Critical residues of alphaX I-domain recognizing fibrinogen central domain

Integrin alphaXbeta2 (CD11c/CD18), which binds several ligands such as fibrinogen and iC3b, has important roles in leukocyte functions including phagocytosis and migration. Establishment of structure and functional relationship in alphaX I-domain, which is a ligand-binding moiety, is important in understanding leukocyte biology and integrin function. Previously we showed that two loops (alpha3-alpha4, betaD-alpha5) around a ligand-binding face of alphaX I-domain are important for the binding of the fibrinogen molecule. In this study, we took the further step of identifying critical residues in these loops and in a supportive loop (betaF-alpha7) for fibrinogen fragment E, the central domain of fibrinogen. The residues S(199) and Q(202) in the alpha3-alpha4 loop and K(243), Y(250) in the betaD-alpha5 loop are critical for the ligand. The residues K(242), D(249), K(251), and D(252) are important but less critical for fibrinogen fragment E. The involvement of the residues in the 3-dimensional model of the I-domain suggests that several amino acid sequences in fibrinogen fragment E are responsible for alphaX I-domain. Sequence comparisons with alphaM I-domain reveal that most of the critical residues shown in alphaX I-domain are also conserved in alphaM and may have important roles in fibrinogen central domain recognition in alphaM I-domain as well.     

Characterization of alphaX I-domain binding to Thy-1

The beta2 integrins are found exclusively in leukocytes and they are composed of a common beta chain, CD18, and one of four unique alpha chains, CD11a (alphaL subunit), CD11b (alphaM subunit), CD11c (alphaX subunit), or CD11d (alphaD subunit). alphaX-beta2 which binds several ligands including fibrinogen and iC3b is expressed in monocytes/macrophages and dendritic cells playing an important role in the host defense. Despite the unique characteristics on expression and regulation, alphaX-beta2 is less functionally characterized than other beta2 integrins. To understand the biological function of alphaX-beta2 more, we tested the possibility that alphaX-beta2 binds Thy-1, a membrane protein involved in cell adhesion and signaling regulation in neurons and T cells. Here we report that a ligand binding moiety of alphaX-beta2, the I-domain, bound Thy-1 in a specific and divalent cation-dependent manner. The dissociation constant (K(D)) of alphaX I-domain binding to Thy-1 was 1.16muM and the affinity of the binding was roughly 2-fold higher than that of alphaM I-domain. Amino acid substitutions on the betaD-alpha5 of alphaX I-domain (D249, KE243/244) showed low affinities for Thy-1 while other point mutations on alpha3-alpha4 and betaE-alpha6 loops of I-domain did not, suggesting that Thy-1 recognizes the portion of a betaD-alpha5 loop, possibly alpha5 helix. Taken together, these results indicate that alphaX-beta2 specifically interacts with Thy-1. Additionally, kinetic analysis reveals a moderate affinity interaction in the presence of divalent cations. Given the reported role of Thy-1 in the regulation of T cell homeostasis and proliferation, it is tempting to speculate that alphaX-beta2 may be involved in Thy-1 function.     

Characteristics of fibrinogen binding to the domain of CD11c, an alpha subunit of p150,95.

beta2 integrins on leukocytes play important roles on cell-cell or cell-matrix adhesion through their ability to bind multiple ligands. The alpha subunits of leukocyte CD11/CD18 integrins contain an approximately 200-amino-acid inserted domain (I-domain) which is implicated in ligand binding function. To understand the characteristics of ligand binding to the alpha subunit of beta2 integrin p150,95 (CD11c/CD18), a recombinant form of the I-domain of CD11c was generated and analyzed for the interaction with fibrinogen, one of the ligands of p150,95. It was found that the CD11c I-domain bound fibrinogen specifically. Fibrinogen binding to the CD11c I-domain was inhibited by a molar excess of fragment E, a central domain of fibrinogen, and not by that of fragment D, a distal domain of fibrinogen, suggesting that CD11c/CD18 recognizes a central domain of fibrinogen. Divalent cations such as Mg(2+) and Mn(2+) were required for fibrinogen binding to the CD11c I-domain. Also alanine substitutions on the putative metal binding sites of the CD11c I-domain such as Asp(242) and Tyr(209) reduced its ability to bind fibrinogen. These data reinforce the fact that the divalent cation is a prerequisite for ligand binding of the CD11c I-domain.     

Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin

The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the alphaXbeta2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.     

Integrin-using rotaviruses bind alpha2beta1 integrin alpha2 I domain via VP4 DGE sequence and recognize alphaXbeta2 and alphaVbeta3 by using VP7 during cell entry

Integrins alpha2beta1, alphaXbeta2, and alphaVbeta3 have been implicated in rotavirus cell attachment and entry. The virus spike protein VP4 contains the alpha2beta1 ligand sequence DGE at amino acid positions 308 to 310, and the outer capsid protein VP7 contains the alphaXbeta2 ligand sequence GPR. To determine the viral proteins and sequences involved and to define the roles of alpha2beta1, alphaXbeta2, and alphaVbeta3, we analyzed the ability of rotaviruses and their reassortants to use these integrins for cell binding and infection and the effect of peptides DGEA and GPRP on these events. Many laboratory-adapted human, monkey, and bovine viruses used integrins, whereas all porcine viruses were integrin independent. The integrin-using rotavirus strains each interacted with all three integrins. Integrin usage related to VP4 serotype independently of sialic acid usage. Analysis of rotavirus reassortants and assays of virus binding and infectivity in integrin-transfected cells showed that VP4 bound alpha2beta1, and VP7 interacted with alphaXbeta2 and alphaVbeta3 at a postbinding stage. DGEA inhibited rotavirus binding to alpha2beta1 and infectivity, whereas GPRP binding to alphaXbeta2 inhibited infectivity but not binding. The truncated VP5* subunit of VP4, expressed as a glutathione S-transferase fusion protein, bound the expressed alpha2 I domain. Alanine mutagenesis of D308 and G309 in VP5* eliminated VP5* binding to the alpha2 I domain. In a novel process, integrin-using viruses bind the alpha2 I domain of alpha2beta1 via DGE in VP4 and interact with alphaXbeta2 (via GPR) and alphaVbeta3 by using VP7 to facilitate cell entry and infection.     

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.     

Domain V of beta2-glycoprotein I binds factor XI/XIa and is cleaved at Lys317-Thr318

The fifth domain (DV) of beta2-glycoprotein I (beta2GPI) is important for binding a number of ligands including phospholipids and factor XI (FXI). Beta2GPI is proteolytically cleaved in DV by plasmin but not by thrombin, VIIa, tissue plasminogen activator, or uPA. Following proteolytic cleavage of DV by plasmin, beta2GPI retains binding to FXI but not to phospholipids. Native beta2GPI, but not cleaved beta2GPI, inhibits activation of FXI by thrombin and factor XIIa, attenuating a positive feedback mechanism for additional thrombin generation. In this report, we have defined the FXI/FXIa binding site on beta2GPI using site-directed mutagenesis. We show that the positively charged residues Lys284, Lys286, and Lys287 in DV are essential for the interaction of beta2GPI with FXI/FXIa. We also demonstrate that FXIa proteolytically cleaves beta2GPI at Lys317-Thr318 in DV. Thus, FXIa cleavage of beta2GPI in vivo during thrombus formation may accelerate FXI activation by decreasing the inhibitory effect of beta2GPI.     

Identification of a negatively charged peptide motif within the catalytic domain of progelatinases that mediates binding to leukocyte beta 2 integrins

The alpha M beta 2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the alpha M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known beta 2 integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified beta 2 integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active beta 2 integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of beta 2 integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the alpha M beta 2 and alpha L beta 2 integrins in leukocytes, and pro-MMP-9 colocalized with alpha M beta 2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked beta 2 integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-beta 2 integrin complex-dependent manner.     

alphavbeta5 integrin sustains growth of human pre-B cells through an RGD-independent interaction with a basic domain of the CD23 protein

