Specific overproduction of very late antigen 1 integrin in two human neuroblastoma cell lines selected for resistance to detachment by an Arg-Gly-Asp-containing synthetic peptide

Very late antigen (VLA) 1 is a member of the family of integral plasma-membrane glycoproteins known as integrins. It is a heterodimer composed of an alpha subunit of Mr 200,000, noncovalently associated with a beta subunit of Mr 110,000 which is shared by other VLA molecules (VLA-2-5). Unlike most of the other VLA proteins which have been shown to be receptors for various extracellular matrix proteins, the ligand for VLA-1 is unknown. Utilizing polyclonal antisera against the human fibronectin receptor as well as alpha subunit-specific monoclonal antibodies and cDNA probes, we have been able to demonstrate that in two human neuroblastoma cell lines, IMR-32 and SK-N-SH, the common beta subunit is associated with alpha 1, alpha 2, alpha 3, and alpha 5 subunits. By culturing these two cell lines in the presence of a synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, which contains the Arg-Gly-Asp cell attachment promotion tripeptide, we have isolated variant cell lines resistant to the detachment effects of this peptide. Peptide-resistant SK-N-SH and IMR-32 neuroblastoma cells exhibit weaker attachment to type I collagen and laminin, but a similar level of attachment to fibronectin as compared to the parental cells. Although the peptide-resistant variant cell lines proliferate at a rate similar to that of the parental cell lines, they stably overproduce (up to 20-fold) the alpha 1 subunit (VLA-1) specifically; and in the IMR-32 variant cells, the common beta 1 subunit is also overproduced. The level of expression of alpha 2 and alpha 3 subunits, however, is considerably reduced and that of the alpha 5 subunit is unchanged relative to the parental cells. These data suggest that the expression of integrin alpha subunits can be regulated differentially and independently of the beta subunit and that the VLA-1 heterodimer has an important function in mediating Arg-Gly-Asp-dependent cell adhesion or other phenotypic properties in human neuroblastoma cells.     

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.     

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.     

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.     

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.     

Analysis of the role of glycosylation of the human fibronectin receptor

1-Deoxymannojirimycin (MNJ), an inhibitor of Golgi alpha-mannosidase IA and IB, was used to assess the possible roles of asparagine-linked oligosaccharides in the structure and function of the integrin fibronectin receptor from cultured human fibroblasts. These cells normally attach well to fibronectin substrates and have only mature forms of the fibronectin receptor on their surfaces. MNJ inhibits the intracellular trimming of high mannose oligosaccharides, and cells treated with 0.2 mg/ml MNJ synthesize only immature precursor forms of both the alpha and beta subunits of the fibronectin receptor. The immature receptor polypeptides were found to be nonfunctional by two criteria: 1) cells treated with MNJ attached poorly to fibronectin substrates; and 2) receptor from the treated cells was defective in binding to fibronectin affinity columns. The precursor forms of the fibronectin receptor subunits were found on the surfaces of cells treated with MNJ, demonstrating that processing of receptor carbohydrates to mature forms was not necessary for receptor insertion into the plasma membrane. A monoclonal antibody that specifically bound the alpha subunit of the fibronectin receptor immunoprecipitated both alpha and beta subunit polypeptides from both control cells and cells treated with MNJ. Similarly, a monoclonal antibody that specifically bound only the beta subunit also immunoprecipitated both alpha and beta subunit polypeptides of the receptor from extracts of both control and MNJ-treated cells. These results indicate that receptor assembly can occur in the absence of complete oligosaccharide processing. Thus, oligosaccharide processing to the mature form of the fibronectin receptor is important for its binding function but not for receptor assembly or insertion into the plasma membrane.     

Arg-Gly-Asp-containing peptides expose novel collagen receptors on fibroblasts: implications for wound healing.

Integrins are a family of cell-surface receptors intimately involved in the interactions of cells with their extracellular matrix. These receptors comprise an alpha and beta subunit in noncovalent association and many have been shown to recognize and bind an arginine-glycine-aspartate (RGD) sequence contained within their specific extracellular matrix ligand. Fibroblasts express integrin receptors belonging to two major subfamilies. Some of the members within the subfamily defined by beta 1 (VLA) are receptors for collagen but, perhaps surprisingly, the other major subfamily of integrins on fibroblasts--that defined by the alpha chain of the vitronectin receptor, alpha v--all appear to bind primarily vitronectin and/or fibronectin. In the present study we show that RGD-containing peptides expose cryptic binding sites on the alpha v-associated integrins enabling them to function as collagen receptors. The addition of RGD-containing peptides to fibroblasts cultured on type I collagen induced dramatic cell elongation and, when the cells were contained within collagen matrices, the peptides induced marked contraction of the gels. These processes were inhibited by Fab fragments of a monoclonal antibody against an alpha v integrin. Also, alpha v-associated integrins from cell lysates bound to collagen I affinity columns in the presence, but not in the absence, of RGD-containing peptides. These data suggest a novel regulatory control for integrin function. In addition, because the cryptic collagen receptors were shown to be implicated in the contraction of collagen gels, the generation of such binding forces suggests that this may be the major biological role for these integrins in processes such as wound healing.     

