Characterization of the integrin alpha v beta 6 as a fibronectin-binding protein.

Integrins are a complex family of divalent cation-dependent cell adhesion receptors composed of one alpha and one beta subunit noncovalently bound to one another. A subset of integrins contains the alpha v subunit in association with one of several beta subunits (e.g. beta 3, beta 5, beta 1). We have recently identified a novel integrin beta subunit, beta 6, that is present in a number of epithelial cell lines. Using a polyclonal antibody raised against the carboxyl-terminal peptide of beta 6, we have now identified the integrin heterodimer, alpha v beta 6, on the surface of two human carcinoma cell lines. Using affinity chromatography of lysates from the pancreatic carcinoma cell line, FG-2, we demonstrate that alpha v beta 6 binds to fibronectin, but not to vitronectin or collagen I. In contrast, the alpha v beta 5 integrin, which is also expressed on FG-2 cells, binds exclusively to vitronectin. Immobilized collagen I does not interact with alpha v integrins, but binds beta 1-containing integrins. Both alpha v beta 6 and alpha v beta 5 are eluted from their respective immobilized ligands by a hexa-peptide containing the sequence Arg-Gly-Asp (RGD). RGD is highly effective in the presence of Ca2+, somewhat less effective in Mg2+, and virtually inactive in Mn2+. These results suggest that alpha v beta 6 functions as an RGD-dependent fibronectin receptor in FG-2 carcinoma cells. In agreement with this notion, cell adhesion assays show that FG-2 cell attachment to fibronectin is only partially inhibited by anti-beta 1 integrin antibodies, implying that other fibronectin receptors may be involved. Taken together with recent reports on the vitronectin receptor function of alpha v beta 5, our results suggest that the previously described carcinoma cell integrin, alpha v beta x (Cheresh, D. A., Smith, J. W., Cooper, H. M., and Quaranta, V. (1989) Cell 57, 59-69), is a mixture of at least two different receptors: alpha v beta 5, mediating adhesion to vitronectin, and alpha v beta 6, mediating adhesion to fibronectin.     

Characterization of zinc-alpha(2)-glycoprotein as a cell adhesion molecule that inhibits the proliferation of an oral tumor cell line.

Zn-alpha(2)-glycoprotein (Znalpha(2)gp) is a soluble protein widely distributed in body fluids and glandular epithelia. We have found it to be expressed in stratified epithelia as well. Znalpha(2)gp is clinically correlated with differentiation in various epithelial tumors, including oral and epidermal tumors. We have cloned epidermal Znalpha(2)gp and report the preparation of the recombinant protein in a Baculovirus expression system. Like the native molecule, recombinant Znalpha(2)gp has RNase activity. Znalpha(2)gp functions as a matrix protein for the Tu-138 oral squamous cell carcinoma cell line. Cell attachment to Znalpha(2)gp is comparable to that for fibronectin and is inhibited by the synthetic RGD peptides RGD, RGDV, and RGDS. Attachment is also inhibited by the antibody to integrin alpha(5)beta(1) (the fibronectin receptor), but not by antibodies to integrins alpha(v)beta(3), alpha(3)beta(1), and alpha(2)beta(1). We find that the proliferation of Tu-138 cells is inhibited on a Znalpha(2)gp matrix, as compared with other matrix proteins (fibronectin, vitronectin, laminin, and collagens I and IV) on which growth resembles that on the BSA control. We believe that the role of Znalpha(2)gp in differentiation and its RNase activity are two likely suspects as agents of the inhibition of proliferation.     

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.     

Integrin-associated protein immunoglobulin domain is necessary for efficient vitronectin bead binding

Integrin-associated protein (IAP/CD47) is physically associated with the alpha v beta 3 vitronectin (Vn) receptor and a functionally and immunologically related integrin on neutrophils (PMN) and monocytes. Anti-IAP antibodies inhibit multiple phagocyte functions, including Arg- Gly-Asp (RGD)-initiated activation of phagocytosis, chemotaxis, and respiratory burst; PMN adhesion to entactin; and PMN transendothelial and transepithelial migration at a step subsequent to tight intercellular adhesion. Anti-IAP antibodies also inhibit binding of Vn- coated particles to many cells expressing alpha v beta 3. However, prior studies with anti-IAP did not directly address IAP function because they could not distinguish between IAP blockade and antibody- induced signaling effects on cells. To better determine the function of IAP, we have characterized and used an IAP-deficient human cell line. Despite expressing alpha v integrins, these cells do not bind Vn-coated particles unless transfected with IAP expression constructs. Increasing the level of alpha v beta 3 expression or increasing Vn density on the particle does not overcome the requirement for IAP. All known splice variants of IAP restore Vn particle binding equivalently. Indeed, the membrane-anchored IAP Ig variable domain suffices to mediate Vn particle binding in this system, while the multiply membrane-spanning and cytoplasmic domains are dispensable. In all cases, adhesion to a Vn- coated surface and fibronectin particle binding through alpha 5 beta 1 fibronectin receptors are independent of IAP expression. These data demonstrate that some alpha v integrin ligand-binding functions are IAP independent, whereas others require IAP, presumably through direct physical interaction between its Ig domain and the 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.     

