An acidic loop within the human soluble CD23 protein may direct the interaction between sCD23 and the αXβ2 integrin

CD23 is involved in a myriad of immune reactions. It is not only a receptor for IgE, but also functions in the regulation of IgE synthesis, isotype switching in B cells, and induction of the inflammatory response. These effector functions of CD23 arise through its interaction with another leukocyte-specific cell surface receptor – the β₂ integrin subfamily. It has been shown that CD23 is also capable of interacting with the β₃ and β₅ integrin β-subunit of integrins via a basic RKC motif in a metal cation-independent fashion. In this study the interaction was probed for whether or not the RKC motif governs the interaction between CD23 and the α_Xβ₂ integrin as well. This was done by performing bioinformatic docking predictions between CD23 and α_Xβ₂ integrin αI domain and SPR spectroscopy analysis of the interaction. This revealed that in the absence of cations, the RKC motif is involved in interaction with the integrin αI domain. However, in the presence of divalent metal cations the interaction showed the involvement of a novel acidic motif within the CD23 protein. This same pattern of interaction was seen in docking predictions between CD23 and the β₃I-like domain. This study thus presents an alternative site as a possible contributor to the CD23-integrin interaction exhibiting cation-dependence.     

Activation mechanisms of αVβ3 integrin by binding to fibronectin: A computational study

Integrin αVβ3 plays an important role in regulating cellular activities and in human diseases. Although the structure of αVβ3 has been studied by crystallography and electron microscopy, the detailed activation mechanism of integrin αVβ3 induced by fibronectin remains unclear. In this study, we investigated the conformational and dynamical motion changes of Mn²⁺‐bound integrin αVβ3 by binding to fibronectin with molecular dynamics simulations. Results showed that fibronectin binding to integrin αVβ3 caused the changes of the conformational flexibility of αVβ3 domains, the essential mode of motion for the domains of αV subunit and β3 subunit and the degrees of correlated motion of residues between the domains of αV subunit and β3 subunit of integrin αVβ3. The angle of Propeller domain with respect to the Calf‐2 domain of αV subunit and the angle of Hybrid domain with respect to βA domain of β3 subunit significantly increased when integrin αVβ3 was bound to fibronectin. These changes could result in the conformational change tendency of αVβ3 from a bend conformation to an extended conformation and lead to the open swing of Hybrid domain relative to βA domain of β3 subunit, which have demonstrated their importance for αVβ3 activation. Fibronectin binding to integrin αVβ3 significantly decreased the relative position of α1 helix to βA domain and that to metal ion‐dependent adhesion site, stabilized Mn²⁺ ions binding in integrin αVβ3 and changed fibronectin conformation, which are important for αVβ3 activation. Results from this study provide important molecular insight into the “outside‐in” activation mechanism of integrin αVβ3 by binding to fibronectin.     

Aggregatibacter actinomycetemcomitans leukotoxin causes activation of lymphocyte function‐associated antigen 1

Repeats‐in‐toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function‐associated antigen 1 (LFA‐1, integrin α_L/β₂)‐dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA‐1‐expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA‐1 with a cyan fluorescent protein‐tagged cytosolic α_L domain and a yellow fluorescent protein‐tagged β₂ domain. Phorbol 12‐myristate 13‐acetate activation of LFA‐1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both α_L and β₂ were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the α_L and β₂ subunits (K_d = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin α_M, α_X, and β₃ subunits (K_d = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of α_L and β₂ show that LtxA binds membrane‐proximal domain of α_L and intermediate domain of β₂.     

