A cluster of basic amino acid residues in the gamma370-381 sequence of fibrinogen comprises a binding site for platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa)

Adhesive interactions of platelet integrin alpha(IIb)beta3 with fibrinogen and fibrin are central events in hemostasis and thrombosis. However, the mechanisms by which alpha(IIb)beta3 binds these ligands remain incompletely understood. We have recently demonstrated that alpha(IIb)beta3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen). This sequence contains neither the AGDV nor the RGD recognition motifs, known to bind alpha(IIb)beta3, suggesting the different specificity of the integrin. Here, using peptide arrays, mutant fibrinogens, and recombinant mutant gammaC-domains, we have examined the mechanism whereby alpha(IIb)beta3 binds gamma365-383. The alpha(IIb)beta3-binding activity was localized within gamma370-381, with two short sequences, gamma370ATWKTR375 and gamma376WYSMKK381, being able to independently bind the integrin. Furthermore, recognition of alpha(IIb)beta3 by gamma370-381 depended on four basic residues, Lys373, Arg375, Lys380, and Lys381. Simultaneous replacement of these amino acids and deletion of the gamma408AGDV411 sequence in the recombinant gammaC-domain resulted in the loss of alpha(IIb)beta3-mediated platelet adhesion. Confirming the critical roles of the identified residues, abnormal fibrinogen Kaiserslautern, in which gammaLys380 is replaced by Asn, demonstrated delayed clot retraction and impaired alpha(IIb)beta3 binding. Also, a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg375 --> Gly) showed delayed clot retraction and reduced binding to purified alpha(IIb)beta3. These results identify the gamma370-381 sequence of fibrin(ogen) as the binding site for alpha(IIb)beta3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.     

Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3

Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.     

Savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi, presents the RGD integrin recognition motif on the Kunitz-BPTI fold

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.     

Identification of a contact domain between echistatin and the integrin alpha(v)beta(3) by photoaffinity cross-linking.

The integrin alpha(v)beta(3) is the major receptor mediating the attachment of osteoclasts to the extracellular matrix in bone and plays a critical role in bone resorption and bone remodeling. Most of the ligands interacting with the alpha(v)beta(3) receptor contain an Arg-Gly-Asp (RGD) motif. Recently, we have identified two small RGD peptides, containing a benzophenone moiety at either the carboxyl or amino terminus, that photo-cross-linked within the beta(3)[99-118] [Bitan, G., et al. (1999) Biochemistry 38, 3414-3420] or the beta(3)[167-171] [Bitan, G., et al. (2000) Biochemistry 39, 11014-11023] sequence, respectively, of the alpha(v)beta(3) receptor in a selective fashion. Here, we report the synthesis of a photoreactive analogue of echistatin (a 49-amino acid peptide), a potent RGD-containing antagonist of the alpha(v)beta(3) receptor both in vitro and in vivo. This bioactive analogue is substituted at position 45 with a p-benzoyl moiety (pBz(2)), located within the flexible C-terminal domain and removed 20 amino acid residues from the R(24)GD(26) triad. This C-terminal domain was reported to contribute to receptor binding affinity by acting as an auxiliary binding site. The radiolabeled (125)I-[Arg(35),Lys(45)(N(epsilon)-pBz(2))]-echistatin photo-cross-links effectively to a site within the beta(3)[209-220] sequence. Residues in this domain have been reported to be part of the metal ion-dependent adhesion site (MIDAS). Receptor fragments overlapping this domain were reported to bind to fibrinogen and block fibrinogen binding to alpha(IIb)beta(3), the platelet integrin receptor. Taken together, position 45 in echistatin, located within an auxiliary binding site in echistatin, cross-links to a site distinct from the two previously reported sites, beta(3)[99-118] and beta(3)[167-171], which cross-link to photophores flanking the RGD triad. These cross-linking data support the hypothesis that the ligand-bound conformation of the integrin beta(3) subunit differs from the known conformation of I domains.     

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.     

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.     

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

Following platelet aggregation, integrin alpha(IIb)beta(3) becomes associated with the platelet cytoskeleton. The conserved NPLY sequence represents a potential beta-turn motif in the beta(3) cytoplasmic tail and has been suggested to mediate the interaction of beta(3) integrins with talin. In the present study, we performed a double mutation (N744Q/P745A) in the integrin beta(3) subunit to test the functional significance of this beta-turn motif. Chinese hamster ovary cells were co-transfected with cDNA constructs encoding mutant beta(3) and wild type alpha(IIb). Cells expressing either wild type (A5) or mutant (D4) alpha(IIb)beta(3) adhered to fibrinogen; however, as opposed to control A5 cells, adherent D4 cells failed to spread, form focal adhesions, or initiate protein tyrosine phosphorylation. To investigate the role of the NPLY motif in talin binding, we examined the ability of the mutant alpha(IIb)beta(3) to interact with talin in a solid phase binding assay. Both wild type and mutant alpha(IIb)beta(3), purified by RGD affinity chromatography, bound to a similar extent to immobilized talin. Additionally, purified talin failed to interact with peptides containing the AKWDTANNPLYK sequence indicating that the talin binding domain in the integrin beta(3) subunit does not reside in the NPLY motif. In contrast, specific binding of talin to peptides containing the membrane-proximal HDRKEFAKFEEERARAK sequence of the beta(3) cytoplasmic tail was observed, and this interaction was blocked by a recombinant protein fragment corresponding to the 47-kDa N-terminal head domain of talin (rTalin-N). In addition, RGD affinity purified platelet alpha(IIb)beta(3) bound dose-dependently to immobilized rTalin-N, indicating that an integrin-binding site is present in the talin N-terminal head domain. Collectively, these studies demonstrate that the NPLY beta-turn motif regulates post-ligand binding functions of alpha(IIb)beta(3) in a manner independent of talin interaction. Moreover, talin was shown to bind through its N-terminal head domain to the membrane-proximal sequence of the beta(3) cytoplasmic tail.     