CD23 is a type II transmembrane glycoprotein synthesized by hematopoietic cells that has biological activity in both membrane-bound and freely soluble forms, acting via a number of receptors, including integrins. We demonstrate here that soluble CD23 (sCD23) sustains growth of human B cell precursors via an RGD-independent interaction with the alphavbeta5 integrin. The integrin recognizes a tripeptide motif in a small disulfide-bonded loop at the N terminus of the lectin head region of CD23, centered around Arg(172), Lys(173), and Cys(174) (RKC). This RKC motif is present in all forms of sCD23 with cytokine-like activity, and cytokine activity is independent of the lectin head, an "inverse RGD" motif, and the CD21 and IgE binding sites. RKC-containing peptides derived from this region of CD23 bind alphavbeta5 and are biologically active. The binding and activity of these peptides is unaffected by inclusion of a short peptide containing the classic RGD sequence recognized by integrins, and, in far-Western analyses, RKC-containing peptides bind to the beta subunit of the alphavbeta5 integrin. The interaction between alphavbeta5 and sCD23 indicates that integrins deliver to cells important signals initiated by soluble ligands without the requirement for interactions with RGD motifs in their common ligands. This mode of integrin signaling may not be restricted to alphavbeta5.     

Structure-function of the putative I-domain within the integrin beta 2 subunit

The central region (residues 125-385) of the integrin beta(2) subunit is postulated to adopt an I-domain-like fold (the beta(2)I-domain) and to play a critical role in ligand binding and heterodimer formation. To understand structure-function relationships of this region of beta(2), a homolog-scanning mutagenesis approach, which entails substitution of nonconserved hydrophilic sequences within the beta(2)I-domain with their homologous counterparts of the beta(1)I-domain, has been deployed. This approach is based on the premise that beta(1) and beta(2) are highly homologous, yet recognize different ligands. Altogether, 16 segments were switched to cover the predicted outer surface of the beta(2)I-domain. When these mutant beta(2) subunits were transfected together with wild-type alpha(M) in human 293 cells, all 16 beta(2) mutants were expressed on the cell surface as heterodimers, suggesting that these 16 sequences within the beta(2)I-domain are not critically involved in heterodimer formation between the alpha(M) and beta(2) subunits. Using these mutant alpha(M)beta(2) receptors, we have mapped the epitopes of nine beta(2)I-domain specific mAbs, and found that they all recognized at least two noncontiguous segments within this domain. The requisite spatial proximity among these non-linear sequences to form the mAb epitopes supports a model of an I-domain-like fold for this region. In addition, none of the mutations that abolish the epitopes of the nine function-blocking mAbs, including segment Pro(192)-Glu(197), destroyed ligand binding of the alpha(M)beta(2) receptor, suggesting that these function-blocking mAbs inhibit alpha(M)beta(2) function allosterically. Given the recent reports implicating the segment equivalent to Pro(192)-Glu(197) in ligand binding by beta(3) integrins, these data suggest that ligand binding by the beta(2) integrins occurs via a different mechanism than beta(3). Finally, both the conformation of the beta(2)I-domain and C3bi binding activity of alpha(M)beta(2) were dependent on a high affinity Ca(2+) binding site (K(d) = 105 microm), which is most likely located within this region of beta(2).     

The leukocyte integrin alpha D beta 2 binds VCAM-1: evidence for a binding interface between I domain and VCAM-1.

The trafficking of leukocytes through tissues is supported by an interaction between the beta 2 (CD18) integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) and their ligand ICAM-1. The most recently identified and fourth member of the beta 2 integrins, alpha D beta 2, selectively binds ICAM-3 and does not appear to bind ICAM-1. We have reported recently that alpha D beta 2 can support eosinophil adhesion to VCAM-1. Here we demonstrate that expression of alpha D beta 2 in a lymphoid cell that does not express alpha 4 integrins confers efficient binding to VCAM-1. In addition, a soluble form of alpha D beta 2 binds VCAM-1 with greater efficiency relative to ICAM-3. The I domain of alpha D contains a binding site for VCAM-1 since recombinant alpha D I domain binds specifically to VCAM-1. In addition, alpha D mAb that block cellular binding to VCAM-1 bind the alpha D I domain. Using VCAM-1 mutants we have determined that the binding site on VCAM-1 for alpha D beta 2 overlaps with that of alpha 4++ integrins. Substitution of VCAM-1 aspartate at position 40, D40, within the conserved integrin binding site, diminishes binding to alpha D beta 2 and abrogates binding to the alpha D I domain. The corresponding integrin binding site residue in ICAM-3 is also essential to alpha D beta 2 binding. Finally, we demonstrate that alpha D beta 2 can support lymphoid cell adhesion to VCAM-1 under flow conditions at levels equivalent to those mediated by alpha 4 beta 1. These results indicate that VCAM-1 can bind to an I domain and that the binding of alpha D beta 2 to VCAM-1 may contribute to the trafficking of a subpopulation of leukocytes that express alpha D beta 2.     

