Antibodies to the vitronectin receptor (integrin alpha V beta 3) inhibit binding and infection of foot-and-mouth disease virus to cultured cells.

The amino acid sequence Arg-Gly-Asp (RGD) is highly conserved on the VP1 proteins of different serotypes and subtypes of foot-and-mouth disease virus (FMDV) and is essential for cell attachment. This sequence is also found in certain extracellular matrix proteins that bind to a family of cell surface receptors called integrins. Within the Picornaviridae family, enterovirus coxsackievirus A9 also has an RGD motif on its VP1 capsid protein and has recently been shown to utilize the vitronectin receptor integrin alpha V beta 3 as a receptor on monkey kidney cells. Competition binding experiments between type A12 FMDV and coxsackievirus A9 using BHK-21 and LLC-MK2 cells revealed shared receptor specificity between these two viruses. Polyclonal anti-serum to the vitronectin receptor and a monoclonal antibody to the alpha V subunit inhibited both FMDV binding and plaque formation, while a monoclonal antibody to the beta 3 subunit inhibited virus binding. In contrast, antibodies to the fibronectin receptor (alpha 5 beta 1) or to the integrin (alpha V beta 5) had no effect on either binding or plaque formation. These data demonstrate that the alpha V beta 3 vitronectin receptor can function as a receptor for FMDV.     

The vitronectin receptor alpha v beta 3 binds fibronectin and acts in concert with alpha 5 beta 1 in promoting cellular attachment and spreading on fibronectin

The vitronectin receptor (alpha v beta 3) is a member of the integrin superfamily of adhesive protein receptors that mediate a wide spectrum of adhesive cellular interactions, including attachment to vitronectin, von Willebrand factor, fibrinogen, and thrombospondin. We have studied the binding of fibronectin to the purified vitronectin receptor, and the role of this receptor in the attachment of cells to fibronectin. A solid-phase microtiter assay was developed to investigate the binding properties of the vitronectin receptor. Purified alpha v beta 3 bound fibronectin with high affinity in a saturable, divalent cation- dependent manner. Binding was inhibited by soluble vitronectin, by RGD- containing peptides, and by LM609, a monoclonal antibody against the vitronectin receptor known to inhibit the binding of adhesive proteins to alpha v beta 3. Immunoinhibition experiments showed that M21 human melanoma cells, which express the fibronectin receptor, alpha 5 beta 1, as well as alpha v beta 3, used both of these integrins to attach and spread on fibronectin. In support of this finding, M21-L cells, a variant cell line that specifically lacks alpha v beta 3 but expresses alpha v beta 1, attached and spread poorly on fibronectin. In addition, alpha v beta 3 from surface-labeled M21 cells was retained, and selectively eluted by RGDS from a fibronectin affinity column. These results indicate that alpha v beta 3 acts in concert with alpha 5 beta 1 in promoting fibronectin recognition by these cells. We conclude that fibronectin binds to the alpha v beta 3 vitronectin receptor specifically and with high affinity, and that this interaction is biologically relevant in supporting cell adhesion to matrix proteins.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells.

Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage-displayed hexapeptide library, and virus-neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C-terminal segment of the alpha2 domain of the heavy chain of human MHC class I molecules (MHC-I alpha2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST-fused MHC-I alpha2- and FNIII-derived oligopeptides interacted with recombinant fibers in a subgroup-specific manner. In vivo, the MHC-I alpha2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In beta2-microglobulin-transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4x10(10)/M. The results suggested that the conserved region of MHC-I alpha2-domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors.     

Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells

Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the alpha 2 beta 1 integrin, beta 2 chain integrins, and vitronectin receptor (alpha v beta 3) were not involved in cellular attachment to invasin.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty-two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose-binding protein (MBP)--invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl-terminal 192 amino acids retained cell binding activity. One carboxyl-terminal invasin fragment and seven MBP--invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl-terminal 192 amino acids of invasin contains the integrin-binding domain, even though this region does not contain the tripeptide sequence Arg-Gly-Asp.     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin

Using mAb technology (Wayner, E. A., W. G. Carter, R. Piotrowicz, and T. J. Kunicki. 1988. J. Cell Biol. 107:1881-1891), we have identified a new fibronectin receptor that is identical to the integrin receptor alpha 4 beta 1. mAbs P3E3, P4C2, and P4G9 recognized epitopes on the alpha 4 subunit and completely inhibited the adhesion of peripheral blood and cultured T lymphocytes to a 38-kD tryptic fragment of plasma fibronectin containing the carboxy-terminal Heparin II domain and part of the type III connecting segment (IIICS). The ligand in IIICS for alpha 4 beta 1 was the CS-1 region previously defined as an adhesion site for melanoma cells. The functionally defined mAbs to alpha 4 partially inhibited T lymphocyte adhesion to intact plasma fibronectin and had no effect on their attachment to an 80-kD tryptic fragment containing the RGD (arg-gly-asp) adhesion sequence. mAbs (P1D6 and P1F8) to the previously described fibronectin receptor, alpha 5 beta 1, completely inhibited T lymphocyte adhesion to the 80-kD fragment but had no effect on their attachment to the 38-kD fragment or to CS-1. Both alpha 4 beta 1 and alpha 5 beta 1 localized to focal adhesions when fibroblasts that express these receptors were grown on fibronectin-coated surfaces. These findings demonstrated a specific interaction of both receptors with fibronectin at focal contacts. In conclusion, these findings show clearly that cultured T lymphocytes use two independent receptors during attachment to fibronectin and that (a) alpha 5 beta 1 is the receptor for the RGD containing cell adhesion domain, and (b) alpha 4 beta 1 is the receptor for a carboxy-terminal cell adhesion region containing the Heparin II and IIICS domains. Furthermore, these data also show that T lymphocytes express a clear preference for a region of molecular heterogeneity in IIICS (CS-1) generated by alternative splicing of fibronectin pre-mRNA and that alpha 4 beta 1 is the receptor for this adhesion site.     

Endothelial cells use alpha 2 beta 1 integrin as a laminin receptor

Human umbilical vein endothelial cells attach and spread on laminin-coated substrates. Affinity chromatography was used to identify the attachment receptor. Fractionation of extracts from surface-iodinated endothelial cells on human laminin-Sepharose yielded a heterodimeric complex, the subunits of which migrated with molecular sizes corresponding to 160/120 kD and 160/140 kD under nonreducing and reducing conditions, respectively. The purified receptor bound to laminin and slightly less to fibronectin and type IV collagen in a radioreceptor assay. This endothelial cell laminin receptor was classified as an alpha 2 beta 1 integrin because monoclonal and polyclonal antibodies directed against the alpha 2 and bet 1 subunits immunoprecipitated the receptor. Cytofluorometric analysis and immunoprecipitation showed that the alpha 2 subunit is an abundant integrin alpha subunit in the endothelial cells and that the alpha subunits associated with laminin binding in other types of cells are expressed in these cells only at low levels. The alpha 2 beta 1 integrin appears to be a major receptor for laminin in the endothelial cells, because an anti-alpha 2 monoclonal antibody inhibited the attachment of the endothelial cells to human laminin. These results define a new role for the alpha 2 subunit in laminin binding and suggest that the ligand specificity of the alpha 2 beta 1 integrin, which is known as a collagen receptor in other types of cells, can be modulated by cell type-specific factors to include laminin binding.     

Alpha 2 beta 1 integrins from different cell types show different binding specificities

Purified alpha 2 beta 1 integrin from human platelets was compared in its function and immunoreactivity to alpha 2 beta 1 from endothelial cells. Both alpha 2 beta 1 integrins bound to type I collagen-Sepharose and had indistinguishable immunoreactivities when analyzed by a panel of monoclonal and polyclonal alpha 2-specific antibodies. However, functional analysis using rechromatography of purified receptors on laminin and collagen-Sepharose showed that endothelial alpha 2 beta 1 was able to bind to laminin, whereas its counterpart from platelets did not. Moreover, a receptor binding assay indicated that, in contrast to platelets, endothelial cells might also use alpha 2 beta 1 to bind to fibronectin. These results suggest that the alpha 2 beta 1 binding specificity may be modulated by cell-type specific factors.     

