinct transcriptional signature that is enriched in metabolic reprogramming and oncogenic signalling. Selective and sustained activation of non-canonical NF-κB signalling in these resistant cells exacerbated their survival by augmenting glycolysis. In response to doxorubicin, p52-RelB complexes transcriptionally activated multiple glycolytic regulators with prognostic significance through increased recruitment at their gene promoters. Targeting p52-RelB and their targets in resistant cells increased doxorubicin sensitivity in vitro and in vivo. Collectively, our study uncovered novel molecular drivers of doxorubicin-induced resistance that are regulated by non-canonical NF-κB pathway. We reveal new avenues of therapeutic targeting for R-CHOP-treated refractory/relapsed DLBCL patients.

Structures and interaction analyses of integrin αMß2 cytoplasmic tails.

Integrins are heterodimeric (α and ß subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin αMß2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of αM and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated αM tails with ß2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of αM or phosphorylated αM are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of αM and ß2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated ß2 tail in dodecylphosphocholine micelles with dansylated αM tail peptides by FRET. These studies revealed enhanced interactions between αM or phosphorylated αM tails with ß2 tail with Kd values ∼5.2±0.6 and ∼4.4±0.7 µm, respectively. Docked structures of tail-tail complexes delineated that the αM/ß2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds.

Integrin alphaMbeta2 clustering triggers phosphorylation and activation of protein kinase C delta that regulates transcription factor Foxp1 expression in monocytes.

Integrins are type I membrane and heterodimeric (alphabeta) cell adhesion receptors. Intracellular signals triggered by ligand-bound integrins are important for cell growth, differentiation, and migration. Integrin alpha(M)beta(2) plays key roles in myeloid cell adhesion, phagocytosis, and degranulation. In this study, we show that protein kinase C (PKC) delta is involved in alpha(M)beta(2) signaling. In human monocytic U937 cells and peripheral blood monocytes, alpha(M)beta(2) clustering induced PKCdelta translocation to the plasma membrane, followed by Tyr(311) phosphorylation and activation of PKCdelta by the src family kinases Hck and Lyn. Interestingly, alpha(M)beta(2)-induced PKCdelta Tyr(311) phosphorylation was not mediated by the tyrosine kinase Syk, which is a well reported kinase in beta(2) integrin signaling. Analysis of the beta(2) cytoplasmic tail showed that the sequence Asn(727)-Ser(734) is important in alpha(M)beta(2)-induced PKCdelta Tyr(311) phosphorylation. It has been shown that alpha(M)beta(2) clustering regulates the expression the transcription factor Foxp1 that has a role in monocyte differentiation. We show that Foxp1 expression was reduced in monocytes that were allowed to adhere to human microvascular endothelial cells. However, the expression of Foxp1 was not affected in monocytes that were treated with PKCdelta-targeting small interfering RNA, suggesting that PKCdelta regulates Foxp1 expression. These results demonstrate a role of PKCdelta in alpha(M)beta(2)-mediated Foxp1 regulation in monocytes.

Structure of human Rack1 protein at a resolution of 2.45†Å.

The crystal structure of human receptor for activated C-kinase 1 (hRack1) protein is reported at 2.45 Šresolution. The crystals belongs to space group P4(1)2(1)2, with three molecules per asymmetric unit. The hRack1 structure features a sevenfold ß-propeller, with each blade housing a sequence motif that contains a strictly conserved Trp, the indole group of which is embedded between adjacent blades. In blades 1-5 the imidazole group of a His residue is wedged between the side chains of a Ser residue and an Asp residue through two hydrogen bonds. The hRack1 crystal structure forms a starting basis for understanding the remarkable scaffolding properties of this protein.

Leukocyte integrin αLß2 transmembrane association dynamics revealed by coarse-grained molecular dynamics simulations.

Integrins are transmembrane (TM) proteins that mediate bidirectional mechanical signaling between the extracellular matrix and the cellular cytoskeletal network. Each integrin molecule consists of non-covalently associated α- and ß-subunits, with each subunit consisting of a large ectodomain, a single-pass TM helix, and a short cytoplasmic tail. Previously we found evidence for a polar interaction (hydrogen bond) in the outer membrane clasp (OMC) of the leukocyte integrin αLß2 TMs that is absent in the platelet integrin αIIß3 OMC. Here, we compare the self-assembly dynamics of αLß2 and αIIß3 TM helices in a model membrane using coarse-grained molecular dynamics simulations. We found that although αIIß3 TM helices associate more easily, packing is suboptimal. In contrast, αLß2 TM helices achieve close-to-optimal packing. This suggests that αLß2 TM packing is more specific, possibly due to the interhelix hydrogen bond. Theoretical association free energy profiles show a deeper minimum at a smaller helix-helix separation for αLß2 compared with αIIß3. The αIIß3 profile is also more rugged with energetic barriers whereas that of αLß2 is almost without barriers. Disruption of the interhelix hydrogen bond in αLß2 via the ß2T686G mutation results in poorer association and a similar profile as αIIß3. The OMC polar interaction in αLß2 thus plays a significant role in the packing of the TM helices.