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.     

Tumor necrosis factor alpha and interferon gamma modulate the expression of the vitronectin receptor (integrin beta 3) in human endothelial cells.

In this paper we show that tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) alter the expression of extracellular matrix receptors (integrins) in cultured human endothelial cells. Endothelial cells express at their surface integrins of the beta 1 and beta 3 groups that include receptors for fibronectin, collagen, laminin, and vitronectin. After treatment for 72 h with a combination of TNF alpha and IFN gamma, the level of the vitronectin receptor (alpha v beta 3) at the cell surface decreases by 70%, whereas the amounts of the beta 1 integrins remain unchanged. The decreased expression of the alpha v beta 3 complex at the cell surface is due to a selective effect of TNF alpha and IFN gamma on the regulation of the beta 3 subunit synthesis at the translational level. In fact, although the steady state levels of the mRNA for the beta 3 subunit are comparable in control and treated cells, the overall synthesis of the beta 3 subunit is decreased by a factor of 70%. No significant alteration of the synthesis of the companion alpha v subunit is detectable in cytokine-treated cells. As a consequence of the decreased expression of the receptor, cytokine-treated cells show decreased ability to adhere to vitronectin but adhere normally to fibronectin. These data show that two important inflammatory mediators, TNF alpha and IFN gamma, can modify the interaction of endothelial cells with the extracellular matrix by selectively altering the expression of specific cell surface integrin complexes.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

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)     

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.     

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.     

Evidence that an RGD-dependent receptor mediates the binding of oligodendrocytes to a novel ligand in a glial-derived matrix

A simple adhesion assay was used to measure the interaction between rat oligodendrocytes and various substrata, including a matrix secreted by glial cells. Oligodendrocytes bound to surfaces coated with fibronectin, vitronectin and a protein component of the glial matrix. The binding of cells to all of these substrates was inhibited by a synthetic peptide (GRGDSP) modeled after the cell-binding domain of fibronectin. The component of the glial matrix responsible for the oligodendrocyte interaction is a protein which is either secreted by the glial cells or removed from serum by products of these cultures; serum alone does not promote adhesion to the same extent as the glial-derived matrix. The interaction of cells with this glial-derived matrix requires divalent cations and is not mediated by several known RGD-containing extracellular proteins, including fibronectin, vitronectin, thrombospondin, type I and type IV collagen, and tenascin.     

Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human epidermal keratinocytes and non-differentiating keratinocytes

We have compared the adhesive properties and integrin expression profiles of cultured human epidermal keratinocytes and a strain of nondifferentiating keratinocytes (ndk). Both cell types adhered to fibronectin, laminin, and collagen types I and IV, but ndk adhered more rapidly and at lower coating concentrations of the proteins. Antibody blocking experiments showed that adhesion of both cell types to fibronectin was mediated by the alpha 5 beta 1 integrin and to laminin by alpha 3 beta 1 in synergy with alpha 2 beta 1. Keratinocytes adhered to collagen with alpha 2 beta 1, but an antibody to alpha 2 did not inhibit adhesion of ndk to collagen. Both cell types adhered to vitronectin by alpha v-containing integrins. Immunoprecipitation of surface-iodinated and metabolically labeled cells showed that in addition to alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1, both keratinocytes and ndk expressed alpha 6 beta 4 and alpha v beta 5. ndk expressed all these integrins at higher levels than normal keratinocytes. ndk, but not normal keratinocytes, expressed alpha v beta 1 and alpha v beta 3; they also expressed alpha 1 beta 1, an integrin that was not consistently detected on normal keratinocytes. Immunofluorescence experiments showed that in stratified cultures of normal keratinocytes integrin expression was confined to cells in the basal layer; terminally differentiating cells were unstained. In contrast, all cells in the ndk population were integrin positive. Our observations showed that the adhesive properties of ndk differ from normal keratinocytes and reflect differences in the type of integrins expressed, the level of expression and the distribution of integrins on the cell surface. ndk thus have a number of characteristics that distinguish them from normal basal keratinocytes.     

Requirement of the integrin beta 3 subunit for carcinoma cell spreading or migration on vitronectin and fibrinogen

FG human pancreatic carcinoma cells use integrin alpha v beta 5 as their primary vitronectin receptor since they fail to express integrin alpha v beta 3. These cells are unable to form focal contacts, spread, or migrate on vitronectin but readily do so on collagen in a beta 1 integrin-dependent manner. Transfection of FG cells with a cDNA encoding the integrin beta 3 subunit results in the surface expression of a functional integrin alpha v beta 3 heterodimer providing these cells with novel adhesive and biological properties. Specifically, FG cells expressing beta 3 acquire the capacity to attach and spread on vitronectin as well as fibrinogen with beta 3 localization to focal contacts. Moreover, these cells gain the capacity to migrate through a porous membrane in response to either vitronectin or fibrinogen. These results demonstrate that the beta 3 and beta 5 integrin subunits when associated with alpha v, promote distinct cellular responses to a vitronectin extracellular environment.     