Identification of integrin beta subunit mutations that alter affinity for extracellular matrix ligand

We examined over 50 mutations in the Drosophila βPS integrin subunit that alter integrin function in situ for their ability to bind a soluble monovalent ligand, TWOW-1. Surprisingly, very few of the mutations, which were selected for conditional lethality in the fly, reduce the ligand binding ability of the integrin. The most prevalent class of mutations activates the integrin heterodimer. These findings emphasize the importance of integrin affinity regulation and point out how molecular interactions throughout the integrin molecule are important in keeping the integrin in a low affinity state. Mutations strongly support the controversial deadbolt hypothesis, where the CD loop in the β tail domain acts to restrain the I domain in the inactive, bent conformation. Site-directed mutations in the cytoplasmic domains of βPS and αPS2C reveal different effects on ligand binding from those observed for αIIbβ3 integrins and identify for the first time a cytoplasmic cysteine residue, conserved in three human integrins, as being important in affinity regulation. In the fly, we find that genetic interactions of the βPS mutations with reduction in talin function are consistent with the integrin affinity differences measured in cells. Additionally, these genetic interactions report on increased and decreased integrin functions that do not result in affinity changes in the PS2C integrin measured in cultured cells.     

Osteopontin mediates macrophage chemotaxis via α4 and α9 integrins and survival via the α4 integrin

Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to α_v‐containing integrins, and the SLAYGLR sequence binding to α₄β₁, α₄β₇, and α₉β₁ integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated‐bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α₄ integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α₄ and α₉ integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL‐12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid‐derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate‐elicited peritonitis. Collectively, these data indicate that α₄ and α₉ integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013. © 2012 Wiley Periodicals, Inc.     

Dissecting intrinsic and ligand-induced structural communication in the β3 headpiece of integrins

BackgroundGraph theory is widely used to dissect structural communication in biomolecular systems. Here, graph theory-based approaches were applied to the headpiece of integrins, adhesion cell-surface receptors that transmit signals across the plasma membranes.MethodsProtein Structure Network (PSN) analysis incorporating dynamic information either from molecular dynamics simulations or from Elastic Network Models was applied to the β3 domains from integrins α_Vβ₃ and α_IIbβ₃ in their apo and ligand-bound states.ResultsClosed and open states of the β headpiece are characterized by distinct allosteric communication pathways involving highly conserved amino acids at the two different α/β interfaces in the βI domain, the closed state being prompted to the closed-to-open transition. In the closed state, pure antagonism is associated with the establishment of communication pathways that start from the ligand, pass through the β1/α3,α4 interface, and end up in the hybrid domain by involving the Y110-Q82 link, which is weakened in the agonist-bound states.ConclusionsAllosteric communication in integrins relies on highly conserved and functionally relevant amino acid residues. The αβα-sandwich architecture of integrin βI domain dictates the structural communication between ligand binding site and hybrid domain. Differently from agonists, pure antagonists are directly involved in allosteric communication pathways and exert long-distance strengthening of the βI/hybrid interface. Release of the structure network in the ligand binding site is associated with the close-to-open transition accompanying the activation process.General significanceThe study strengthens the power of graph-based analyses to decipher allosteric communication intrinsic to protein folds and modified by functionally different ligands.     

The roles of αV integrins in lens EMT and posterior capsular opacification

Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α‐smooth muscle actin (α‐SMA)‐expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of α_V integrin and its interacting β‐subunits β1, β5, β6, β8 are up‐regulated concomitant with α‐SMA in LCs following surgery. To test the hypothesis that α_V integrins are functionally important in PCO pathogenesis, we created mice lacking the α_V integrin subunit in all lens cells. Adult lenses lacking α_V integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up‐regulated the expression of α‐SMA, β1‐integrin, fibronectin, tenascin‐C and transforming growth factor beta (TGF‐β)–induced protein within 48 hrs, while LCs lacking α_V integrins exhibited much less cell proliferation and little to no up‐regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of α_V integrins in latent TGF‐β activation as α_V integrin null lenses do not exhibit detectable SMAD‐3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF‐β in fibrotic PCO. These data suggest that therapeutics antagonizing α_V integrin function could be used to prevent fibrotic PCO following cataract surgery.     

