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W T Chen

Publications and source records attributed to W T Chen.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia.

We have examined the contribution of adhesion mechanisms to cell invasiveness by growing chicken embryo fibroblasts (CEF) or Rous sarcoma virus-transformed cells (RSVCEF) on fibronectin-coated crosslinked gelatin beads (FN-beads). RSVCEF attached more readily and spread more rapidly on FN-beads than CEF, suggesting an increase in the adhesion-related motility of the transformed cells. In addition, RSVCEF invaded the FN-beads, but CEF did not, by extending specialized cell surface protrusions called invadopodia at sites of cell invasion. FN removal by RSVCEF cultured on prelabeled fluorescent FN-beads (FL-FN) was evident at sites of invadopodia, and internalized FL-FN occurred in vacuoles near the ventral membrane of cells at sites of FN removal. The precise distribution of FN and integrins in cells invading FN-beads was determined by immunofluorescence and immunoelectron microscopy of frozen thin-sections. In both CEF and RSVCEF, beta 1 integrins and FN occupied separate intracellular compartments during the early stage of spreading on FN-beads. Later, beta 1 integrins were largely localized at the ventral cell surface of both CEF and RSVCEF. Polyclonal anti-integrin antibody recognizing beta 1 and several alpha chains, however, labeled both ventral and dorsal cell surfaces. During invasion by RSVCEF, beta 1 integrins were concentrated at extended invadopodia and also colocalized with internalized FL-FN material in phagocytic vesicles. Furthermore, secreted FN was deposited by RSVCEF at the base of invadopodia colocalizing with beta 1 integrin. Both FL-FN matrix removal and formation of the invadopodia were found to be resistant to treatment with GRGDS at concentrations that inhibit the interaction between cells and FN-beads. Thus, the localization of beta 1 integrins to the plasma membrane contacting immobilized FN results in an extremely tight cellular adherence to the matrix bead, that stabilizes invadopodia and also mediates endocytic clearance of degraded FN-matrix material.

Animals↗
PubMed1990

Transmembrane interactions at cell adhesion and invasion sites.

Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) invade into a film of the extracellular matrix (ECM) by extending membrane protrusions, termed the invadopodia. The invadopodia share similar cytoskeletal components and membrane receptors for ECM components as adhesion sites. However, the organization of these transmembrane components at invadopodia and adhesion sites differs. In addition, degradation of the ECM occurs at sites of the invadopodia, but not at focal adhesions. Thus, the protease and integrin molecules on invadopodia are available for dynamic interactions with the ECM, cleaving established adhesion complexes as well as reconstituting new adhesion sites.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibody and synthetic peptide inhibitors of human tumor cell migration.

The processes of migration and invasion by human tumor cells are likely to involve specific cell surface receptors, such as receptors for the extracellular matrix molecules fibronectin, laminin, and collagen. We have examined the roles of several of these receptors using a set of monoclonal antibodies directed against the beta 1 integrin family, as well as a series of synthetic peptides reported to inhibit various interactions of each of these proteins with the cell surface. The most general inhibitor of tumor cell migration was found to be the anti-beta 1 monoclonal antibody 13, which inhibited the migration of human HT-1080 fibrosarcoma cells, 5637 bladder carcinoma cells, VA13 viral transformants, and HCT 116 colon carcinoma cells when fibronectin was the migration substrate. Moreover, this antibody was particularly effective in blocking cell migration on laminin, as well as migration within 3-dimensional collagen gels. It also inhibited in vitro invasiveness in a reconstituted basement membrane invasion assay (Matrigel assay) at concentrations as low as 1 microgram/ml. Integrins of the beta 1 class thus appear to play a central role in several types of migration by a variety of human tumor cell lines. Anti-alpha 5 fibronectin receptor monoclonal antibody 16 also significantly inhibited migration on fibronectin, but not on other substrates, in 3 of the 4 cell lines. Conversely, anti-alpha 2 monoclonal antibody F17 strikingly inhibited migration in 3-dimensional collagen gels, but not on other substrates, implicating the alpha 2 beta 1 integrin system in migration of tumor cells within collagenous matrices. A series of synthetic peptides previously reported to inhibit interactions of normal cells with fibronectin, laminin, and collagen were also tested as inhibitors of tumor cell migration. Peptides containing the Arg-Gly-Asp adhesive recognition signal were partially inhibitory, but with occasional exceptions, most other peptides had no effects on migration. Our results indicate the central importance of several specific beta 1 integrins in human tumor cell migration and show the effectiveness of monoclonal antibody treatment in blocking this process in vitro.