Subunit structure of a laminin-binding integrin and localization of its binding site on laminin

A laminin receptor was isolated from human MG-63 osteosarcoma cells by affinity chromatography on human laminin. The isolated receptor was defined as the alpha 3 beta 1 integrin by immunoprecipitation with subunit-specific antibodies. A previously unclassified laminin-binding integrin from rat cells was shown also to contain the alpha 3 subunit. Both receptors bound to human and mouse laminin in a radioreceptor assay. They also both bound to some extent to fibronectin in this assay, but only the MG-63 cell receptor showed binding to type IV collagen. The binding of the radiolabeled receptor to insoluble laminin was inhibited by unlabeled receptor, by soluble laminin, and by chymotryptic fragments of laminin that have previously been shown to contain neurite-promoting and cell attachment-promoting activities. Moreover, the receptor binding was also inhibited by monoclonal antibodies capable of inhibiting the neurite-promoting activity of laminin and known to bind to laminin near the junction of the long arm and its terminal globule. One of these antibodies was reactive with fusion proteins expressed from laminin cDNA clones. The immunoreactive clones corresponded to the COOH-terminal end of the B1 subunit. These results identify the integrin-type laminin receptor isolated from the osteosarcoma cells as the alpha 3 beta 1 integrin and localize its binding site in close proximity of the B1 subunit COOH terminus.     

Adhesion of neutrophils to fibronectin: role of the cd66 antigens

Adhesion of neutrophils to substrate is initiated by receptor-ligand interactions that induce outside-in signaling. Inside-out signals and lateral interactions between surface molecules further fine tune the response. This study investigates the role of CD66 in adhesion of neutrophils to fibronectin, using domain-mapped monoclonal antibodies to CD66. Neutrophils express CD66a, CD66b, and CD66c on their surface. The neutrophil surface molecules that bind to fibronectin are the alpha(4)beta(1) and alpha(5)beta(1) integrins. Our results show that the monoclonal antibody Kat4c, which recognizes the AB domain of CD66a, b, and c and the polyclonal anti-CD66 (anti-carcinoembryonic antigen), augments neutrophil adhesion to fibronectin, while monoclonal antibodies to the individual CD66 antigens, the Fab fragment of Kat4c, and a mixture of the individual antibodies to CD66 antigens were unable to affect the adhesion. Thus heterodimerization of CD66a, b, and c is required for promoting neutrophil adhesion to fibronectin. The increased adhesion in presence of Kat4c was inhibited by antibodies to the beta(1) and beta(2) integrins. Antibody ligation of CD66 antigens causes their clustering and concomitant coclustering of the alpha(M) subunit of the beta(2) integrin, thereby activating the integrin. The sugar alpha-methyl mannoside inhibited anti-CD66-mediated clustering, indicating that a carbohydrate-lectin interaction may exist between CD66 and alpha(M) integrin. It also reduced the increased adhesion of neutrophils to fibronectin, suggesting that beta(2) integrin activation precedes beta(1) integrin activation. Further, the anti-CD66-mediated adhesion to fibronectin is accompanied by increased localization of Src family kinases (lyn and hck) to the cytoskeleton and an increase in their kinase activity. These results suggest that crosslinking of CD66a, CD66b, and CD66c promotes activation of the beta(2) integrin and in turn an alteration in the affinity of the beta(1) integrin, which enhances the adhesion of neutrophils to fibronectin.     