Structural basis of integrin transmembrane activation

Integrins are cell adhesion receptors that transmit bidirectional signals across plasma membrane and are crucial for many biological functions. Recent structural studies of integrin transmembrane (TM) and cytoplasmic domains have shed light on their conformational changes during integrin activation. A structure of the resting state was solved based on Rosetta computational modeling and experimental data using intact integrins on mammalian cell surface. In this structure, the α_IIb GXXXG motif and their β₃ counterparts of the TM domains associate with ridge‐in‐groove packing, and the α_IIb GFFKR motif and the β₃ Lys‐716 in the cytoplasmic segments play a critical role in the α/β association. Comparing this structure with the NMR structures of the monomeric α_IIb and β₃ (represented as active conformations), the α subunit helix remains similar after dissociation whereas β subunit helix is tilted by embedding additional 5–6 residues into the lipid bilayer. These conformational changes are critical for integrin activation and signaling across the plasma membrane. We thus propose a new model of integrin TM activation in which the recent NMR structure of the α_IIbβ₃ TM/cytoplasmic complex represents an intermediate or transient state, and the electrostatic interaction in the cytoplasmic region is important for priming the initial α/β association, but not absolutely necessary for the resting state. J. Cell. Biochem. 109: 447–452, 2010. © 2009 Wiley‐Liss, Inc.     

Cross-talk between integrins alpha1beta1 and alpha2beta1 in renal epithelial cells

The collagen-binding integrins α1β1 and α2β1 have profoundly different functions, yet they are often co-expressed in epithelial cells. When both integrins are expressed in the same cell, it has been suggested that α1β1 negatively regulates integrin α2β1-dependent functions. In this study we utilized murine ureteric bud (UB) epithelial cells, which express no functionally detectable levels of endogenous integrins α1β1 and α2β1, to determine the mechanism whereby this regulation occurs. We demonstrate that UB cells expressing integrin α2β1, but not α1β1 adhere, migrate and proliferate on collagen I as well as form cellular cords in 3D collagen I gels. Substitution of the transmembrane domain of the integrin α2 subunit with that of α1 results in decreased cell adhesion, migration and cord formation. In contrast, substitution of the integrin α2 cytoplasmic tail with that of α1, decreases cell migration and cord formation, but increases proliferation. When integrin α1 and α2 subunits are co-expressed in UB cells, the α1 subunit negatively regulates integrin α2β1-dependent cord formation, adhesion and migration and this inhibition requires expression of both α1 and α2 tails. Thus, we provide evidence that the transmembrane and cytoplasmic domains of the α2 integrin subunit, as well as the α1 integrin subunit, regulate integrin α2β1 cell function.     

Mutagenesis studies of the β I domain metal ion binding sites on integrin αVβ3 ligand binding affinity

Three divalent cation binding sites in the integrin β I domain have been shown to regulate ligand binding and adhesion. However, the degree of ligand binding and adhesion varies among integrins. The αLβ2 and α4β7 integrins show an increase in ligand binding affinity and adhesion when one of their ADMIDAS (adjacent to MIDAS, or the metal ion-dependent adhesion site) residues is mutated. By contrast, the α2β1, α5β1, and αIIbβ3 integrins show a decrease in binding affinity and adhesion when their ADMIDAS is mutated. Our study here indicated that integrin αVβ3 had lower affinity when the ADMIDAS was mutated. By comparing the primary sequences of these integrin subunits, we propose that one residue associated with the MIDAS (β3 Ala(252)) may account for these differences. In the β1 integrin subunit, the corresponding residue is also Ala, whereas in both β2 and β7 integrin subunits, it is Asp. We mutated the β3 residue Ala(252) to Asp and combined this mutant with mutations of one or two ADMIDAS residues. The mutant A252D showed reduced ligand binding affinity and adhesion. The ligand binding affinity and adhesion were increased when this A252D mutant was paired with mutations of one ADMIDAS residue. But when paired with mutations of two ADMIDAS residues the mutant nearly abolished ligand-binding ability, which was restored by the activating glycosylation mutation. Our study suggests that the variation of this residue contributes to the different ligand binding affinities and adhesion abilities among different integrin families.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin α_Lβ₂ (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β₂ integrin. Cells expressing α_Mβ₂ (CD11b/CD18) or α_Xβ₂ (CD11c/CD18) were killed as efficiently as cells expressing α_Lβ₂. Erythrocytes, which do not express β₂ integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β₂ chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins α_Mβ₂ and α_Xβ₂ as novel receptors for LtxA.     

