Characterization of a major fibroblast cell surface glycoprotein

Yamada, Kenneth M.; Schlesinger, David H.; Kennedy, Dorothy W.; Pastan, Ira

doi:10.1021/bi00644a025

Cited by 178 publications

(65 citation statements)

References 50 publications

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“…1). Cellular fibronectin is not soluble under the conditions usually used to evaluate antibody specificity (17,56). We therefore first solubilized the fibronectin by elevating the pH to 11.0 (32,56), then assayed for specificity at neutral pH.…”

Section: Specificity Of the Antibodymentioning

confidence: 99%

“…Cellular fibronectin is not soluble under the conditions usually used to evaluate antibody specificity (17,56). We therefore first solubilized the fibronectin by elevating the pH to 11.0 (32,56), then assayed for specificity at neutral pH. In Ouchterlony double immunodiffusion gels, confrontation of either antibody (antifibronectin dimer or anti-electrophoretically purified fibronectin) with either isolated fibronectin or homogenates of cultures of chick embryo fibroblasts resulted in a single immunoprecipitin line of identity (Fig.…”

Section: Specificity Of the Antibodymentioning

confidence: 99%

“…Isolated cellular fibronectin is not soluble under conditions usually used to evaluate antibody specificity (56), which necessitated the use of modified double immunodiffusion and immunoprecipitation procedures in which fibronectin or cell homogenates are first solubilized at pH 11, then assayed at neutral pH. We have also used a new procedure for detecting antigens separated in SDS gels (31).…”

Section: Specificity Of the Antibodymentioning

confidence: 99%

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Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

Yamada¹

1978

The Journal of Cell Biology

171

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The major cell surface glycoprotein of chick embryo fibroblasts, cellular fibronectin (formerly known as CSP or LETS protein), was purified and used to produce monospecific antisera. After affinity purification, the anti-fibronectin was used to investigate fibronectin's localization, its transfer from intracellular to extracellular pools, its antibody-induced redistribution on the cell surface, and its role in cell shape.Anti-fibronectin localizes to extracellular fibrils located under and between sparse cells, and to a dense matrix that surrounds confluent cells. Cellular fibronectin is also present in granular intracytoplasmic structures containing newly synthesized fibronectin before secretion. This intracellular staining disappears 2 h after treatment with cycloheximide or puromycin, and returns after removal of these protein synthesis inhibitors. In pulse-chase experiments using cycloheximide, fibronectin was sequentially transferred from the intracellular to the fibrillar extracellular forms.Transformation of chick fibroblasts results in decreases in both extracellular and intracellular fibronectin, and in altered cell shape. Treatment of untransformed chick fibroblasts with anti-fibronectin results in rapid (30 min) alteration to a rounder cell shape resembling that of many transformed cells. These rapid shape changes are followed by a slow, antibody-induced redistribution of fibronectin to supranuclear caplike structures. This "capping" is inhibited by metabolic inhibitors.Reconstitution of cell surface fibronectin onto transformed cells restores a more normal fibroblastic phenotype. The reconstituted fibronectin on these cells organizes into fibrillar patterns similar to those of untransformed cells. As with untransformed cells, treatment of these reconstituted cells with anti-fibronectin also results in cell rounding and "capping" of fibronectin. KEY WORDS fibronectinA glycoprotein of high molecular weight has been immunofluorescence antibody-induced identified on the surface of a variety of fibroblastic redistribution 9 secretion adhesion and epithelial cells in vitro, using procedures that 520 TnE JOURNAL OF CELL BIOLOGY 9 VOLUME 78, 1978 9 pages 520-541on May 10, 2018 jcb.rupress.org Downloaded from http://doi.org/10.1083/jcb.78.2.520 Published Online: 1 August, 1978 | Supp Info: label cell surface proteins by radioactive compounds or by specific antibodies (reviewed in references 15, 44, 55: see also reference 7). This glycoprotein is known as cell surface protein (CSP), large, external, transformation-sensitive (LETS) protein, or cellular fibronectin (16,21,58). This protein is often decreased after establishment of a permanent cell line (61), and is usually further decreased after neoplastic transformation (15,44,55). Cells missing this glycoprotein are generally tumorigenic (7,12), although in vitro experiments indicate that it does not directly control growth rates (43,60).This glycoprotein, or the immunologically indistinguishable plasma glycoprotein "cold insoluble globulin" (plasma f...