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.     

Motility of fibronectin receptor-deficient cells on fibronectin and vitronectin: collaborative interactions among integrins.

Cells are capable of adhering to and migrating on protein components of the extracellular matrix. These cell-matrix interactions are thought to be mediated largely through a family of cell surface receptors termed integrins. However, the manner in which individual integrins are involved in cell adhesion and motility has not been fully determined. To explore this issue, we previously selected a series of CHO variants that are deficient in expression of the integrin alpha 5 beta 1, the "classical" fibronectin receptor. Two sets of subclones of these variants were defined which respectively express approximately 20% or 2% of fibronectin receptor on the cell surface when compared to wild-type cells (Schreiner, C. L., J. S. Bauer, Y. N. Danilov, S. Hussein, M. M. Sczekan, and R. L. Juliano. 1989. J. Cell Biol. 109:3157-3167). In the current study, the variant clones were tested for haptotactic motility on substrata coated with fibronectin or vitronectin. Data from assays using fibronectin show that cellular motility of the 20% variants was substantially decreased (30-75% of wild type), while the motility of the 2% variants was nearly abolished (2-20% of wild type). Surprisingly, a similar pattern was seen for haptotactic motility of both 2% and 20% variants when vitronectin was used (approximately 20-30% of wild type). The reduced haptotactic motility of the fibronectin receptor-deficient variant clones on vitronectin was shown not to be due to reduced vitronectin receptor (alpha v beta 3) expression nor to a failure of these variants to adhere to vitronectin substrata. Transfection of the deficient variants with a cDNA for the human alpha 5 subunit resulted in normal levels of fibronectin receptor expression (as a human alpha 5/hamster beta 1 chimera) and restored the motility of the CHO variants on fibronectin and vitronectin. This indicates that expression of the alpha 5 subunit is required for normal haptotactic motility on vitronectin substrata and suggests that the fibronectin receptor (alpha 5 beta 1) plays a cooperative role with vitronectin receptors in cell motility.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Purification and characterization of mammalian integrins expressed by a rat neuronal cell line (PC12): evidence that they function as alpha/beta heterodimeric receptors for laminin and type IV collagen

Cells of the rat neuronal line, PC12, adhere well to substrates coated with laminin and type IV collagen, but attach poorly to fibronectin. Adhesion and neurite extension in response to these extracellular matrix proteins are inhibited by Fab fragments of an antiserum (anti-ECMR) that recognizes PC12 cell surface integrin subunits of Mr 120,000, 140,000, and 180,000 (Tomaselli, K. J., C. H. Damsky, and L. F. Reichardt. 1987. J. Cell Biol. 105:2347-2358). Here we extend our study of integrin structure and function in PC12 cells using integrin subunit-specific antibodies prepared against synthetic peptides corresponding to the cytoplasmic domains of the human integrin beta 1 and the fibronectin receptor alpha (alpha FN) subunits. Anti-integrin beta 1 immunoprecipitated a 120-kD beta 1 subunit and two noncovalently associated integrin alpha subunits of 140 and 180 kD from detergent extracts of surface-labeled PC12 cells. Immunodepletion studies using anti-integrin beta 1 demonstrated that these two putative alpha/beta heterodimers are identical to those recognized by the adhesion-perturbing ECMR antiserum. Anti-alpha FN immunoprecipitated fibronectin receptor heterodimers in human and rat fibroblastic cells, but not in PC12 cells. Thus, low levels of expression of the integrin alpha FN subunit can explain the poor attachment of PC12 cells to FN. The PC12 cell integrins were purified using a combination of lectin and ECMR antibody affinity chromatography. The purified integrins: (a) completely neutralize the ability of the anti-ECMR serum to inhibit PC12 cell adhesion to laminin and collagen IV; (b) have hydrodynamic properties that are very similar to those of previously characterized integrin alpha/beta heterodimeric receptors for ECM proteins; and (c) can be incorporated into phosphatidylcholine vesicles that then bind specifically to substrates coated with laminin or collagen IV but not fibronectin. Thus, the ligand-binding specificity of the liposomes containing the purified PC12 integrins closely parallels the substrate-binding preference of intact PC12 cells. These results demonstrate that mammalian integrins purified from a neuronal cell line can, when incorporated into lipid vesicles, function as receptors for laminin and type IV collagen.     