Isolation of a highly specific ligand for the alpha 5 beta 1 integrin from a phage display library

Our previous studies showed that the alpha 5 beta 1 integrin selects cysteine pair-containing RGD peptides from a phage display library based on a random hexapeptide. We have therefore searched for more selective peptides for this integrin using a larger phage display library, where heptapeptides are flanked by cysteine residues, thus making the inserts potentially cyclic. Most of the phage sequences that bound to alpha 5 beta 1 (69 of 125) contained the RGD motif. Some of the heptapeptides contained an NGR motif. As the NGR sequence occurs in the cell-binding region of the fibronectin molecule, this sequence could contribute to the specific recognition of fibronectin by alpha 5 beta 1. Selection for high affinity peptides for alpha 5 beta 1 surprisingly yielded a sequence RRETAWA that does not bear obvious resemblance to known integrin ligand sequences. The synthetic cyclic peptide GACRRETAWACGA (*CRRETAWAC*) was a potent inhibitor of alpha 5 beta 1-mediated cell attachment to fibronectin. This peptide is nearly specific for the alpha 5 beta 1 integrin, because much higher concentrations were needed to inhibit the alpha v beta 1 integrin, and there was no effect on alpha v beta 3- and alpha v beta 5-mediated cell attachment to vitronectin. The peptide also did not bind to the alpha IIb beta 3 integrin. *CRRETAWAC* appears to interact with the same or an overlapping binding site in alpha 5 beta 1 as RGD, because cell attachment to *CRRETAWAC* coated on plastic was divalent cation dependent and could be blocked by an RGD-containing peptide. These results reveal a novel binding specificity in the alpha 5 beta 1 integrin.     

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.     

Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.     

Role of ADMIDAS cation-binding site in ligand recognition by integrin alpha 5 beta 1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and multiple cation-binding sites are found in both alpha and beta integrin subunits. A key cation-binding site that lies in the beta subunit A-domain is known as the metal-ion dependent adhesion site (MIDAS). Recent x-ray crystal structures of integrin alpha V beta 3 have identified a novel cation binding site in this domain, known as the ADMIDAS (adjacent to MIDAS). The role of this novel site in ligand recognition has yet to be elucidated. Using the interaction between alpha 5 beta 1 and fibronectin as a model system, we show that mutation of residues that form the ADMIDAS site inhibits ligand binding but this effect can be partially rescued by the use of activating monoclonal antibodies. The ADMIDAS mutants had decreased expression of activation epitopes recognized by 12G10, 15/7, and HUTS-4, suggesting that the ADMIDAS is important for stabilizing the active conformation of the integrin. Consistent with this suggestion, the ADMIDAS mutations markedly increased the dissociation rate of the integrin-fibronectin complex. Mutation of the ADMIDAS residues also reduced the allosteric inhibition of Mn2+-supported ligand binding by Ca2+, suggesting that the ADMIDAS is a Ca2+-binding site involved in the inhibition of Mn2+-supported ligand binding. Mutations of the ADMIDAS site also perturbed transduction of a conformational change from the MIDAS through the C-terminal helix region of the beta A domain to the underlying hybrid domain, implying an important role for this site in receptor signaling.     

Selection and structure of ion-selective ligands for platelet integrin alpha IIb(beta) 3.

Integrins contain a number of divalent cation binding sites that control ligand binding affinity. Ions such as Ca(2+) and Mg(2+) bind to distinct sites on integrin and can have opposing effects on ligand binding. These effects are presumably brought about by alterations of the shape of the ligand binding pocket. To gain insight into the nature of these structural differences, we probed the integrin ligand binding site with an RGD-based library of unparalleled complexity. A cysteine-constrained phage library containing six random amino acids and the RGD motif present in seven different registers was used to select for ligands that exhibit ion-selective binding to integrin alpha(IIb)beta(3). The library was used to select for peptides that bind to the integrin alpha(IIb)beta(3) preferentially in Ca(2+) versus Mg(2+). Peptides were identified which bound selectively in each ion. The Ca(2+)-selective peptides had a range of sequences, with the only obvious consensus involving a motif that had four cysteine residues bonded in a 1,4:2,3 arrangement. Interestingly though, the Mg(2+)-selective peptides exhibited a well defined consensus motif containing Cys-X-aromatic-L/G-R-G-D-hydrophobic-R-R/K-Cys. As a first step toward understanding the structural basis for this selectivity, solution NMR structures were obtained for representatives of both sets of peptides. All peptides formed turns, with the RGD motif at the apex. The Mg(2+)-selected peptides contained a unique basic patch that protrudes from the base of the turn.     

