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.     

Identification of critical residues for plasminogen binding by the alphaX I-domain of the beta2 integrin, alphaXbeta2

The beta2 integrins on leukocytes play important roles in cell adhesion, migration and phagocytosis. One of the beta2 integrins, alphaXbeta2 (CD11c/CD18), is known to bind ligands such as fibrinogen, Thy-1 and iC3b, but its function is not well characterized. To understand its biological roles, we attempted to identify novel ligands. The functional moiety of alphaXbeta2, the alphaX I-domain, was found to bind plasminogen, the zymogen of plasmin, with moderate affinity (1.92 X 10-(6) M) in the presence of Mg(2+) or Mn(2+). The betaD-alpha5 loop of the alphaX I-domain proved to be responsible for binding, and lysine residues (Lys(242), Lys(243)) in the loop were the most important for recognizing plasminogen. An excess amount of the lysine analog, 6-aminohexanoic acid, inhibited alphaX I-domain binding to plasminogen, indicating that binding is lysine-dependent. The results of this study indicate that leukocytes regulate plasminogen activation, and consequently plasmin activities, through an interaction with alphaXbeta2 integrin.     

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.     

Specific integrin subunits in bovine oocytes, including novel sequences for alpha 6 and beta 3 subunits

Integrins facilitate attachment of cells to the extra-cellular matrix, often binding the arginine-glycine-aspartic acid tri-peptide motif, thus facilitating cell migration, mediating cell-cell adhesion, linking the extracellular matrix (ECM) with cytoskeletal elements, and acting as signaling molecules. Adhesion activates signaling mechanisms that regulate integrin function, cytoskeletal assembly, cell behavior, and protein synthesis. Immunofluorescence was used to determine the presence of integrin alpha and beta subunits on the surface of bovine oocytes using a panel of monoclonal antibodies (mAbs) specific for alphaL, alphaM, alphaX, alphaV, alpha2, alpha4, alpha6, beta1, beta2, and beta3 antigens, with multiple antibodies for each subunit. Confocal microscopy indicated the presence of alphaV, alpha6, alpha4, alpha2, ss1, and ss3 integrin subunits on the plasma membrane of bovine oocytes. The presence of these subunits was verified by RT-PCR analysis using primers designed based on known gene sequences of bovine integrin subunits, or by using sequence information using bovine expressed sequence tags (EST) compared with known human and murine integrin subunit gene sequence information. Previously unpublished sequence information for bovine alpha6 and beta3 integrins was determined. The presence of these integrin subunits on the bovine oocyte vitelline membrane supports the hypothesis that sperm-oocyte interactions in the bovine are mediated by integrins.     

"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.     

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.     

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.     

Distinct roles for the alpha and beta subunits in the functions of integrin alphaMbeta2

Integrin alphaMbeta2 (Mac-1, CD11b/CD18) is a noncovalently linked heterodimer of alphaM and beta2 subunits on the surface of leukocytes, where it plays a pivotal role in the adhesion and migration of these cells. Using HEK293 cells expressing alphaMbeta2 or the individual constituent chains on their surface, we analyzed the contributions of the alphaM or beta2 subunits to functional responses mediated by the integrin. In cells expressing only alphaM or beta2, the individual subunits were not associated with the endogenous integrins of the cells, and other partners for the subunits were not detected by surface labeling and immunoprecipitation under a variety of conditions. The alphaM cells mediated adhesion and spreading on a series of alphaMbeta2 ligands (fibrinogen, Factor X, iC3b, ICAM-1 (intercellular adhesion molecule-1), and denatured ovalbumin) but could not support cell migration to any of these. The spreading of the alphaM cells suggested an unanticipated linkage of this subunit to the cytoskeleton. The beta2 cells supported migration and attachment but not spreading on a subset of the alphaMbeta2 ligands. The heterodimeric receptor and its individual subunits were purified from the cells by affinity chromatography and recapitulated the ligand binding properties of the corresponding cell lines. These data indicate that each subunit of alphaMbeta2 contributes distinct properties to alphaMbeta2 and that, in most but not all cases, the response of the integrin is a composite of the functions of its individual subunits.     

Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function

Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.     

Crystal structure of the alpha1beta1 integrin I-domain: insights into integrin I-domain function.

The alpha1beta1 integrin is a major cell surface receptor for collagen. Ligand binding is mediated, in part, through a 200 amino acid inserted 'I'-domain contained in the extracellular part of the integrin alpha chain. Integrin I-domains contain a divalent cation binding (MIDAS) site and require cations to interact with integrin ligands. We have determined the crystal structure of recombinant I-domain from the rat alpha1beta1 integrin at 2.2 A resolution in the absence of divalent cations. The alpha1 I-domain adopts the dinucleotide binding fold that is characteristic of all I-domain structures that have been solved to date and has a structure very similar to that of the closely related alpha2beta1 I-domain which also mediates collagen binding. A unique feature of the alpha1 I-domain crystal structure is that the MIDAS site is occupied by an arginine side chain from another I-domain molecule in the crystal, in place of a metal ion. This interaction supports a proposed model for ligand-induced displacement of metal ions. Circular dichroism spectra determined in the presence of Ca2+, Mg2+ and Mn2+ indicate that no changes in the structure of the I-domain occur upon metal ion binding in solution. Metal ion binding induces small changes in UV absorption spectra, indicating a change in the polarity of the MIDAS site environment.     

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).     

Specific activation of leukocyte beta2 integrins lymphocyte function-associated antigen-1 and Mac-1 by chemokines mediated by distinct pathways via the alpha subunit cytoplasmic domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (alphaMbeta2) but not lymphocyte function-associated antigen-1 (LFA-1; alphaLbeta2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1alpha in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1alpha were confirmed by expression of alphaM or alphaL in alphaL-deficient Jurkat cells. Moreover, expression of chimeras containing alphaL and alphaM cytoplasmic domain exchanges indicated that alpha cytoplasmic tails conferred the specific mode of regulation. Coexpressing alphaM or chimeras in mutant Jurkat cells with a "gain of function" phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the alphaL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of beta2 integrins. Our data suggest that a specific regulation of beta2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the alpha subunit cytoplasmic domains.     

Genomic structure of an integrin alpha subunit, the leukocyte p150,95 molecule

The genomic structure of integrins is important to our understanding of the evolution of this complex family. The alpha subunit of the leukocyte integrin p150,95 (CD11c) is a transmembrane polypeptide of 1144 residues whose long extracellular region contains three putative divalent cation binding repeats and a 200- amino acid inserted or "I" domain. The p150,95 alpha subunit gene extends over 25 kilobases and is comprised of at least 31 exons grouped in five clusters. The I domain, which is only present in some integrins and is homologous to domains in von Willebrand factor, cartilage matrix protein, complement factor B and the alpha 1 and alpha 2 chains of collagen type VI, is distributed in four exons. Each one of the three divalent cation binding repeats is encoded by a separate exon. Surprisingly, a sequence homologous to the first two putative divalent cation binding repeats is present in an inverted orientation in the intron following the last exon of the I domain. Both the signal peptide and the transmembrane domain are split in two exons. Putative proteolytic cleavage sequences in other integrin alpha subunits align as inserts within the p150,95 alpha subunit gene falling at exon boundaries. The organization of the p150,95 alpha subunit gene provides further insights into the structure and evolution of the integrins.     

Effect of integrin beta 2 subunit truncations on LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) assembly, surface expression, and function

LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) are members of the beta2 integrins involved in leukocyte function during immune and inflammatory responses. We aimed to determine a minimized beta2 subunit that forms functional LFA-1 and Mac-1. Using a series of truncated beta2 variants, we showed that the subregion Q23-D300 of the beta2 subunit is sufficient to combine with the alphaL and alphaM subunits intracellularly. However, only the beta2 variants terminating after Q444 promote cell surface expression of LFA-1 and Mac-1. Thus, the major cysteine-rich region and the three highly conserved cysteine residues at positions 445, 447, and 449 of the beta2 subunit are not required for LFA-1 and Mac-1 surface expression. The surface-expressed LFA-1 variants are constitutively active with respect to ICAM-1 adhesion and these variants express the activation reporter epitope of the mAb 24. In contrast, surface-expressed Mac-1, both the wild type and variants, require 0. 5 mM MnCl2 for adhesion to denatured BSA. These results suggest that the role of the beta2 subunit in LFA-1- and Mac-1-mediated adhesion may be different.     

Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen

The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.     

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.     

A cell-free electrochemiluminescence assay for measuring beta1-integrin-ligand interactions

We have developed a cell-free assay for binding of solubilized beta1 integrins to their physiologically relevant ligands using an electrochemiluminescent detection method. The method utilizes ruthenium-conjugated monoclonal antibodies for detection of either purified integrins or, more conveniently, integrin-expressing cell lysates, which are captured on beads coated with extracellular matrix or vascular ligand proteins. For the interaction of alpha1beta1 integrin with collagen IV, a signal of 10-fold over background was generated with samples containing only 10 ng (0.05 pmol) of integrin. This interaction is cation-dependent and can be inhibited by blocking antibodies to the alpha1 subunit. The method was extended to studies of ligand binding by integrins alpha2beta1, alpha4beta1, alpha5beta1, and alpha6beta1. For each integrin-ligand pair, the specificity of the interaction was verified with neutralizing antibodies against the specific integrin. The specific binding signal correlated with the activating ability of the labeled antibody used for detection, although the ability of divalent cations (Mn2+, Mg2+, Ca2+) to support integrin-ligand binding varied dramatically among the various integrin-ligand pairs. The assay provides a simple method for investigating integrin-ligand interactions without avidity and/or signaling effects which can complicate conventional cell-based assay methods.     

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.     

Model of the alphaLbeta2 integrin I-domain/ICAM-1 DI interface suggests that subtle changes in loop orientation determine ligand specificity

The interaction of the alphaLbeta2 integrin with its cellular ligand the intercellular adhesion molecule-1 (ICAM-1) is critical for the tight binding interaction between most leukocytes and the vascular endothelium before transendothelial migration to the sites of inflammation. In this article we have modeled the alphaL subunit I-domain in its active form, which was computationally docked with the D1 domain of the ICAM-1 to probe potential protein-protein interactions. The experimentally observed key interaction between the carboxylate of Glu 34 in the ICAM-1 D1 domain and the metal ion-dependent adhesion site (MIDAS) in the open alphaL I-domain was consistently reproduced by our calculations. The calculations reveal the nature of the alphaLbeta2/ICAM-1 interaction and suggest an explanation for the increased ligand-binding affinity in the "open" versus the "closed" conformation of the alphaL I-domain. A mechanism for substrate selectivity among alphaL, alphaM, and alpha2 I-domains is suggested whereby the orientation of the loops within the I-domain is critical in mediating the interaction of the Glu 34 carboxylate of ICAM-1 D1 with the MIDAS.     

Beta 1 integrins isolated from embryonic chicken fibroblasts bind to monomers and polymers of type I collagen.

The avian integrin beta 1 subfamily consists of multiple alpha-beta subunit heterodimers. We employed two different physical states of type I collagen, monomers and fibrils, in the isolation and characterization of avian collagen integrins. Affinity chromatography showed that three integrins, tentatively designated alpha 155 beta 1 (band 1), alpha 5a beta 1, and alpha 3 beta 1 (band 2), bind fibrillar and monomeric collagen under physiological ionic conditions and require divalent cations for binding activity. Sodium chloride gradients (0-0.5 M) were used to assess the functional ability of the integrins to remain bound to the two forms of type I collagen. The results show that integrins elute from the two forms of collagen with distinct fractionation profiles. One integrin, alpha 155 beta 1, binds fibrillar collagen with relatively higher affinity than the other beta 1 receptors. This same avian integrin, alpha 155 beta 1, is immunoreactive with an antiserum (Hynes et al., 1989) raised against a peptide that corresponds to the entire alpha 5 cytoplasmic domain, and coincidently, part of the alpha 6 cytoplasmic domain (de Curtis et al., 1991). Cell biological studies employing double immunofluorescence show that integrins recognized by this antiserum co-localize with extracellular deposits of type I collagen.