Activation of the alpha 4 beta 1 integrin through the beta 1 subunit induces recognition of the RGDS sequence in fibronectin

Lymphocyte attachment to fibronectin is mainly mediated by the interaction of alpha 5 beta 1 and alpha 4 beta 1 integrins with the RGD and CS-1/Hep II sites, respectively. We have recently shown that the anti-beta 1 mAb TS2/16 can convert the partly active alpha 4 beta 1 present on certain hemopoietic cells that recognizes CS-1 but not Hep II, to a high avidity form that binds both ligands. In this report we have studied whether mAb TS2/16 also affects alpha 4 beta 1 ligand specificity. Incubation of the B cell lines Ramos and Daudi (which lack alpha 5 beta 1) with mAb TS2/16 induced specific attachment to an 80-kD fragment which lacks CS-1 and Hep II and contains the RGD sequence. mAbs anti-alpha 4 and the synthetic peptides CS-1 and IDAPS inhibited adhesion to the 80-kD fragment thus implying alpha 4 beta 1 as the receptor for this fragment. Interestingly, the synthetic peptide GRGDSPC and a 15-kD peptic fibronectin fragment containing the RGD sequence also inhibited B cell adhesion to the 80-kD fragment. Because we have previously shown that RGD peptides do not affect the constitutive function of alpha 4 beta 1, we tested whether TS2/16- activated alpha 4 beta 1 acquired the capacity to recognize RGD. Indeed RGD peptides inhibited TS2/16-treated B cell adhesion to a 38-kD fragment containing CS-1 and Hep II but did not affect binding of untreated cells to this fragment. An anti-fibronectin mAb reactive with an epitope on or near the RGD sequence also efficiently inhibited cell adhesion to the 80-kD fragment, indicating that the RGD sequence is a novel adhesive ligand for activated alpha 4 beta 1. These results emphasize the role of alpha 4 beta 1 as a receptor with different ligand specificities according to the activation state, a fact that may be important for lymphocyte migration, localization, and function.     

Integrins alpha v beta 3 and alpha v beta 5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface

We investigated the role of the integrins alpha v beta 3 and alpha v beta 5 in mediating vitronectin adhesion of three phenotypically distinct cell types. M21 human melanoma cells and H2981 lung carcinoma cells use both alpha v-containing integrins in adhering to vitronectin while UCLA-P3 lung carcinoma cells adhere exclusively with alpha v beta 5. Specifically, monoclonal antibodies directed to functional epitopes on both receptors were required to block adhesion of M21 or H2981 cells while adhesion of UCLA-P3 cells to vitronectin could be blocked with a monoclonal antibody to alpha v beta 5. Although both receptors are involved in M21 and H2981 cell adhesion to vitronectin, only alpha v beta 3 can be detected in focal contacts, colocalizing with vinculin, talin, and the ends of actin filaments, while alpha v beta 5 shows a distinct, nonfocal contact, distribution on the cell surface. These results provide the first evidence that two homologous integrins that recognize the same ligand distribute differentially on the cell surface.     

Novel role of presenilins in maturation and transport of integrin beta 1

Presenilins (PSs) play important roles in modulating the trafficking and maturation of several membrane proteins. However, the target membrane proteins whose trafficking and maturation are regulated by PS are largely unknown. By characterizing PS-deficient fibroblasts, we found that integrin beta1 maturation is promoted markedly in PS1 and PS2 double-deficient fibroblasts and moderately in PS1- or PS2-deficient fibroblasts; in contrast, nicastrin maturation is completely inhibited in PS1 and PS2 double-deficient fibroblasts. Subcellular fractionation analysis demonstrated that integrin beta1 maturation is promoted in the Golgi apparatus. The mature integrin beta1 with an increased expression level was delivered to the cell surface, which resulted in an increased cell surface expression level of mature integrin beta1 in PS1 and PS2 double-deficient fibroblasts. PS1 and PS2 double-deficient fibroblasts exhibited an enhanced ability to adhere to culture dishes coated with integrin beta1 ligands, namely, fibronectin and laminin. The inhibition of gamma-secretase activity enhances neither integrin beta1 maturation nor the adhesion of wild-type cells. Moreover, PS deficiency also promoted the maturation of integrins alpha3 and alpha5 and the cell surface expression of integrin alpha3. Integrins alpha3 and alpha5 were coimmunoprecipitated with integrin beta1, suggesting the formation of the functional heterodimers integrins alpha3beta1 and alpha5beta1. Note that integrin beta1 exhibited features opposite those of nicastrin in terms of maturation and trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus in PS1 and PS2 double-deficient fibroblasts. Our results therefore suggest that PS regulates the maturation of membrane proteins in opposite directions and cell adhesion by modulating integrin maturation.     