Amino Acid Sequence↗
PubMed1990

A 170-kDa membrane-bound protease is associated with the expression of invasiveness by human malignant melanoma cells.

Malignant spreading of cancer cells requires cell surface proteases that cleave the crosslinked collagenous matrix of connective tissues. From correlating the morphologically defined invasiveness of tumor cells with the presence of specific membrane-associated proteases, we have identified a malignant human melanoma cell line, LOX, that invades crosslinked gelatin films in vitro and contains uniquely a neutral 170-kDa gelatinase in the cell membrane. A similar gelatinase was found in membranes recovered from culture media conditioned with LOX. The 170-kDa gelatinase is a wheat germ agglutinin-binding protein. The proteolytic activity is maximal at neutral pH, enhanced by EDTA and dithiothreitol, inhibited by the cysteine protease inhibitors N-ethylmaleimide, HgCl2, and phenylmethylsulfonyl fluoride, and can bind to an organomercurial adsorbent, suggesting that it is a neutral sulfhydryl-sensitive protease. This 170-kDa gelatinase of LOX cells was not found in a control melanoma cell line, SK-MEL28, or in 32 other tumor cell lines that did not show extracellular gelatin degradation. Thus, we have identified a large membrane-bound protease that may be a specific marker molecule for melanoma cell invasiveness.

Cell Fractionation↗
PubMed1989

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells.

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.

Animals↗
PubMed1989

Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.

Antibodies, Monoclonal↗
PubMed1989

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin.

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.

Animals↗
PubMed1989

Diagnostic value of adenosine deaminase and gamma-interferon in tuberculous and malignant pleural effusions.

It has been found that both adenosine deaminase (ADA) and gamma-interferon (r-IFN) are higher in tuberculous pleural effusions than in other types of pleural effusion. Both mechanisms may be related to the T-lymphocytes in effusions. We studied 39 pleural effusions: 19 tuberculous and 20 malignant [The ADA was determined according to the Giusti method and r-IFN by ELISA]. We compared the ADA and r-IFN levels in both groups of effusions and tried to determine if there was any correlation between them. The results showed: (1) The ADA enzyme level was significantly higher in tuberculous effusions than in malignant effusions as was expected (133.0 +/- 50.4 vs 32.0 +/- 17.7 U/L). (2) The r-IFN was also significantly higher in tuberculous effusions than in malignant effusions (30.4 +/- 17.4 vs 2.9 +/- 2.8 U/ml). (3) The coefficients of regression for the ADA and r-IFN levels were poor. In conclusion, both tests for measuring ADA and r-IFN levels are excellent methods for differentiating tuberculous and malignant effusions, and especially measurement of the r-IFN level could serve as a more specific test for differentiating malignant pleural effusions with high ADA levels. However, no strong correlation was found between the ADA and r-IFN levels.

Adenosine Deaminase↗
PubMed1988

Enhanced fibronectin receptor expression in Rous sarcoma virus-induced tumors.

The mechanisms by which cells acquire the capacity to invade interstitial connective tissues during malignancy are as yet uncertain. Since the fibronectin receptor complex has been implicated in transient, developmentally regulated steps of migration and morphogenesis in embryogenesis, we examined whether this receptor might be reexpressed at elevated levels in tumors. Immunofluorescence revealed increased expression of the receptor throughout frozen sections of Rous sarcoma virus-induced tumors in chickens, and expression was enhanced 4.7-fold after such malignant transformation of fibrocytes in vivo. Frozen thin sections showed that the increased antigen was localized diffusely in the plasma membrane. Western immunoblotting with monoclonal antibodies and immunoprecipitation analyses indicated that the tumor cell receptor contained all three known avian receptor subunits, i.e., Bands 1, 2, and 3. This type of induction of a key extracellular matrix receptor involved in cell migration may be a prerequisite for tumor cell invasion.

