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PubMed1988

Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain.

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.

Binding Sites↗
PubMed1994

Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.

Urokinase receptors are distributed on surfaces of many cell types where they are thought to focus plasminogen-dependent proteolysis important to migration and tissue remodeling to the immediate pericellular space. In addition to its well characterized role in proteolysis, urokinase receptor binding per se promotes the adhesiveness of leukemic cell lines exposed to differentiating cytokines in vitro. We sought to determine if a serum or matrix component is involved in urokinase-dependent adhesion. We now report that cytokine-stimulated human myelomonocytic cells express a divalent cation- and Arg-Gly-Asp-independent high affinity receptor for urea-purified vitronectin (Kd < 10 nM). Soluble native vitronectin does not effectively bind to the receptor, while cellular adhesion was noted to both urea-purified and native vitronectin when adsorbed to plastic. The activity of this receptor is tightly coupled to urokinase receptor occupancy. Urokinase receptor binding thus induces selective and reversible cellular adhesion to the matrix form of vitronectin. Because transfer of vitronectin-bound plasminogen activator inhibitor type 1 to urokinase promotes rapid turnover of receptor-bound enzyme, these results illuminate a novel binding cycle by which urokinase receptor occupancy coordinately regulates cellular adhesiveness and pericellular proteolysis.

Adenocarcinoma↗
PubMed1994

Relative topography of biologically active domains of human vitronectin. Evidence from monoclonal antibody epitope and denaturation studies.

A panel of monoclonal antibodies to human vitronectin was used to define some epitopes for the multifunctions of this protein. Separate antibodies were identified which strongly inhibited cell spreading activity and which prevented binding to collagen. A third group interfered with the ability of vitronectin to inhibit complement-mediated guinea pig erythrocyte reactive lysis. None of the antibodies from these three groups prevented heparin binding, providing evidence that this reaction occurs at a fourth location; a different monoclonal antibody partially inhibited the binding of heparin. The relative accessibility of each biologically active epitope was assessed by the differential binding of the monoclonals to native and denatured vitronectin. Reactivity of the antibody which inhibited heparin binding greatly increased upon denaturation of vitronectin, implying that this region is normally inaccessible in the native form of the molecule. By contrast, epitopes for cell spreading, collagen binding, and inhibition of terminal complement complex lysis were destroyed by denaturation. On the basis of denaturation data and epitope mapping by competitive exclusion of monoclonal antibodies, a Venn diagram was constructed to represent overlap of monoclonal antibody epitopes in the tertiary structure. Linear epitopes for the antibodies were identified using cyanogen bromide and plasmin-derived peptides from vitronectin. The antibody which strongly inhibited cell binding reacted with a region containing the RGD site, whereas linear epitopes for collagen binding and complement inhibition appeared to reside in a 43-kDa peptide representing the internal region of the amino acid sequence, excluding the heparin-binding site. The latter two epitopes were differentiated from each other by reactivity of the antibody which inhibited collagen binding toward a smaller 20-kDa plasmin-derived peptide.

Animals↗
PubMed1994

Identification of the urokinase receptor as an adhesion receptor for vitronectin.

Urokinase receptors, expressed on surfaces of many cell types, focus to the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. We now report that the urokinase receptor (uPAR) is also a high affinity (Kd < 30 nM) receptor for vitronectin. Recombinant uPAR binds vitronectin in the absence of urokinase, but vitronectin binding is promoted by concurrent receptor binding of either urokinase or fragments thereof containing its uPAR binding domain. Stable epithelial cell transfectants expressing membrane-anchored uPAR, but not cells expressing soluble uPAR, become strongly adhesive with altered morphology in the absence of urokinase. These observations identify a new class of vitronectin receptor and imply a duality in function for the receptor that intrinsically links matrix adhesion to regulation of protease activity. Increases in urokinase receptor expression known to be associated with cellular activation and malignant transformation could modulate cellular trafficking and function by promoting attachment to vitronectin.

Adsorption↗
PubMed1995

Cell surface proteolysis by serine proteinases enhances RGD-sensitive melanoma cell adhesion on fibrinogen and vitronectin.