A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha 3 beta 1 integrin

The purpose of this study was to identify the binding site(s) within laminin for the alpha 3 beta 1 integrin receptor. It has been previously shown, using proteolytic fragments and anti-laminin antibodies, that the region in laminin for alpha 3 beta 1 integrin binding is localized to the carboxy-terminal region at the end of the long arm (Gehlsen, K. R., E. Engvall, K. Dickerson, W. S. Argraves, and E. Ruoslahti. 1989. J. Biol. Chem. 264:19034-19038; Tomaselli, K. J., D. E. Hall, L. T. Reichardt, L. A. Flier, K. R. Gehlsen, D. C. Turner, and S. Carbonetto. 1990. Neuron. 5:651-662). Using synthetic peptides, we have identified an amino acid sequence within the carboxy-terminal region of the laminin A chain that is recognized by the alpha 3 beta 1 integrin. The amino acid sequence represented by the synthetic peptide GD-6 (KQNCLSSRASFRGCVRNLRLSR residues numbered 3011 to 3032) of the globular domain of the murine A chain supports cell attachment and inhibits cell adhesion to laminin-coated surfaces. By affinity chromatography, peptide GD-6-Sepharose specifically bound solubilized alpha 3 beta 1 from extracts of surface-iodinated cells in a cation-dependent manner, while it did not bind other integrins. In addition, exogenous peptide GD-6 specifically eluted bound alpha 3 beta 1 from laminin-Sepharose columns but did not elute the alpha 3 beta 1 integrin from a fibronectin-Sepharose column. Using integrin subunit-specific monoclonal antibodies, only those antibodies against the alpha 3 and beta 1 subunits inhibited cell adhesion to peptide GD-6-coated surfaces. Finally, a polyclonal antibody made against peptide GD-6 reacted specifically with both murine and human laminin and significantly inhibited cell adhesion to laminin-coated surfaces but not those coated with other matrix proteins. These results identify the laminin A chain amino acid sequence of peptide GD-6 as representing a binding site in laminin for the alpha 3 beta 1 integrin.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Human B lymphocytes define an alternative mechanism of adhesion to fibronectin. The interaction of the alpha 4 beta 1 integrin with the LHGPEILDVPST sequence of the type III connecting segment is sufficient to promote cell attachment

In this report we have studied the mechanism of human B lymphocyte adhesion to fibronectin and to proteolytic fragments of this protein. B cells adhered to fibronectin and to a 38-kDa fragment, derived from the A chain, containing the Hep II domain and most of the type III connecting segment, IIICS, of fibronectin. Cells did not bind to an 80-kDa fragment containing the RGD adhesive sequence of fibronectin. Attachment to fibronectin or to the 38-kDa fragment was not affected by the 80-kDa fragment, the GRGDSPC synthetic peptide, or by a mAb specific for the alpha chain of the RGD-dependent fibronectin receptor, alpha 5 beta 1. However, B cell adhesion to fibronectin was inhibited by the synthetic peptides CS-1, comprising the first 25 amino acids of IIICS and B12, containing the sequence LHGPEILDVPST of CS-1 (residues 14-25). Moreover, this sequence was shown to be sufficient to induce stable cell adhesion when coated on plastic surfaces. A mAb specific for the alpha-subunit of the alpha 4 beta 1 integrin, completely inhibited B cell attachment to B12, CS-1, 38 kDa, and fibronectin coated substrata. These data clearly indicate that adhesion of B lymphocytes to fibronectin is exclusively mediated by the interaction of alpha 4 beta 1 with residues 14-25 of the IIICS region in fibronectin. Therefore this interaction constitutes an alternative pathway of adhesion to fibronectin, independent of RGD and alpha 5 beta 1.     

Purification and functional characterization of integrin alpha v beta 5. An adhesion receptor for vitronectin.