Animals↗
PubMed1988

Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts.

Transforming growth factors of the beta-class (TGFs-beta) stimulate extracellular matrix synthesis and have been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury. Because cells communicate with several extracellular matrix components via specific cell membrane receptors, we hypothesized that TGFs-beta may also regulate the expression of such receptors. We confirmed that TGF-beta 1 increases the expression of fibronectin, an adhesive glycoprotein expressed during embryogenesis and tissue remodeling. Based upon the 48-72-h period required for a maximal fibroproliferative response to dermal injections of TGF-beta 1, we exposed human fetal lung fibroblasts (IMR-90) to TGF-beta 1 for periods up to 48 h in vitro. We observed as much as 6-fold increases in fibronectin synthesis by 24 h as previously reported for fibroblastic cells (Ignotz, R. A., and Massagué, J. (1986) J. Biol. Chem. 261, 4337-4345; Ignotz, R. A., Endo, T., and Massagué, J. (1987) J. Biol. Chem. 262, 6443-6446; Raghow, R., Postlethwaithe, A. E., Keski-Oja, J., Moses, H. L., and Kang, A. H. (1987) J. Clin. Invest. 79, 1285-1288), but up to 30-fold increases by 48 h. These increases are accompanied by similar increases in fibronectin mRNA levels which are prevented by actinomycin D treatment. Using a monospecific antibody raised to the human placental fibronectin receptor complex, we found that TGF-beta 1 stimulated fibronectin receptor synthesis up to 20-40-fold and increases mRNA levels encoding both the alpha- and beta-subunits up to 3-fold, compared to control IMR-90 in serum-free medium. Actinomycin D blocks TGF-beta 1-mediated increases in receptor mRNA levels. The earliest detectable TGF-beta 1-mediated increases in fibronectin receptor complex protein synthesis and mRNA levels occur at 8 h, whereas the earliest increases in fibronectin protein synthesis and mRNA levels occur at 12 h. These results demonstrate that TGF-beta 1 stimulates fibronectin receptor synthesis, extending the diverse stimulatory activities of this polypeptide to matrix receptors. In addition, because fibronectin matrix assembly may involve the fibronectin cell adhesive receptor complex, increased receptor expression may help drive fibronectin deposition into matrix.

Cell Line↗
PubMed1988

Transmembrane orientation of the fibronectin receptor complex (integrin) demonstrated directly by a combination of immunocytochemical approaches.

The avian 140-KD cell adhesion receptor or "integrin," a complex of three glycoproteins with molecular masses averaging 140 KD, interacts with extracellular fibronectin and forms a linkage complex that co-localizes with intracellular actin. To probe the molecular interactions involved in this linkage complex, we used monoclonal antibodies and a combination of immunolocalization approaches to determine whether any component was transmembrane. Immunoadsorption and immunoblotting experiments demonstrated that anti-120-KD Mabs recognized the band 3 component of integrin isolated from chicken embryo fibroblasts (CEF) by JG22E immunoaffinity chromatography, and they co-localize with anti-fibronectin and polyclonal anti-integrin at cell contact sites in double-labeling experiments. Immunofluorescence experiments involved comparisons of double-labeled intact cells or substrate-attached, ventral plasma membrane "rip-off" fragments, using anti-fibronectin and each of the anti-120-KD Mabs. The extracellular faces of living intact cells were strongly labeled by a majority (approximately 70%) of the anti-120-KD Mabs at fibronectin-membrane attachment sites. The remainder (approximately 30%) labeled intact cells weakly or not at all. However, although the anti-120-KD Mab ES186 did not stain living cells, it did demonstrate positive staining above substratum contact sites over entire isolated rip-off membranes. In contrast, Mabs directed against putative extracellular epitopes and anti-fibronectin antibodies did not label these sites at the center of rip-offs unless the membranes were detergent permeabilized. Proteolysis experiments suggested that the ES186 epitope was located at one end of the molecule, since removal of short fragments from integrin band 3 concomitantly removed or destroyed the ES186 epitope, whereas the extracellular epitopes still remained. These experiments directly demonstrate that integrin band 3 is a transmembrane polypeptide with at least one epitope recognized by anti-120-KD Mabs on the cytoplasmic side of the plasma membrane and at least one epitope on the extracellular cell surface.