Tumor cells avidly secrete various proteinases, and cascades of proteolytic activation occur around the cells. Therefore, cell surface receptors of tumor cells are under the constant influence of proteinases. In this study, the effects of serine proteinases on integrin-medicated cell-matrix interactions were studied in C32TG and Mewo human melanoma cells. These melanoma cells were pretreated with proteinases and their adhesive properties on various substrata were evaluated by cell adhesion assays. Paradoxically, appropriate cell surface proteolysis enhanced the RGD-sensitive cell adhesion on fibrinogen and vitronectin, but not the RGD-insensitive adhesion on type I collagen or laminin. Pretreatment of these cells with 0.1 to 1 microM of trypsin, chymotrypsin, or plasmin for 30 min at 37 degrees C increased the number of spread cells on fibrinogen and vitronectin by 200-300%. The enhancement of cell spreading was not accompanied by up-regulation of the relevant RGD-sensitive integrin expression. Analysis of the cell surface receptor by GRGDSPK-Sepharose affinity chromatography showed that trypsin treatment did not up-regulate alpha v beta 3 integrin, an RGD-sensitive receptor for fibrinogen and vitronectin in the melanoma cells, nor the induced appearance of novel receptors. Treatment of cells with 100 nM proteinases increased cell binding of both monoclonal and polyclonal antibodies against alpha v beta 3 integrin subunits by 70%, but not that of monoclonal antibody against alpha 2, alpha 3, or alpha 6 subunit, indicating that cell surface proteolysis exposed more alpha v beta 3 integrin on the cell surface. These results suggest that exposure of alpha v beta 3 integrin is a part of the mechanisms underlying the serine proteinase-induced enhancement of melanoma cell adhesion on fibrinogen and vitronectin.

Amino Acid Sequence↗
PubMed1999

alphavbeta3 and vitronectin expression by normal ovarian surface epithelial cells: role in cell adhesion and cell proliferation.

The alphavbeta3 integrin and its ligand vitronectin are expressed by differentiated epithelial ovarian carcinomas and carcinoma cell lines in culture. Moreover, alphavbeta3/vitronectin interaction influences adhesion and migration of ovarian carcinoma cells in culture. For a better understanding of the behavior of these carcinomas, it appeared necessary to study the characteristics of their normal counterpart, the ovarian surface epithelium (OSE). The present study showed that normal cultured human OSE cells, like the carcinoma cells, have the ability to synthesize vitronectin. The vitronectin receptor, alphavbeta3 integrin, is also expressed by OSE cells and is localized in focal contacts close to paxillin, a focal contact-specific protein, and p125(FAK), a cytoskeletal and signaling molecule. This localization suggested an active participation of the integrin in the adhesion and/or proliferation of OSE cells. Indeed, the use of a blocking antibody demonstrated that alphav integrins promote OSE cell adhesion on vitronectin but not on fibronectin and that these integrins are required for maximal proliferative activity. The results suggest a role of the alphavbeta3/vitronectin system in normal OSE physiology and demonstrate that the expression of this system by well-differentiated ovarian carcinomas reflects the retention of normal cell properties.

Adult↗
PubMed1989

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.

Adenocarcinoma↗
PubMed1991

Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins.

Extracellular matrix (ECM) glycoproteins regulate neuronal development and axonal growth. In this paper, the ECM glycoprotein vitronectin was identified and localized in the embryonic chick neuroretina. To identify potentially important neurite outgrowth-promoting molecules, responses of embryonic chick retinal neurons to vitronectin and thrombospondin, another retinal ECM constituent, were examined. These neurons were shown to attach and extend neurites on either glycoprotein. Integrins containing the alpha v or beta 1 subunits mediate both responses to vitronectin and neurite outgrowth on thrombospondin. Attachment to thrombospondin was inhibited by heparin, suggesting that neurons also utilize a proteoglycan or sulfated glycolipid as a receptor for this glycoprotein. Thus, retinal neurons use specific receptors to interact with vitronectin and thrombospondin, two glycoproteins present in the embryonic neuroretina, suggesting roles for these ligands and their receptors in retinal development.

Animals↗
PubMed1992

Vitronectin-binding surface proteins of Staphylococcus aureus.

S. aureus strain ISP 546 was selected (of 55 strains tested) to define optimal conditions for expression of vitronectin binding. High binding was expressed when the strain was grown on blood agar and in Todd-Hewitt broth. Binding was optimal in the 6.0 to 7.2 pH range and was unaffected by divalent cations and ionic strength. Binding was partially inhibited by D-mannose, heparin, types I and IV collagen, fibronectin, fibrinogen and vitronectin, but was not affected by other carbohydrates or glycoproteins tested. Cell surface binding components were extracted with the aid of 1 M LiCl (pH 5.0) from strain ISP 546 grown in Todd Hewitt broth. Vitronectin binding proteins were purified by affinity chromatography on heparin-Sepharose. Fractions inhibiting binding of 125I-labelled vitronectin to strain ISP 546 were eluted by 0.01 M NaOH, dialysed, concentrated and subjected to SDS-PAGE. Silver staining revealed one major band (70 kDa) and two minor bands (34 and 36 kDa).

Binding, Competitive↗
PubMed1998

Expression of alphav integrins and vitronectin receptor identity in breast cancer cells.

