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.     

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.     

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.     

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.     

Alpha v beta 3 and alpha v beta 5 integrins and their role in muscle precursor cell adhesion

BACKGROUND INFORMATION: Functional adaptation of skeletal muscle is a requirement for different muscle groups (e.g. craniofacial, ocular and limb) to undergo site-specific changes. Such tissue remodelling depends on dynamic interactions between muscle cells and their extracellular matrix, via participation of multifunctional molecules such as integrins. In view of data suggesting a role in fundamental muscle biology and muscle development in other systems, the present study has focused on expression and function of alpha v integrins, in cultured adult human craniofacial muscle (masseter) precursor cells and myotubes, and the predominantly fibroblastic IC (interstitial cells) population. RESULTS AND CONCLUSIONS: Flow-cytometric phenotyping and immunofluorescence phenotyping show that alpha v, alpha v beta 3 and alpha v beta 5 are expressed in all mononuclear cells (muscle precursors and IC) seeded on muscle extracellular molecules such as gelatin, VN (vitronectin) and FN (fibronectin). In this system, blockade of alpha v activity using a function-perturbing antibody abrogates cell migration on VN and FN. alpha v integrins act predominantly as VN receptors as cell-substrate attachment is diminished when alpha v neutralizing agents are introduced into cultures seeded on VN, and this inhibition is reversible; these integrins also appear to be minor FN receptors. These results demonstrate that the alpha v subset of integrins present on both myogenic precursors and IC is an essential cohort of VN and, to a lesser extent, FN receptors mediating cell adhesion and, either directly or indirectly, arbiters of cell motility.     

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 CK2 phosphorylation of vitronectin. Promotion of cell adhesion via the alpha(v)beta 3-phosphatidylinositol 3-kinase pathway

Phosphorylation of vitronectin (Vn) by casein kinase II was previously shown to occur at Thr50 and Thr57 and to augment a major physiological function of vitronectin-cell adhesion and spreading. Here we show that this phosphorylation increases cell adhesion via the alpha(v)beta3 (not via the alpha(v)beta5 integrin), suggesting that alpha(v)beta3 differs from alpha(v)beta5 in its biorecognition profile. Although both the phospho (CK2-PVn) and non-phospho (Vn) analogs of vitronectin (simulated by mutants Vn(T50E,T57E), and Vn(T50A,T57A), respectively) trigger the alpha(v)beta3 as well as the alpha(v)beta5 integrins, and equally activate the ERK pathway, these two forms are different in their activation of the focal adhesion kinase/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) pathway. Specifically, we show (i) that, upon exposure of cells to Vn/CK2-PVn, their PKB activation depends on the availability of the alpha(v)beta3 integrin on their surface; (ii) that upon adhesion of the beta3-transfected cells onto the CK2-PVn, the extent of PKB activation coincides with the enhanced adhesion of these cells, and (iii) that both the PKB activation and the elevation in the adhesion of these cells is PI3K-dependent. The occurrence of a cell surface receptor that specifically distinguishes between a phosphorylated and a non-phosphorylated analog of Vn, together with the fact that it preferentially activates a distinct intra-cellular signaling pathway, suggest that extra-cellular CK2 phosphorylation may play an important role in the regulation of cell adhesion and migration.     

Induction of alpha v beta 3 integrin-mediated attachment to extracellular matrix in beta 1 integrin (CD29)-negative B cell lines.

beta 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the beta 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the beta 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the alpha v beta 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the alpha v beta 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ alpha v beta 3, and these lines similarly lacked expression of beta 1 integrin. These results indicate that alpha v beta 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated alpha v beta 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.     

Distinct ligand-binding modes for integrin alpha(v)beta(3)-mediated adhesion to fibronectin versus vitronectin.

Cell surface integrins can adopt distinct conformations in response to ligand binding and intracellular signals. Several integrins including alpha(v)beta(3) can bind to multiple ligands. The binding of alpha(v)beta(3) to fibronectin and vitronectin was used as a model to determine whether the same or distinct forms of the receptor were utilized in strong binding to the two different ligands. A spinning-disc device was used to measure the relative strength of the alpha(v)beta(3)-ligand bonds. The initial binding reaction for both ligands occurred in the absence of metabolic energy and resulted in a strong adhesion to fibronectin but a weak adhesion to vitronectin. Increases in the strength of the alpha(v)beta(3)-vitronectin bond required phosphorylation of the beta(3) cytoplasmic domain, intracellular signals, and the binding of cytoskeletal proteins to cytoplasmic domains of beta(3) controlled by Tyr-747 and Tyr-759. In contrast, alpha(v)beta(3)-mediated adhesion to fibronectin was unaffected by phorbol 12-myristate 13-acetate, mutations of Tyr-747 and Tyr-759 to phenylalanine, or availability of metabolic energy. This suggests that strong adhesion to fibronectin used the initial binding conformation, whereas strong binding to vitronectin required signaling-induced changes in the conformation of alpha(v)beta(3).     

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.     