Animals↗
PubMed1987

Fibronectin-degrading proteases from the membranes of transformed cells.

The local degradation of fibronectin substrata by Rous sarcoma virus-transformed chick embryonic fibroblasts requires cell-contact-related metalloendoprotease and serine-protease activities. Using fibronectin-containing SDS gels, two large proteases with apparent molecular weights of 120K and 150K were found only in the membrane fraction of transformed cells and were absent in normal cells. Both 120K and 150K proteases were active at neutral pH, but showed preferential inhibitor sensitivities of serine and metal proteases, respectively. The 150K protease appeared to account for most of the proteolytic activity since metalloendoprotease inhibitors completely blocked proteolytic activity of the 150K in fibronectin gels, more than 80% of the fibronectin-degrading activity of solubilized membranes, and largely suppressed the appearance of fibronectin degradation spots in cultures of transformed cells.

Animals↗
PubMed1987

pH-dependent function, purification, and intracellular location of a major collagen-binding glycoprotein.

A major collagen-binding heat shock protein of molecular mass 47,000 D was found to bind to collagen by a pH-dependent interaction; binding was abolished at pH 6.3. Native 47-kD protein could therefore be purified from chick embryo homogenates in milligram quantities by gelatin-affinity chromatography and gentle acidic elution. Rat monoclonal and rabbit polyclonal antibodies were generated against the purified 47-kD protein. Immunofluorescence microscopy of cultured chick embryo fibroblasts with these antibodies revealed bright, granular perinuclear staining as well as a weaker reticular network structure towards the cell periphery, suggesting that this protein was located in the endoplasmic reticulum. No immunofluorescence staining was detected on the cell surface. Double-staining experiments with these antibodies and fluorescently labeled wheat-germ agglutinin suggested that the 47-kD protein was absent from the Golgi apparatus. Localization of the 47-kD protein in the endoplasmic reticulum but not in the Golgi complex was confirmed by immunoelectron microscopy. In vivo localization studies using immunohistochemistry of cryostat sections of chick liver revealed that the 47-kD protein was present in fibrocytes, Kupffer cells, and smooth muscle cells. It was absent from hepatocytes and the epithelia of bile ducts or sinusoidal endothelium. This major transformation- and heat shock-regulated glycoprotein is thus localized intracellularly, is expressed in only certain cells, and functions in a pH-regulated manner. These findings suggest that this glycoprotein is not likely to be a general cell-surface collagen receptor, but may instead play roles in intracellular protein processing or translocation.

Animals↗
PubMed1986

Cell adhesion and migration in the early vertebrate embryo: location and possible role of the putative fibronectin receptor complex.

Using a combined in vivo and in vitro approach, we have analyzed the immunofluorescent localization and function of a 140,000-mol-wt glycoprotein complex implicated in cell adhesion to fibronectin (FN), with particular emphasis on neural crest cell adhesion and migration. This putative fibronectin receptor complex (FN-receptor) was detectable in almost all tissues derived from each of the three primary germ layers. It was present in both mesenchymal and epithelial cells, and was particularly enriched at sites close to concentrations of FN, e.g., at the basal surfaces of epithelial cells. It was also present on neural crest cells. The distribution and function of this putative receptor was then analyzed on individual cells in vitro. It was diffusely organized on highly locomotory neural crest cells and somitic fibroblasts. Both motile cell types also displayed relatively low numbers of focal contacts and microfilament bundles and limited amounts of localized vinculin, alpha-actinin, and endogenous FN. In contrast, the FN-receptor in stationary embryonic cells, i.e., somitic cells after long-term culture or ectodermal cells, existed in characteristic linear patterns generally co-distributed with alpha-actinin and fibers of endogenous FN. Anti-FN-receptor antibodies inhibited the adhesion to FN of motile embryonic cells, but not of stationary fibroblasts. However, these same antibodies adsorbed to substrata readily mediated adhesion and spreading of cells, but were much less effective for cell migration. Our results demonstrate a widespread occurrence in vivo of the putative FN-receptor, with high concentrations near FN. Embryonic cell migration was associated with a diffuse organization of this putative receptor on the cell surface in presumably labile adhesions, whereas stationary cells were anchored to the substratum at specific sites linked to the cytoskeleton near local concentrations of FN-receptor.