In the present study we have used fluorocytometry and immunoprecipitation to characterize the expression of alphav-containing integrins in a panel of eight human breast cancer cell lines and one normal human mammary epithelial line. We show that the classical vitronectin receptor alphavbeta3 is expressed in only one cell line (MDA-MB-231), whereas alphavbeta5 is expressed on all breast cancer cell lines and alphavbeta1 is expressed on the majority. Using adherence assays to purified ligands in the presence and absence of function-blocking monoclonal antibodies, we have demonstrated that alphavbeta5 mediates adhesion to vitronectin in the majority of these cells. In one cell line, ZR75-1, alphavbeta1 contributes significantly to adhesion to immobilized vitronectin. The formation of focal adhesions containing the alphav and beta1 subunits on vitronectin is also demonstrated by indirect immunofluorescence.

Breast Neoplasms↗
PubMed2001

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).

Actins↗
PubMed1991

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.

Actin Cytoskeleton↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1993

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.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Vitronectin regulates smooth muscle contractility via alphav and beta1 integrin.

Previous work from this laboratory has established a method for maintaining physiological contractility of dissociated avian smooth muscle in a defined medium at low density. The present report emphasizes the dramatic potency of serum to alter smooth muscle phenotype and induce a loss of contractility. Vitronectin, a molecule purified from plasma, mimicked these effects of serum via an integrin that is RGD-sensitive. Studies utilizing blocking antibodies against vitronectin demonstrated that the presence of this specific adhesion molecule was necessary for the serum-induced loss of contractility. Based on the actions of function-blocking antibodies and RGD-containing peptides, the integrin alphavbeta1 appears to be the primary receptor involved in vitronectin's ability to induce phenotypic transformation in amniotic smooth muscle. The influence of vitronectin on smooth muscle contractility is particularly relevant, because this molecule is abundant in whole blood and plasma (approx. 400 microg/ml). The results suggest that smooth muscle needs to be continually protected from normal blood constituents in vivo. The implications of these results for smooth muscle-related diseases like atherosclerosis, restenosis and Kaposi's sarcoma are discussed.

Amnion↗
PubMed1997

Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells.

PURPOSE: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay. RESULTS: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS. CONCLUSIONS: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.

Animals↗
PubMed1996

Fibulin-1, vitronectin, and fibronectin expression during avian cardiac valve and septa development.

BACKGROUND: Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873-882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process. METHODS: Immunofluorescence microscopy was used to examine the distribution of fibulin-1, vitronectin, and fibronectin in the embryonic chicken heart over a broad developmental time frame (Hamburger and Hamilton stages 14 to 44), emphasizing stages that illustrate endocardial cushion formation, growth, fusion, and development into valvuloseptal components. RESULTS AND CONCLUSIONS: Fibulin-1 immunolabeling was concentrated in endocardial cushions, notably at boundaries with the myocardium, during stages when the cushions are differentiating into valvular and septal components. Fibulin-1 was detected in the endocardial cushions prior to their seeding with cushion cells, but became undetectable by early midgestation. Vitronectin expression was similar to fibulin-1, but less restricted in its distribution. Vitronectin was observed before endocardial cushion cell migration commenced and persisted until the formation of prevalvular structures (early midgestation) in the atrioventricular cushions. Vitronectin remained detectable in the semilunar valves until late midgestation. Fibronectin was present in the endocardial cushion region and in portions of the endocardium and myocardium throughout the stages presented. Our data suggests that the ECM of the endocardial cushions undergoes remodelling in a regionally and temporally specific manner which corresponds with morphogenetic changes during valvuloseptal development.

Animals↗
PubMed1997

The role of vitronectin in the attachment and spatial distribution of bone-derived cells on materials with patterned surface chemistry.

In recent years a central objective of tissue engineering has been understanding the interaction of cells with biomaterial surfaces. In this study we examined the protein adsorption events necessary to control the attachment and the subsequent spatial distribution of bone-derived cells exposed to chemically modified surfaces. Silane chemistry and photolithography techniques were used to create substrates with alternating regions of an aminosilane, N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS), along side an alkylsilane, dimethyldichlorosilane (DMS), on quartz surfaces. Sera depleted of fibronectin (Fn), vitronectin (Vn), or both were used to determine if these proteins were necessary for the initial attachment and spatial distribution of bone-derived cells exposed to modified surfaces in vitro. The kinetics and mechanisms of the spatial distribution of cells were examined using light microscopy and digital image acquisition and subsequently were analyzed. Compared to complete serum, the use of serum depleted of fibronectin with vitronectin included had minimal effect on the cell attachment, spreading, and spatial distribution on the EDS regions of the surface. However, the use of serum depleted of vitronectin with or without fibronectin included resulted in greatly reduced cell attachment and spreading. Thus the presence of vitronectin was required for the attachment, spreading, and spatial distribution of bone-derived cells exposed to EDS/DMS-patterned surfaces.

Adsorption↗