A novel vitronectin receptor integrin (alpha v beta x) is responsible for distinct adhesive properties of carcinoma cells

Carcinoma cells express a novel integrin involved in cell adhesion to vitronectin, but not to fibrinogen or von Willebrand factor, whereas melanoma and endothelial cells express a vitronectin receptor (alpha v beta 3) that promotes cell attachment to all of these matrix components. The integrin responsible for this adhesive phenotype of carcinoma cells is composed of an alpha subunit that is indistinguishable from the alpha v of the vitronectin receptor and a beta subunit (beta x) that is distinct from any known integrin beta subunit. Accordingly, Northern blot analysis identifies an mRNA for alpha v, but not for beta 3 in carcinoma cells. This receptor appears to mediate cell adhesion to vitronectin as well as fibronectin since an antibody directed to its alpha subunit blocked carcinoma cell adhesion to both of these matrix proteins. These results suggest that homologous integrins with identical alpha subunits and structurally distinct beta subunits can account for the functional recognition of different matrixes by two cell types.     

The glycobiology of gametes and fertilization

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.     

Adhesion of a chicken myeloblast cell line to fibrinogen and vitronectin through a beta 1-class integrin.

The adhesive interactions of circulating blood cells are tightly regulated, receptor-mediated events. To establish a model for studies on regulation of cell adhesion, we have examined the adhesive properties of the HD11 chick myeloblast cell line. Function-perturbing antibodies were used to show that integrins containing the beta 1 subunit mediate HD11 cell attachment to several distinct extracellular matrix proteins, specifically fibronectin, collagen, vitronectin, and fibrinogen. This is the first evidence that an integrin heterodimer in the beta 1 family functions as a receptor for fibrinogen. While the alpha v beta 1 heterodimer has been shown to function as a vitronectin receptor on some cells, this heterodimer could not be detected on HD11 cells. Instead, results suggest that the beta 1 subunit associates with different, unidentified alpha subunit(s) to form receptors for vitronectin and fibrinogen. Results using function-blocking antibodies also demonstrate that on these cells, additional receptors for vitronectin are formed by alpha v beta 3 and alpha v associated with an unidentified 100-kD beta subunit. The adhesive interactions of HD11 cells with these extracellular matrix ligands were shown to be regulated by lipopolysaccharide treatment, making the HD11 cell line attractive for studies of mechanisms regulating cell adhesion. In contrast to primary macrophage which rapidly exhibit enhanced adhesion to laminin and collagen upon activation, activated HD11 cells exhibited reduced adhesion to most extracellular matrix constituents.     

Human parechovirus 1 utilizes integrins alphavbeta3 and alphavbeta1 as receptors

Human parechovirus 1 (HPEV1) displays an arginine-glycine-aspartic acid (RGD) motif in the VP1 capsid protein, suggesting integrins as candidate receptors for HPEV1. A panel of monoclonal antibodies (MAbs) specific for integrins alphavbeta3, alphavbeta1, and alphavbeta5, which have the ability to recognize the RGD motif, and also a MAb specific for integrin alpha2beta1, an integrin that does not recognize the RGD motif, were tested on A549 cells. Our results showed that integrin alphav-specific MAb reduced infectivity by 85%. To specify which alphav integrins the virus utilizes, we tested MAbs specific to integrins alphavbeta3 and alphavbeta1 which reduced infectivity significantly, while a MAb specific for integrin alphavbeta5, as well as the MAb specific for alpha2beta1, showed no reduction. When a combination of MAbs specific for integrins alphavbeta3 and alphavbeta1 were used, virus infectivity was almost completely inhibited; this shows that integrins alphavbeta3 and alphavbeta1 are utilized by the virus. We therefore proceeded to test whether alphav integrins' natural ligands fibronectin and vitronectin had an effect on HPEV1 infectivity. We found that vitronectin reduced significantly HPEV1 infectivity, whereas a combination of vitronectin and fibronectin abolished infection. To verify the use of integrins alphavbeta3 and alphavbeta1 as HPEV1 receptors, CHO cells transfected and expressing either integrin alphavbeta3 or integrin alphavbeta1 were used. It was shown that the virus could successfully infect these cells. However, in immunoprecipitation experiments using HPEV1 virions and allowing the virus to bind to solubilized A549 cell extract, we isolated and confirmed by Western blotting the alphavbeta3 heterodimer. In conclusion, we found that HPEV1 utilises both integrin alphavbeta3 and alphavbeta1 as receptors; however, in cells that express both integrins, HPEV1 may preferentially bind integrin alphavbeta3.     