Actinin↗
PubMed1986

Coupled expression and colocalization of 140K cell adhesion molecules, fibronectin, and laminin during morphogenesis and cytodifferentiation of chick lung cells.

We have analyzed the expression and distribution of fibronectin, laminin, and the 140K cell adhesion molecules (140K complex) in embryonic chick lung cells by a combination of biochemical and immunofluorescent approaches. The 140K complex was identified by monoclonal antibody JG22E as a complex of glycoproteins averaging 140,000 Mr and has been implicated in vitro as a receptor for fibronectin and laminin. Our studies provide the first description that the 140K complex is developmentally regulated, and that the 140K complex appears to be involved in adhesion of epithelial and endothelial cells during morphogenesis. We have shown that the 140K complex is expressed in high quantity in embryonic lung cell types, but is markedly reduced in all of the differentiated cell types except smooth muscle. Embryonic lung cells are enriched in 140K complex on portions of cells in close proximity to areas rich in fibronectin. For example, during the formation of airways and alveolar tissues, 140K complex is concentrated at the basal surfaces of epithelial cells adjacent to fibronectin. Likewise, during the angiogenic invasion of capillaries into lung mesenchyme, the 140K complex becomes localized at sites on the basal surfaces of endothelial cells in close contact with fibronectin. Finally, cytodifferentiating lung smooth muscle cells show unusually high levels of 140K complex, fibronectin, and laminin that persist into the adult. In contrast to fibronectin, laminin is found to be uniformly distributed in the basement membranes of differentiating epithelial cells. It becomes prominent in adult alveolar epithelium and airway epithelium concomitant with a reduction or loss of 140K complex and fibronectin at cell-basement membrane attachment sites. Surprisingly, laminin is also present in a punctate pattern in the mesenchyme of early lung buds, however, laminin, fibronectin, and 140K complex are greatly reduced or lost during mesenchymal maturation. Our results are consistent with the active participation of the 140K complex in cell-to-matrix adhesion during morphogenesis of alveolar walls and cytodifferentiation of mesenchymal and smooth muscle cells.

Animals↗
PubMed1986

Regulation of fibronectin receptor distribution by transformation, exogenous fibronectin, and synthetic peptides.

Recent studies have shown that fibronectin and its 140K membrane receptor complex are spatially associated with microfilaments to form cell surface linkage complexes which are thought to mediate adhesive interactions between fibroblasts and their substrata. We examined the regulation of the organization of these cell surface structures in transformed and fibronectin-reconstituted cells as well as in cells treated with a competitive synthetic peptide inhibitor of fibronectin binding to its receptor. Correlative localization experiments with interference reflection microscopy and double-label or triple-label immunofluorescence revealed a concomitant loss of fibronectin, 140K receptor, and alpha-actinin colocalization at cell substratum extracellular matrix contact sites after transformation of chick fibroblasts by wild-type or temperature-sensitive Rous sarcoma viruses (RSV). Western and dot immunoblot analyses established that although similar total quantities of intact 140K molecules were present in the transformed cell cultures, significantly more was released into the culture medium of transformed cells. The 140K molecules on transformed cells were available for interaction with exogenously added fibronectin, which could reconstitute fibronectin-140K linkage complexes. In such fibronectin reconstitution experiments, many cells expressed both fibronectin-140K-actin linkage complexes and RSV pp60src, indicating that the morphological reversion could occur even in the continued presence of RSV transformation. The synthetic peptide Gly-Arg-Gly-Asp-Ser derived from the sequence of the cell-binding region of fibronectin could also prevent the organization of fibronectin-140K linkage complexes. Our results suggest that fibronectin interaction with cells regulates the organization of fibronectin receptor complexes and cytoskeletal components at the cell surface.

Actinin↗
PubMed1985

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin.

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.

Amino Acids↗