A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C.

We demonstrate that a novel integrin beta subunit is present in association with the vitronectin receptor (VNR) alpha subunit on the surface of MG-63 human osteosarcoma cells. This beta subunit and the glycoprotein IIIa beta subunit (beta 3) were both found complexed with VNR alpha on MG-63 cells and in at least two other human cell types we examined. Tryptic peptide mapping indicated that the two beta subunits are related but distinct. The novel beta chain, referred to here as beta s, was not recognized by the monoclonal antibody AP3, which recognizes GPIIIa, nor by an antiserum raised against a peptide from the COOH-terminal cytoplasmic domain of beta 3. Both receptor complexes bound to and were specifically eluted from a column containing the cell adhesion peptide GRGDSP. The unique beta subunit became phosphorylated at high stoichiometry when MG-63 cells or AG1523 human fibroblasts were treated with the phorbol-ester tumor promoter phorbol 12-myristate 13-acetate. This phosphorylation occurred mainly on serine and probably at one major site, as determined by phosphotryptic peptide mapping. Protein kinase C phosphorylated the beta s subunit of intact receptor in vitro, at the same site phosphorylated in treated cells, indicating that protein kinase C is likely to be responsible for this phosphorylation in vivo.     

Beta 8 integrins mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin.

Integrins are major receptors used by cells to interact with extracellular matrices. In this paper, we identify the first ligands for the beta 8 family of integrins, presenting evidence that integrin heterodimers containing the beta 8 subunit mediate interactions of chick sensory neurons with laminin-1, collagen IV, and fibronectin. A polyclonal antibody, anti-beta 8-Ex, was prepared to a bacterial fusion protein expressing an extracellular portion of the chicken beta 8 subunit. In nonreducing conditions, this antibody immunoprecipitated from surface-labeled embryonic dorsal root ganglia neurons a M(r) 100 k protein, the expected M(r) of the beta 8 subunit, and putative alpha subunit(s) of M(r) 120 k. Affinity-purified anti-beta 8-Ex strongly inhibited sensory neurite outgrowth on laminin-1, collagen IV, and fibronectin-coated substrata. Binding sites were identified in a heat-resistant domain in laminin-1 and in the carboxyl terminal, 40-kDa fibronectin fragment. On substrates coated with the carboxyl terminal fragment of fibronectin, antibodies to beta 1 and beta 8 were only partially effective alone, but were completely effective in combination, at inhibiting neurite outgrowth. Results thus indicate that the integrin beta 8 subunit in association with one or more alpha subunits forms an important set of extracellular matrix receptors on sensory neurons.     

Basic fibroblast growth factor modulates integrin expression in microvascular endothelial cells.

During angiogenesis capillary endothelial cells undergo a coordinated set of modifications in their interactions with extracellular matrix components. In this study we have investigated the effect of the prototypical angiogenic factor basic fibroblast growth factor (bFGF) on the expression and function of several integrins in microvascular endothelial cells. Immunoprecipitation experiments with antibodies to individual subunits indicated that microvascular cells express at their surface several integrins. These include the alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1 laminin/collagen receptors; the alpha 6 beta 1 laminin receptor; the alpha 5 beta 1 and alpha v beta 1 fibronectin receptors; the alpha 6 beta 4 basement membrane receptor; and the alpha v beta 3 and alpha v beta 5 vitronectin receptors. Treatment with bFGF caused a significant increase in the surface expression of the alpha 2 beta 1, alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1, alpha 6 beta 4, and alpha v beta 5 integrins. In contrast, the level of expression of the alpha 1 beta 1 and alpha v beta 3 integrins was decreased in bFGF-treated cells. Immunoprecipitation of metabolically labeled cells indicated that bFGF increases the biosynthesis of the alpha 3, alpha 5, alpha 6, beta 4, and beta 5 subunits and decreases the production of the alpha v and beta 3 subunits. These results suggest that bFGF modulates integrin expression by altering the biosynthesis of individual alpha or beta subunits. In accordance with the upregulation of several integrins observed in bFGF-treated cells, these cells adhered better to fibronectin, laminin, vitronectin, and type I collagen than did untreated cells. The largest differences in beta 1 integrin expression occurred approximately 72 h after exposure to bFGF, at a time when the expression of the endothelial cell-to-cell adhesion molecule endoCAM was also significantly upregulated. In contrast, a shorter exposure to bFGF (24-48 h) was required for the maximal induction of plasminogen activator production in the same cells. Taken together, these results show that bFGF causes significant changes in the level of expression and function of several integrins in microvascular endothelial cells.     

Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.