Annexin-V binds to the intracellular part of the beta(5) integrin receptor subunit

Bovine lactadherin binds to the alpha(v)beta(3) and alpha(v)beta(5) integrins in an RGD-dependent manner and also to anionic phospholipids. During the affinity purification of lactadherin binding receptors, a 35-kDa protein persistently coeluted with the alpha(v)beta(5) integrin receptor. Subsequently, peptide mapping, amino acid sequencing, and mass spectrometry analysis identified this protein as bovine annexin-V. Annexin-V accompanied the integrin receptor eluted with either RGD peptide or with EDTA suggesting that annexin-V bound specifically to the alpha(v)beta(5) integrin. To further investigate this putative interaction of annexin-V with the alpha(v)beta(5) integrin receptor, human annexin-V and intracellular domains of the human alpha(v)beta(5) integrin subunits were used in ligand blotting assays. Radiolabeled annexin-V showed weak binding to the intracellular part of beta(5) integrin subunit. However, by adding the aminophospholipid, phosphatidyl serine, the interaction with the beta(5) cytoplasmic peptide was enhanced many fold. Furthermore, the interaction was shown to be independent of phosphorylation, as annexin-V bound to unphosphorylated beta(5) peptide at a similar level to the phosphorylated peptide. Since binding of annexin-V to the alpha(v) integrin subunit tail was not detected, annexin-V was shown to associate specifically with the beta(5) cytoplasmic tail. Together these findings suggest a novel link between annexins and the integrin receptor family.     

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.     

Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1

Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.     

Kinetic regulation of beta 3 integrin tyrosine phosphorylation

Tyrosine phosphorylation of beta(3) integrins is a permissive stage in the activation of alpha(IIb)beta(3) and alpha(v)beta(3) in platelets and leukocytes, respectively. In this study we demonstrated direct phosphorylation of beta(3) integrins as a result of interaction with soluble monomeric ligand, and we characterized the differential kinetics of beta(3) phosphorylation as a consequence of alpha subunit pairing. We found that beta(3) phosphorylation is initiated by RGD peptide binding in a dose-dependent and saturable fashion with alpha(IIb)beta(3) becoming phosphorylated and dephosphorylated more rapidly than alpha(v)beta(3). Site mapping of phosphate incorporation reveals significant phosphorylation at Tyr-747 in both beta(3) integrin species with incorporation at Tyr-759 found at significant levels only in alpha(IIb)beta(3). Mutation of cytoplasmic beta(3) tyrosine residues in a transfection model prevents cell adhesion via these integrins. These data demonstrate that recognition of ligand is sufficient to induce beta(3) tyrosine phosphorylation and suggests that this event is regulated by the alpha subunit pairing of beta(3).     

Regulation of integrin αIIbβ3 ligand binding and signaling by the metal ion binding sites in the β I domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.     

Interaction of West Nile virus with alpha v beta 3 integrin mediates virus entry into cells

The functional receptor for the flavivirus West Nile (WNV) infection has been characterized in this study with a combination of biochemical and molecular approaches. A 105-kDa protease-sensitive glycoprotein that binds WNV was isolated from the plasma membrane of cells permissive to WNV infection. The protein was subjected to peptide sequencing, and this glycoprotein was identified as a member of the integrin superfamily. Infection of WNV was shown to be markedly inhibited in Vero cells pretreated with blocking antibodies against alpha(v)beta(3) integrin and its subunits by receptor competition assay. It was also noted that cells pretreated with antibodies against alpha(v)beta(3) integrin can effectively inhibit flavivirus Japanese encephalitis but to a lesser extent flavivirus dengue infections. West Nile virus entry is independent of divalent cations and is not highly blocked by arginine-glycine-aspartic acid (RGD) peptides, suggesting that the interaction between the virus and alpha(v)beta(3) integrin is not highly dependent on the classical RGD binding motif. In addition, gene silencing of the beta(3) integrin subunit in cells has resulted in cells largely resistant to WNV infection. In contrast, expression of recombinant human beta(3) integrin substantially increased the permissiveness of CS-1 melanoma cells for WNV infection. Soluble alpha(v)beta(3) integrin can also effectively block WNV infection in a dose-dependent manner. Furthermore, WNV infection also triggered the outside-in signaling pathway via the activation of integrin-associated focal adhesion kinase. The identification of alpha(v)beta(3) integrin as a receptor for WNV provides insight into virus-receptor interaction, hence creating opportunities in the development of anti-viral strategies against WNV infection.