Role of the α1 and α2 Integrin Cytoplasmic Domains in Cell Morphology,Motility and Responsiveness to Stimulation by the Protein Kinase C Pathway

The α₁β₁ and α₂β₁ integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the α subunits. We expressed either full-length α₁ integrin subunit cDNA (XICI), full-length α₂ integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of Q₂ subunit and the cytoplasmic domain of α₁ (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of α₁ subunit and the, cytoplasmic domain of α₂ (X1C2), α₁ cDNA truncated after the GFFKR sequence (X1C0) or α₂ cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the a_x and α₂ subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the α₁ or α₂ cytoplasmic tail. Spreading was also influenced by the presence of an α subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the α₂ cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, α₂-cytoplasmic tail-dependent migration.     

Conformational equilibria and intrinsic affinities define integrin activation

We show that the three conformational states of integrin α₅β₁ have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000‐fold higher affinity of the extended‐open state than the bent‐closed and extended‐closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α₅β₁. On the surface of K562 cells, α₅β₁ is 99.8% bent‐closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N‐glycans and leg domains in each subunit that connect the ligand‐binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single‐span transmembrane domains.     

The C-terminal αI domain linker as a critical structural element in the conformational activation of αI integrins

The activation of α/β heterodimeric integrins is the result of highly coordinated rearrangements within both subunits. The molecular interactions between the two subunits, however, remain to be characterized. In this study, we use the integrin α(L)β(2) to investigate the functional role of the C-linker polypeptide that connects the C-terminal end of the inserted (I) domain with the β-propeller domain on the α subunit and is located at the interface with the βI domain of the β chain. We demonstrate that shortening of the C-linker by eight or more amino acids results in constitutively active α(L)β(2) in which the αI domain is no longer responsive to the regulation by the βI domain. Despite this intersubunit uncoupling, both I domains remain individually sensitive to intrasubunit conformational changes induced by allosteric modulators. Interestingly, the length and not the sequence of the C-linker appears to be critical for its functionality in α/β intersubunit communication. Using two monoclonal antibodies (R7.1 and CBR LFA-1/1) we further demonstrate that shortening of the C-linker results in the gradual loss of combinational epitopes that require both the αI and β-propeller domains for full reactivity. Taken together, our findings highlight the role of the C-linker as a spring-like element that allows relaxation of the αI domain in the resting state and controlled tension of the αI domain during activation, exerted by the β chain.     

Physiological evidence of integrin-antibody reactive proteins influencing the innate cellular immune responses of larval Galleria mellonella hemocytes

