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Biomedical subjects

F G Giancotti

Publications and source records attributed to F G Giancotti.

14 recordsLinked to original sources
PubMed1992

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

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

Animals↗
PubMed1992

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.

Amino Acid Sequence↗
PubMed1990

A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1).

The integrins are a family of heterodimeric cell surface receptors for extracellular matrix molecules. An analysis of integrin subunits expressed by a number of cell lines identified a novel heterodimer. The alpha subunit of this integrin was immunologically and electrophoretically indistinguishable from the vitronectin receptor alpha subunit (alpha v) and the beta subunit was indistinguishable from beta 1. Affinity chromatography experiments and cell adhesion assays indicated that this receptor complex is a new fibronectin receptor. Its unexpected subunit composition demonstrates the importance of the beta subunit in determining the ligand specificity of integrins and suggests that the current integrin classification scheme needs revision.

Antibodies, Monoclonal↗
PubMed1990

Elevated levels of the alpha 5 beta 1 fibronectin receptor suppress the transformed phenotype of Chinese hamster ovary cells.

We report here on gene transfer studies designed to investigate the function of the alpha 5 beta 1 integrin and its role in transformation. Transfection of the human alpha 5 and beta 1 cDNAs into transformed Chinese hamster ovary (CHO) cells followed by methotrexate-induced amplification yielded clonal cell lines overexpression this fibronectin receptor. The overexpressors deposited more fibronectin in their extracellular matrix and migrated less than control cells. In addition, they showed reduced saturation density and reduced ability to grow in soft agar. The overexpressor cells, unlike the control CHO cells, were nontumorigenic when injected subcutaneously into nude mice. The results indicate that extracellular matrix recognition by the alpha 5 beta 1 integrin plays a role in the control of cell proliferation and suggest that a reduction of this fibronectin receptor may be responsible for the acquisition of anchorage independence by transformed cells.

Animals↗
PubMed1989

Integrins and tumor cell dissemination.

The ability of a tumor cell to metastasize is determined, in part, by its adhesive interactions with other cells and with components of the extracellular matrix. An ever-expanding family of cell surface receptors, the integrins, is an essential player in these interactions. Research efforts aimed at understanding the precise role of individual integrins in normal and transformed cells may offer a general framework for future therapeutic approaches. Here we review recent progress toward this goal.

Cell Adhesion↗
PubMed1988

Purification of the beta subunit of the fibronectin receptor.

In this work we describe a method for purification of the beta subunit of the mouse fibronectin receptor (GP135). Cellular glycoproteins were isolated from a detergent extract of SR-Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin-Sepharose and wheat-germ-agglutinin--agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS-PAGE analysis of 125I-labelled material.

Animals↗
PubMed1988

Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts.

Rous sarcoma virus-transformed baby hamster kidney fibroblasts (RSV/B4-BHK) adhere to a fibronectin-coated substratum by means of dot-like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.: Exp Cell Res 159:141, 1985). Podosomes concentrate tyrosine-phosphorylated proteins, including pp60v-src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10-100 microM) induced in a time- and dose-dependent manner the redistribution of F-actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylated proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine-phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may be primarily induced by the tyrosine phosphorylation of unknown target(s) operated by endogenous kinases.

Actin Cytoskeleton↗
PubMed1987

Platelets express a membrane protein complex immunologically related to the fibroblast fibronectin receptor and distinct from GPIIb/IIIa.

We have previously identified and characterized a membrane glycoprotein complex (GP150/135) that functions as fibronectin receptor (FN-R) in fibroblast adhesion. Here we report that an immunologically related protein complex is expressed at the surface of human platelets. Antibodies monospecific for the smaller subunit (GP135) of the fibroblast FN-R in fact specifically stained the platelet surface, as determined by FACS analysis, and reacted with a component of molecular weight (mol wt) 138,000 as shown in western blots of platelet membranes. Moreover, the same antibodies precipitated the 138,000 component together with a 160,000 protein, suggesting that the two molecules are associated in a supramolecular complex. A comparative analysis indicated that this protein complex is distinct from the GPIIb/IIIa complex, known to function as a receptor of wide specificity for fibrinogen, fibronectin, and von Willebrand factor. Differential extraction experiments revealed that the platelet 138,000 component is an integral membrane protein.

Antibodies↗
PubMed1986

A 135,000 molecular weight plasma membrane glycoprotein involved in fibronectin-mediated cell adhesion. Immunofluorescence localization in normal and RSV-transformed fibroblasts.

Monospecific antibodies to GP135, a plasma membrane glycoprotein involved in cell attachment and spreading on fibronectin-coated substratum (Giancotti et al., Exp cell res 156 (1985) 182), were affinity-purified from a broad-spectrum antiserum using the GP135 molecule immobilized on nitrocellulose filters. The purified antibodies specifically inhibited attachment and spreading of normal as well as RSV-transformed fibroblasts on fibronectin-coated dishes indicating that GP135 is involved in adhesion of both normal and transformed cells. Immunofluorescence experiments showed that GP135 has a discrete distribution on control 3T3 fibroblasts. On cells in the process of spreading GP135 was concentrated in 'close contact' sites, while on fully spread cells the glycoprotein was concentrated in 'adhesion plaques' and in streaks co-aligning with portions of stress fibres. In RSV-transformed cells the discrete distribution of GP135 was lost. The protein appeared in a diffuse distribution at the plasma membrane and was not concentrated in the dot- or rosette-shaped substratum contacts sites peculiar to these cells. It is concluded that GP135 mediates adhesion of both normal and RSV-transformed fibroblasts to fibronectin. While in normal cells this glycoprotein participates in the organization of specialized adhesion structures, in RSV-transformed fibroblasts recruiting of GP135 molecules within cell-substratum contact sites is perturbed.

Animals↗
PubMed1986

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells.

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.

Animals↗
PubMed1985

Cleavage of a 135 kD cell surface glycoprotein correlates with loss of fibroblast adhesion to fibronectin.

We have previously described a group of three plasma membrane glycoproteins that are recognized by an adhesion-disrupting antiserum and that are involved in fibronectin-mediated BHK cell adhesion. A peculiar property of these molecules is their resistance to tryptic digestion. We have now extended this study in the attempt to identify the active component within this group of molecules. SR/BALB mouse fibroblasts, used in this work, expose at their surface only two trypsin-resistant glycoproteins, gp1 (150 K) and gp2 (135 K), that are recognized by the adhesion-disrupting anti-BHK serum. Controlled proteolysis of the cell surface in the presence of a reducing agent results in the loss of cell adhesion to fibronectin-coated substratum. gp2 is selectively cleaved under these conditions. Moreover, cells treated with trypsin and reducing agent can no longer adsorb the adhesion-relevant antibodies from the anti-BHK serum. These data indicate that gp2 plays a critical role in the adhesion of SR/BALB fibroblasts to fibronectin-coated substratum, and that disulfide bonds are important in the conformation and function of this molecule.

Animals↗
PubMed1985

Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes.

Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.

Actins↗