Manifestations of inflammatory arthritis are critically dependent on LFA-1

Leukocyte infiltration of synovial fluid and tissues is the hallmark of inflammatory arthritis. Selectins and beta2 integrins have been implicated in the multistep process of leukocyte adhesion to vascular endothelium. However, previous work has revealed disparate requirements for leukocyte recruitments to specific anatomic locales. Moreover, the mechanisms regulating recruitment of leukocytes to the joint in inflammatory arthritis models are not fully understood. We hypothesized that beta2 integrins, expressed on leukocytes, might play a pathogenic role in synovial inflammation. Using mice deficient in all beta2 integrins (CD18 null mice), we demonstrate that expression of these heterodimeric adhesion molecules is critical for arthritis induction in the K/B x N serum transfer model. Using null-allele mice and blocking mAbs, we demonstrate specifically that CD11a/CD18 (LFA-1) is absolutely required for the development of arthritis in this model. Blocking mAbs further revealed an ongoing requirement for LFA-1 I-domain adhesive function in disease perpetuation. These findings suggest that the LFA-1 I-domain forms an attractive target for treatment of human inflammatory arthritis.     

"RKKH" peptides from the snake venom metalloproteinase of Bothrops jararaca bind near the metal ion-dependent adhesion site of the human integrin alpha(2) I-domain.

Integrin alpha(1)beta(1) and alpha(2)beta(1) are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their alpha subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the alpha(2) I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the alpha(2) I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the "metal ion-dependent adhesion site" (MIDAS) of the I-domain, when mutated, show significantly impaired binding of the peptide. Removal of helix alphaC, located along one side of the MIDAS and suggested to be involved in collagen-binding in these I-domains, does not affect peptide binding. This study supports the notion that the metalloproteinase initially binds to the alpha(2) I-domain at a location distant from the active site of the protease, thus blocking collagen binding to the adhesion molecule in the vicinity of the MIDAS, while at the same time leaving the active site free to degrade nearby proteins, the closest being the beta(1) subunit of the alpha(2)beta(1) cell-surface integrin itself.     

Nonlysine-analog plasminogen modulators promote autoproteolytic generation of plasmin(ogen) fragments with angiostatin-like activity.

We recently discovered several nonlysine-analog conformational modulators for plasminogen. These include SMTP-6, thioplabin B and complestatin that are low molecular mass compounds of microbial origin. Unlike lysine-analog modulators, which increase plasminogen activation but inhibit its binding to fibrin, the nonlysine-analog modulators enhance both activation and fibrin binding of plasminogen. Here we show that some nonlysine-analog modulators promote autoproteolytic generation of plasmin(ogen) derivatives with its catalytic domain undergoing extensive fragmentation (PMDs), which have angiostatin-like anti-endothelial activity. The enhancement of urokinase-catalyzed plasminogen activation by SMTP-6 was followed by rapid inactivation of plasmin due to its degradation mainly in the catalytic domain, yielding PMD with a molecular mass ranging from 68 to 77 kDa. PMD generation was observed when plasmin alone was treated with SMTP-6 and was inhibited by the plasmin inhibitor aprotinin, indicating an autoproteolytic mechanism in PMD generation. Thioplabin B and complestatin, two other nonlysine-analog modulators, were also active in producing similar PMDs, whereas the lysine analog 6-aminohexanoic acid was inactive while it enhanced plasminogen activation. Peptide sequencing and mass spectrometric analyses suggested that plasmin fragmentation was due to cleavage at Lys615-Val616, Lys651-Leu652, Lys661-Val662, Lys698-Glu699, Lys708-Val709 and several other sites mostly in the catalytic domain. PMD was inhibitory to proliferation, migration and tube formation of endothelial cells at concentrations of 0.3-10 microg.mL(-1). These results suggest a possible application of nonlysine-analog modulators in the treatment of cancer through the enhancement of endogenous plasmin(ogen) fragment formation.     