We have purified a novel member of the integrin gene family from placenta that serves as a vitronectin receptor. This integrin is composed of the alpha v subunit and a beta subunit that we designate beta 5. Purification was accomplished by immunodepleting a placental extract of integrin alpha v beta 3, allowing us to purify alpha v beta 5 from the remaining extract by monoclonal antibody affinity chromatography on LM 142-Sepharose, which binds to the alpha v subunit. Purification to homogeneity was subsequently achieved by affinity chromatography on wheat germ lectin-Sepharose. Western blot analysis with antibodies raised against alpha v beta 5 and alpha v beta 3 demonstrated that beta 3 and beta 5 were distinct but confirmed that the alpha subunit of the two integrins were immunologically identical. Similarly, antibodies that bind beta 3 proximal to the ligand-binding site failed to react with beta 5, indicating an architectural difference at the ligand-binding site of these related integrins. This structural difference apparently results in a functional distinction, since purified alpha v beta 3 bound to vitronectin, fibrinogen, von Willebrand factor, and fibronectin, whereas integrin alpha v beta 5 bound preferentially to vitronectin. Finally, we demonstrate by three criteria that beta 5 and beta x, the latter of which was identified in lung carcinoma cells (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), are identical. First, peptide maps of beta x and beta 5 are identical. Secondly, polyclonal antibodies raised against alpha v beta 5 immunoprecipitate both beta 5 and beta x, and finally, the amino-terminal amino acid sequences of beta x and beta 5 are identical.     

HIV-1 infection of human T lymphocytes results in enhanced alpha 5 beta 1 integrin expression.

Altered T cell adherence after human immunodeficiency virus 1 (HIV-1) infection may contribute to viral pathogenesis in the acquired immune deficiency syndrome. To address this hypothesis, we assessed mechanisms of T cell adherence to extracellular matrix proteins in vitro. We found that after HIV-1 infection, both chronically infected H9 CD4+ T cells and acutely infected primary peripheral blood lymphocytes acquired the ability to adhere to the extracellular matrix glycoprotein fibronectin, to a lesser extent to type IV collagen and laminin, but not to type I collagen. H9 cells chronically infected with two of the three HIV-1 strains studied showed approximately a sevenfold increase in attachment to fibronectin, while the same cells infected with the human retrovirus HIV-2 did not. Adhesion was accompanied by changes in morphology, including marked spreading and increased filopodia. These alterations were not blocked by the protein kinase C inhibitor H-7, which did inhibit TPA-induced T cell attachment to fibronectin. Monoclonal antibodies against both the alpha 5 and the beta 1 subunits of the classical fibronectin receptor as well as an Arg-Gly-Asp (RGD) peptide inhibited attachment, whereas anti-alpha 4 monoclonal antibodies and the CS1 peptide did not. Binding to collagen IV was also inhibited by the anti-beta 1 monoclonal antibody, but not the other antibodies. Cells metabolically labeled with [35S]methionine and analyzed by immunoprecipitation with polyclonal anti-beta 1 integrin antibody showed a 2.5-fold increase in integrin synthesis in infected cells compared to uninfected controls. This increase in synthesis was associated with an increase in cell surface expression of both alpha 5 and beta 1 integrins by FACS (registered trademark of Becton Dickinson for a fluorescence-activated cell sorter) analysis. Enhanced expression of integrins such as alpha 5 beta 1 may cause T cell adherence to a variety of tissues, where released viral gene products may induce some of the tissue-specific manifestations of HIV-1 infection.     

GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through enhanced integrin alpha5beta1-fibronectin interaction

Carcinoembryonic antigen (CEA) and CEA family member CEACAM6 are glycophosphatidyl inositol (GPI)-anchored, intercellular adhesion molecules that are up-regulated in a wide variety of human cancers, including colon, breast, and lung. When over-expressed in a number of cellular systems, these molecules are capable of inhibiting cellular differentiation and anoikis, as well as disrupting cell polarization and tissue architecture, thus increasing tumorigenicity. The present study shows that perturbation of the major fibronectin receptor, integrin alpha5beta1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co-cluster with integrin alpha5beta1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin alpha5beta1 receptor to its ligand fibronectin, without changing its cell surface levels, resulting in increased adhesion of CEA/CEACAM6-expressing cells to fibronectin. More tenacious binding of free fibronectin to cells led to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. Disruption of this process with specific monoclonal antibodies against either fibronectin or integrin alpha5beta1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells.     

Laminin alpha1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alpha2beta1

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.