P21-activated kinase 4 interacts with integrin alpha v beta 5 and regulates alpha v beta 5-mediated cell migration

p21-activated kinase 1 (PAK1) can affect cell migration (Price et al., 1998; del Pozo et al., 2000) and modulate myosin light chain kinase and LIM kinase, which are components of the cellular motility machinery (Edwards, D.C., L.C. Sanders, G.M. Bokoch, and G.N. Gill. 1999. Nature Cell Biol. 1:253-259; Sanders, L.C., F. Matsumura, G.M. Bokoch, and P. de Lanerolle. 1999. SCIENCE: 283:2083-2085). We here present a novel cell motility pathway by demonstrating that PAK4 directly interacts with an integrin intracellular domain and regulates carcinoma cell motility in an integrin-specific manner. Yeast two-hybrid screening identified PAK4 binding to the cytoplasmic domain of the integrin beta 5 subunit, an association that was also found in mammalian cells between endogenous PAK4 and integrin alpha v beta 5. Furthermore, we mapped the PAK4 binding to the membrane-proximal region of integrin beta 5, and identified an integrin-binding domain at aa 505-530 in the COOH terminus of PAK4. Importantly, engagement of integrin alpha v beta 5 by cell attachment to vitronectin led to a redistribution of PAK4 from the cytosol to dynamic lamellipodial structures where PAK4 colocalized with integrin alpha v beta 5. Functionally, PAK4 induced integrin alpha v beta 5-mediated, but not beta1-mediated, human breast carcinoma cell migration, while no changes in integrin cell surface expression levels were observed. In conclusion, our results demonstrate that PAK4 interacts with integrin alpha v beta 5 and selectively promotes integrin alpha v beta 5-mediated cell migration.     

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 neural cell adhesion molecule L1 and rat homologue NILE are ligands for integrin alpha v beta 3

Integrin alpha v beta 3 is distinct in its capacity to recognize the sequence Arg-Gly-Asp (RGD) in many extra-cellular matrix (ECM) components. Here, we demonstrate that in addition to the recognition of ECM components, alpha v beta 3 can interact with the neural cell adhesion molecule L1-CAM; a member of the immunoglobulin superfamily (IgSF). M21 melanoma cells displayed significant Ca(++)-dependent adhesion and spreading on immunopurified rat L1 (NILE). This adhesion was found to be dependent on the expression of the alpha v-integrin subunit and could be significantly inhibited by an antibody to the alpha v beta 3 heterodimer. M21 cells also displayed some alpha v beta 3-dependent adhesion and spreading on immunopurified human L1. Ligation between this ligand and alpha v beta 3 was also observed to promote significant haptotactic cell migration. To map the site of alpha v beta 3 ligation we used recombinant L1 fragments comprising the entire extracellular domain of human L1. Significant alpha v beta 3-dependent adhesion and spreading was evident on a L1 fragment containing Ig-like domains 4, 5, and 6. Importantly, mutation of an RGD sequence present in the sixth Ig-like domain of L1 abrogated M21 cell adhesion. We conclude that alpha v beta 3-dependent recognition of human L1 is dependent on ligation of this RGD site. Despite high levels of L1 expression the M21 melanoma cells did not display significant adhesion via a homophilic L1-L1 interaction. These data suggest that M21 melanoma cells recognize and adhere to L1 through a mechanism that is primarily heterophilic and integrin dependent. Finally, we present evidence that melanoma cells can shed and deposit L1 in occluding ECM. In this regard, alpha v beta 3 may recognize L1 in a cell-cell or cell- substrate interaction.     

Substrate specificity of alpha(v)beta(3) integrin-mediated cell migration and phosphatidylinositol 3-kinase/AKT pathway activation

The alpha(v)beta(3) integrin has been shown to bind several ligands, including osteopontin and vitronectin. Its role in modulating cell migration and downstream signaling pathways in response to specific extracellular matrix ligands has been investigated in this study. Highly invasive prostate cancer PC3 cells that constitutively express alpha(v)beta(3) adhere and migrate on osteopontin and vitronectin in an alpha(v)beta(3)-dependent manner. However, exogenous expression of alpha(v)beta(3) in noninvasive prostate cancer LNCaP (beta(3)-LNCaP) cells mediates adhesion and migration on vitronectin but not on osteopontin. Activation of alpha(v)beta(3) by epidermal growth factor stimulation is required to mediate adhesion to osteopontin but is not sufficient to support migration on this substrate. We show that alpha(v)beta(3)-mediated cell migration requires activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB/AKT) pathway since wortmannin, a PI 3-kinase inhibitor, prevents PC3 cell migration on both osteopontin and vitronectin; furthermore, alpha(v)beta(3) engagement by osteopontin and vitronectin activates the PI 3-kinase/AKT pathway. Migration of beta(3)-LNCaP cells on vitronectin also occurs through activation of the PI 3-kinase pathway; however, AKT phosphorylation is not increased upon engagement by osteopontin. Furthermore, phosphorylation of focal adhesion kinase (FAK), known to support cell migration in beta(3)-LNCaP cells, is detected on both substrates. Thus, in PC3 cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin and osteopontin; in beta(3)-LNCaP cells, alpha(v)beta(3) mediates cell migration and PI 3-kinase/AKT pathway activation on vitronectin, whereas adhesion to osteopontin does not support alpha(v)beta(3)-mediated cell migration and PI 3-kinase/AKT pathway activation. We conclude therefore that alpha(v)beta(3) exists in multiple functional states that can bind either selectively vitronectin or both vitronectin and osteopontin and that can differentially activate cell migration and intracellular signaling pathways in a ligand-specific manner.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.     