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Section: Specificity Of the Antibodymentioning

confidence: 99%

Section: Specificity Of the Antibodymentioning

confidence: 99%

Section: Specificity Of the Antibodymentioning

confidence: 99%

See 1 more Smart Citation

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

Yamada¹

1978

The Journal of Cell Biology

171

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“…In this way, we have discovered that fibronectin, a collagen-binding protein, is present on platelet membranes. "Fibronectin" refers to a family of related proteins including the cold-insoluble globulin (CIG) of plasma and the large external transformation-sensitive (LETS) protein found in membranes of many cell types (12).t Both forms bind to collagen and crossreact immunologically (13)(14)(15), have a molecular weight of 440,000, consist of two similar chains of 220,000 daltons crosslinked by a disulfide bond (16)(17)(18), and have similar amino acid compositions (17,18). One of the few known differences is that LETS is less soluble at neutral pH than CIG (17).…”

mentioning

confidence: 99%

Evidence that fibronectin is the collagen receptor on platelet membranes.

Bensusan¹,

Koh²,

Henry³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

113

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Evidence is presented that fibronectin is present on the platelet cell membrane and that it is a receptor for collagen in the platelet-collagen interaction. First, sodium dodecyl sulfate/acrylamide gel electrophoresis was performed on the proteins remaining attached to the surface of collagen after the removal of the remainder of the platelet by sonication. The material was relatively enriched in a glycoprotein that comigrated with cold-insoluble globulin (CIG), a form of fibronectin, and in other proteins which comigrated with myosin, actin, and tropomyosin. The presumptive presence of contractile proteins is consistent with the presence of microfibrillar proteins. Second, the collagen-attached material was shown to contain a protein that reacted'with anti-GIG serum by immunoelectrophoresis. Third, when CIG was preincubated with fibrous collagen, the platelet-collagen interaction was inhibited. Fourth, rabbit anti-human CIG stimulated human platelets to secrete the contents of their dense granules. The stimulation was not due to antibody complexes present in the solution. Fifth, a protein was extracted from wel -washed platelets and purified on affinity columns of anti-GIG antibodies. The isolated protein was found to bind to fibrous collagen. When the endothelial lining of blood vessels is breached, blood platelets come in contact with the components of connective tissue. The platelets adhere to the collagen in the connective tissue and are stimulated to release the contents of their dense granules, including ADP, Ca2+, and serotonin (1, 2). The Ca2+ and ADP are required for the subsequent platelet-platelet interactions, which lead to the formation of aggregates on a nucleus of the platelets bound to collagen (3). The aggregates form a platelet plug as an initial step in hemostasis. Because the collagen-platelet interaction is an important event in hemostasis, a great deal of interest has centered on the determination of the mechanism of the interaction. A major stimulus to interest in the collagen-platelet interaction came as a result of the investigations of Barber and Jamieson (4, 5). They presented evidence that membrane-bound glucosyl transferases on the surface of platelets bind to the galactose and glucose residues of collagen in an enzyme-substrate and enzyme-product complex, respectively. Further investigations by others tended to support this hypothesis (6, 7). However, evidence to the contrary has been presented. Menashi et al. (8) have shown that only denatured collagen can act as a substrate for the platelet glucosyl transferase, whereas only native, fibrous collagen serves as a platelet substrate in the collagen-platelet interaction (9, 10). In addition, others have shown that the carbohydrate residues of collagen can be completely destroyed by periodate oxidation without affecting the ability of the fibrous form of the oxidized collagen to interact normally with platelets (10, 11).Because the only current hypothesis as to the nature of the collagen receptor of platelets has been severe...

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“…Cellular fibronectin purified from conditioned medium of WI-3 8 human fibroblasts was kindly provided by Dr W. G. Carter. Cell-surface fibronectin of chick embryo fibroblasts was isolated as in [ 15]. The domain fragments of human plasma fibronectin were isolated after thermolysin digestion as in [6].…”

Section: Purification Of Fibronectin and Its Proteolytic Fragmentsmentioning

confidence: 99%

Monoclonal antibodies directed to two different domains of human plasma fibronectin: their specificities

et al. 1982

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Characterization of a major fibroblast cell surface glycoprotein

Cited by 178 publications

References 50 publications

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape.

Evidence that fibronectin is the collagen receptor on platelet membranes.

Monoclonal antibodies directed to two different domains of human plasma fibronectin: their specificities

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