Angiopoietin-like 4 interacts with integrins beta1 and beta5 to modulate keratinocyte migration.

Adipose tissue secretes adipocytokines for energy homeostasis, but recent evidence indicates that some adipocytokines also have a profound local impact on wound healing. Upon skin injury, keratinocytes use various signaling molecules to promote reepithelialization for efficient wound closure. In this study, we identify a novel function of adipocytokine angiopoietin-like 4 (ANGPTL4) in keratinocytes during wound healing through the control of both integrin-mediated signaling and internalization. Using two different in vivo models based on topical immuno-neutralization of ANGPTL4 as well as ablation of the ANGPTL4 gene, we show that ANGPTL4-deficient mice exhibit delayed wound reepithelialization with impaired keratinocyte migration. Human keratinocytes in which endogenous ANGPTL4 expression was suppressed by either siRNA or a neutralizing antibody show impaired migration associated with diminished integrin-mediated signaling. Importantly, we identify integrins ß1 and ß5, but not ß3, as novel binding partners of ANGPTL4. ANGPTL4-bound integrin ß1 activated the FAK-Src-PAK1 signaling pathway, which is important for cell migration. The findings presented herein reveal an unpredicted role of ANGPTL4 during wound healing and demonstrate how ANGPTL4 stimulates intracellular signaling mechanisms to coordinate cellular behavior. Our findings provide insight into a novel cell migration control mechanism and underscore the physiological importance of the modulation of integrin activity in cancer metastasis.

Binary and ternary complexes of FLNa-Ig21 with cytosolic tails of αMß2 integrin reveal dual role of filamin mediated regulation.

BACKGROUND: Cytoskeletal protein filamin A is critical for the outside-in signaling of integrins. Although molecular mechanisms of filamin-integrin interactions are not fully understood. Mostly, the membrane distal (MD) part of the cytosolic tail (CT) of ß subunit of integrin is known to interact with filamin A domain 21 (FLNa-Ig2). However, binary and ternary complexes of full-length CTs of leucocyte specific ß2 integrins with FLNa-Ig21 are yet to be elucidated. METHODS: Binding interactions of the CTs of integrin αMß2 with FLNa-Ig21 are extensively investigated by NMR, ITC, cell-based functional assays and computational docking. RESULTS: The αM CT demonstrates interactions with FLNa-Ig21 forming a binary complex. Filamin/αM interface is mediated by sidechain-sidechain interactions among non-polar and aromatic residues involving MP helix of αM and the canonical CD face of FLNa-Ig21. Functional assays delineated an interfacial residue Y1137 of αM CT is critical for in-cell binding to FLNa-Ig2. In addition, full-length ß2 CT occupies two distinct binding sites in complex with FLNa-Ig21. A ternary complex of FLNa-Ig21 with CTs has been characterized. In the ternary complex, αM CT moves away to a distal site of FLNa-Ig21 with fewer interactions. CONCLUSION: Our findings demonstrate a plausible dual role of filamin in integrin regulation. The molecular interactions of the ternary complex are critical for the resting state of integrins whereas stable FLNa-Ig21/αM CT binary complex perhaps be required for the activated state. GENERAL SIGNIFICANCE: Filamin binding to both α and ß CTs of other integrins could be essential in regulating bidirectional signaling mechanisms.

Functional and structural characterization of the talin F0F1 domain.

The globular head domain of talin, a large multi-domain cytoplasmic protein, is required for inside-out activation of the integrins, a family of heterodimeric transmembrane cell adhesion molecules. Talin head contains a FERM domain that is composed of F1, F2, and F3 subdomains. A F0 subdomain is located N-terminus to F1. The F3 contains a canonical phosphotyrosine binding (PTB) fold that directly interacts with the membrane proximal NPxY/F motif in the integrin beta cytoplasmic tail. This interaction is stabilized by the F2 that interacts with the lipid head-groups of the plasma membrane. In comparison to F2 and F3, the properties of the F0F1 remains poorly characterized. Here, we showed that F0F1 is essential for talin-induced activation of integrin alphaLbeta2 (LFA-1). F0F1 has a high content of beta-sheet secondary structure, and it tends to homodimerize that may provide stability against proteolysis and chaotrope induced unfolding.