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.     

Individual embryonic fibroblasts express multiple beta chains in association with the alpha v integrin subunit. Loss of beta 3 expression with cell confluence.

The alpha chain of the vitronectin receptor, alpha v, has been found in association with the integrin subunits beta 1, beta 3, or beta 5 on different cell types. We show here that cultured embryonic fibroblasts simultaneously display alpha v beta 3, alpha v beta 1, and alpha v in association with two other beta subunits, one of which is probably beta 5. Polymerase chain reaction analysis of single cells isolated by micromanipulation identified mRNA for alpha v, beta 1, beta 3, and beta 5 in six of eight clones. Immunoprecipitation of iodinated cell surface proteins with a monoclonal antibody to alpha v indicated that the relative proportions of the different beta chains in association with alpha v varied, particularly between two different cell lines. The cytokines platelet-derived growth factor, transforming growth factor beta 1, and tumor necrosis factor alpha did not appear to alter this ratio although tumor necrosis factor alpha increased the surface expression of the alpha v-associated integrins; but overnight culture in basic fibroblast growth factor caused a lower expression of alpha v beta 1 and alpha v beta 5 with no reduction in alpha v beta 3 expression. When the cell cultures were grown to complete confluence, surface expression of beta 3 was abolished, and the expression of an unknown beta chain (beta u) became more prominent. This effect was not overcome by culturing confluent cells with basic fibroblast growth factor. Affinity column chromatography showed that alpha v beta 5 bound to vitronectin but alpha v beta 1 did not, whereas alpha v beta 1 but not alpha v beta 5 bound to fibronectin. These results suggest that, on individual cells, the beta subunits found in association with alpha v may vary according to the proliferative capacity of the cell and that the promiscuous beta 3 subunit is progressively replaced by beta subunits of individual ligand specificity.     

Adhesive properties of the beta 3 integrins: comparison of GP IIb-IIIa and the vitronectin receptor individually expressed in human melanoma cells

Glycoprotein IIb-IIIa (alpha IIb beta 3) and the vitronectin receptor (alpha v beta 3), two integrins that share the common beta 3 subunit, have been reported to function as promiscuous receptors for the RGD-containing adhesive proteins fibrinogen, vitronectin, fibronectin, von Willebrand factor, and thrombospondin. The present study was designed to establish a cell system for the expression of either GP IIb-IIIa or the vitronectin receptor in an otherwise identical cellular environment and to compare the adhesive properties of these two integrins with those of native GP IIb-IIIa and the vitronectin receptor constitutively expressed in HEL cells or platelets. M21 human melanoma cells lack GP IIb-IIIa and use the vitronectin receptor to attach to vitronectin, fibrinogen, fibronectin, and von Willebrand factor. To study the functional properties of GP IIb-IIIa in these cells, we transfected GP IIb into M21-L cells, a variant of M21 cells (Cheresh, D.A., and R.C. Spiro. 1987. J. Biol. Chem. 262:17703-17711), which lack the expression of functional alpha v and are therefore unable to attach to vitronectin, fibrinogen, and von Willebrand factor. Transfectants expressing GP IIb were isolated by immunomagnetic beads and surface expression of the GP IIb-IIIa complex was documented by FACS analysis and immunoprecipitation experiments performed with 125I-labeled M21-L/GP IIb cells. Comparative functional studies demonstrated that GP IIb-IIIa expressed in M21-L/GPIIb cells as well as native GP IIb-IIIa constitutively expressed in HEL-5J20 cells (an HEL variant lacking alpha v beta 3) mediated cell attachment to immobilized fibrinogen, but not to vitronectin or von Willebrand factor, whereas the vitronectin receptor expressed in M21 cells and HEL-AD1 cells (an HEL variant expressing alpha v beta 3) mediated cell attachment to fibrinogen, vitronectin, and von Willebrand factor. Similarly, PGl2-treated resting platelets attached to immobilized fibrinogen but not to vitronectin or von Willebrand factor, and this attachment could be inhibited by mAb A2A9 (directed against a functional site on the GP IIb-IIIa complex). However, in contrast to platelets, which adhered to vitronectin and von Willebrand factor after stimulation by thrombin or PMA, activation of the protein kinase C pathway in M21-L/GP IIb or HEL cells did not induce cell adhesion to vitronectin or von Willebrand factor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterisation of alpha3beta1 and alpha(v)beta3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of beta1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, alpha3beta1 and alpha(v)beta3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with beta1,6-branches and short polylactosamine chains. In WM9 cells, alpha3beta1 integrin was more variously glycosylated than alpha(v)beta3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and alpha3beta1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of alpha(v)beta3 integrin glycans in melanoma or in any cancer cells.