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.     

Beta1 integrins are distributed in adhesion structures with fibronectin and caveolin and in coated pits.

Integrins are found in adhesion structures, which link the extracellular matrix to cytoskeletal proteins. Here, we attempt to further define the distribution of beta1 integrins in the context of their association with matrix proteins and other cell surface molecules relevant to the endocytic process. We find that beta1 integrins colocalize with fibronectin in fibrillar adhesion structures. A fraction of caveolin is also organized along these adhesion structures. The extracellular matrix protein laminin is not concentrated in these structures. The alpha4beta1 integrin exhibits a distinct distribution from other beta1 integrins after cells have adhered for 1 h to extracellular matrix proteins but is localized in adhesion structures after 24 h of adhesion. There are differences between the fibronectin receptors: alpha5beta1 integrins colocalize with adaptor protein-2 in coated pits, while alpha4beta1 integrins do not. This parallels our earlier observation that of the two laminin receptors, alpha1beta1 and alpha6beta1, only alpha1beta1 integrins colocalize with adaptor protein-2 in coated pits. Calcium chelation or inhibition of mitogen-activated protein kinase kinase, protein kinase C, or src did not affect localization of alpha1beta1 and alpha5beta1 integrins in coated pits. Likewise, the integrity of coated-pit structures or adhesion structures is not required for integrin and adaptor protein-2 colocalization. This suggests a robust and possibly constitutive interaction between these integrins and coated pits.     

Manganese ion elicits a binding activity of placenta vitronectin receptor to fibronectin cell-binding domain.

Some members of the integrin family recognize the RGD sequence which is common to cell adhesive proteins in a divalent cation-dependent manner. In the presence of Ca2+ and Mg2+, the fibronectin receptor of placenta recognizes the RGD sequence of fibronectin, but not that of vitronectin, while the vitronectin receptor of placenta recognizes the RGD sequence of vitronectin, but not that of fibronectin, although both receptors recognize the same RGD sequence. We have found by performing an enzyme-linked immunosorbent assay (ELISA) using receptor-specific monoclonal antibodies and by electrophoretic analysis that in the presence of Mn2+ a vitronectin receptor of placenta binds to an affinity column coupled with the cell-binding domain of fibronectin. By replacing divalent cations from Mn2+ to Ca2+ and Mg2+, the vitronectin receptor was completely eluted from the column. When the synthetic peptides GRGDSP and GRGESP were applied to the column as competitors, the Mn(2+)-dependent binding was inhibited by both peptides. These results suggest that Mn2+ elicits a binding activity of the placenta vitronectin receptor to the RGD site of fibronectin. The modulation of ligand specificity by Mn2+ will provide an important clue in the elucidation of the cause of individual ligand specificity of RGD-recognizing integrins.     

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) recognizes multiple sites in fibronectin.

The binding of fibronectin (Fn) to several integrins involves the Arg-Gly-Asp (RGD) tripeptide sequence. However, linear synthetic RGD peptides do not completely mimic the cell attachment activity of intact Fn or certain large Fn fragments. This suggests that the integrin-Fn interaction involves a more extended surface of Fn than that provided by the RGD sequence. To test this possibility, three novel monoclonal anti-Fn antibodies that inhibit its binding to a purified integrin, alpha IIb beta 3, were developed. The epitopes of these three antibodies mapped to a region at least 55 residues amino-terminal of the RGD sequence. Further, recombinant fragments of Fn containing these epitopes and lacking the RGD site also inhibited the binding of Fn to purified alpha IIb beta 3. These fragments, which spanned Fn residues 1359-1436, bound to alpha IIb beta 3 in a divalent cation-dependent manner. In addition, this region of Fn bound specifically to alpha IIb beta 3 on thrombin-stimulated but not resting platelets. These results demonstrate the presence of additional sequences in Fn that interact with integrin alpha IIb beta 3 and suggest that multiple sites in Fn are involved in its recognition by this integrin.