Evaluation of specificity and effects of monoclonal antibodies submitted to the Eighth Human Leucocyte Differentiation Antigen Workshop on rotavirus-cell attachment and entry

Rotavirus infection of permissive cells is a multi-step process that requires interaction with several cell surface receptors. Integrins alpha2beta1, alpha4beta1, alphaXbeta2, and alphavbeta3 are involved in the attachment and entry into permissive cells for many rotavirus strains. However, possible roles of known partners of these integrins in this process have not been studied. Here, the specificities of new monoclonal antibodies directed to beta1 and beta2 integrins were determined using integrin-transfected cells. The ability of monoclonal antibodies to integrin partners CD82, CD151, CD321, and CD322 to bind rotavirus-permissive cell lines (MA104, Caco-2, and RD) and K562 cells expressing or lacking alpha4beta1 also was investigated. CD82 and CD151 were expressed on K562, alpha4-K562, and RD cells. CD321-specific antibodies bound K562, alpha4-K562, MA104, and Caco-2 cells. CD322 expression was detected on MA104 but not Caco-2 cells. Antibodies to CD82, CD151, CD321, and CD322 that bound these cells were investigated for their ability to inhibit cellular attachment and entry by rotaviruses. Antibody blockade of these integrin-associated proteins did not affect cell attachment or entry of the integrin-using rhesus rotavirus RRV or porcine rotavirus CRW-8, which uses alpha4beta1 integrin for infection. Antibody blockade of CD322 did not alter cell attachment or infectivity by human rotavirus strain RV-3, so RV-3 infection was independent of CD322. Overall, these studies indicate that CD82, CD151, CD321, and CD322 are unlikely to play a role in rotavirus-cell binding or entry.     

Delineation of the key amino acids involved in neutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacity of integrin alphaMbeta 2 to recognize multiple ligands

To gain insight into the mechanism by which the alpha(M)I-domain of integrin alpha(M)beta(2) interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the alpha(L)I-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp(149), Arg(151), Gly(207), Tyr(252), and Glu(258) as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the alpha(L)I-domain, the chimera bound NIF with high affinity. Another ligand of alpha(M)beta(2), C3bi, which is known to use the same segments of the alpha(M)I-domain in engaging the receptor, failed to bind to the chimeric alpha(L)I-domain. Thus, the alpha(M)I-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.     

Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.     

Regulated unmasking of the cryptic binding site for integrin alpha M beta 2 in the gamma C-domain of fibrinogen

Fibrinogen is a ligand for leukocyte integrin alpha(M)beta2 (CD11b/CD18, Mac-1) and mediates adhesion and migration of leukocytes during the immune-inflammatory responses. The binding site for alpha(M)beta2 resides in gammaC, a constituent subdomain in the D-domain of fibrinogen. The sequence gamma383-395 (P2-C) in gammaC was implicated as the major binding site for alpha(M)beta2. It is unknown why alpha(M)beta2 on leukocytes can bind to immobilized fibrinogen in the presence of high concentrations of soluble fibrinogen in plasma. In this study, we have investigated the accessibility of the binding site in fibrinogen for alpha(M)beta2. We found that the alpha(M)beta2-binding site in gammaC is cryptic and identified the mechanism that regulates its unmasking. Proteolytic removal of the small COOH-terminal segment(s) of gammaC, gamma397/405-411, converted the D100 fragment of fibrinogen, which contains intact gammaC and is not able to inhibit adhesion of the alpha(M)beta2-expressing cells, into the fragment D98, which effectively inhibited cell adhesion. D98, but not D100, bound to the recombinant alpha(M)I-domain, and the alpha(M)I-domain recognition peptide, alpha(M)(Glu253-Arg261). Exposure of the P2-C sequence in fibrinogen, D100, and D98 was probed with a site-specific mAb. P2-C is not accessible in soluble fibrinogen and D100 but becomes exposed in D98. P2-C is also unmasked by immobilization of fibrinogen onto a plastic and by deposition of fibrinogen in the extracellular matrix. Thus, exposure of P2-C by immobilization and by proteolysis correlates with unmasking of the alpha(M)beta2-binding site in the D-domain. These results demonstrate that conformational alterations regulate the alpha(M)beta2-binding site in gammaC and suggest that processes relevant to tissue injury and inflammation are likely to be involved in the activation of the alpha(M)beta2-binding site in fibrinogen.     

Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2

Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2).     

A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP- 1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.