Larval Galleria melonella(L.)hemocytes form microaggregates in response to stimulation by Gram-positive bacteria Hemocyte adhesion to foreign materials is mediated by the CAMP/protein kinase A pathway and the B-subunit of cholera toxin using a cAMP-independent mechanism.Cholera toxin-induced microaggregation was inhibited by the integrin inhibitory RGDS peptide,implying integrins may be part of the mechanism.Based on the types of mammalian integrin-antibody reactive proteins affecting hemocyte adhesion and bacterial-induced responses ars,ory,Ai,and B3 subunits occred on both granular cell and plasmatocyte hemocyte subtypes.A fluorescent band representing the binding of rabbit as-integrin subunit antibodies occurred between adhering heterotypic hemocytes.The frequency of the bands was increased by cholera toxin.The as andβrabbit integrin subunit antibodies inhibited removal of Bacillus subtilis(Cohn)from the hemolymph in vivo,A as ir-specific synthetic peptide blocker similarly diminished hemocyte function whereas the 0v Bs-specific inhibitory peptide and the corresponding integrin subunit antibodies did not influence nonself hemocyte activities.Western blots revealed several proteins reacting with a given integrin-antibody subtype.Thus integrin-antibody reactive proteins(which may include integrins)with possible as and B epitopes modulate immediate hemocyte function.Confocal microscopy established plasmatocyte adhesion to and rosetting over substrata followved by granular cell microaggregate adhesion to plasmatocytes during early stage nodulation.     

Effect of hypoxia on integrin-mediated adhesion of endothelial progenitor cells

Homing of endothelial progenitor cells (EPCs) is crucial for neoangiogenesis, which might be negatively affected by hypoxia. We investigated the influence of hypoxia on fibronectin binding integrins for migration and cell‐matrix‐adhesion. AMP‐activated kinase (AMPK) and integrin‐linked kinase (ILK) were examined as possible effectors of hypoxia.Human EPCs were expanded on fibronectin (FN) and integrin expression was profiled by flow cytometry. Cell‐matrix‐adhesion‐ and migration‐assays on FN were performed to examine the influence of hypoxia and AMPK‐activation. Regulation of AMPK and ILK was shown by Western blot analysis. We demonstrate the presence of integrin β₁, β₂ and α₅ on EPCs. Adhesion to FN is reduced by blocking β₁ and α₅ (49% and 2% of control, P < 0.05) whereas α₄‐blockade has no effect. Corresponding effects were shown for migration. Hypoxia and AMPK‐activation decrease adhesion on FN. Although total AMPK‐expression remains unchanged, phospho‐AMPK increases eightfold.The EPCs require α₅ for adhesion on FN. Hypoxia and AMPK‐activation decrease adhesion. As α₅ is the major adhesive factor for EPCs on FN, this suggests a link between AMPK and α₅‐integrins. We found novel evidence for a connection between hypoxia, AMPK‐activity and integrin activity. This might affect the fate of EPCs in ischaemic tissue.     

Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Structure of a double ubiquitin‐like domain in the talin head: a role in integrin activation

Talin is a 270‐kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N‐terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for β‐integrin tails, F0 and F1 are also required for activation of β1‐integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin‐like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin‐mediated activation of β1‐integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.     

Structural requirements for activation in alphaIIb beta3 integrin

Integrins are postulated to undergo structural rearrangement from a low affinity bent conformer to a high affinity extended conformer upon activation. However, some reports have shown that a bent conformer is capable of binding a ligand, whereas another report has shown that integrin extension does not absolutely lead to activation. To clarify whether integrin affinity is indeed regulated by the so-called switchblade-like movement, we have engineered a series of mutant αIIbβ3 integrins that are constrained specifically in either a bent or an extended conformation. These mutant αIIbβ3 integrins were expressed in mammalian cells, and fibrinogen binding to these cells was examined. The bent integrins were created through the introduction of artificial disulfide bridges in the β-head/β-tail interface. Cells expressing bent integrins all failed to bind fibrinogen unless pretreated with DTT to disrupt the disulfide bridges. The extended integrins were created by introducing N-glycosylation sites in amino acid residues located close to the α-genu, where the integrin legs fold backward. Among these mutants, activation was maximized in one integrin with an N-glycosylation site located behind the α-genu. This extension-induced activation was completely blocked when the swing-out of the hybrid domain was prevented. These results suggest that the bent and extended conformers represent low affinity and high affinity conformers, respectively, and that extension-induced activation depends on the swing-out of the hybrid domain. Taken together, these results are consistent with the current hypothesis that integrin affinity is regulated by the switchblade-like movement of the integrin legs.