Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization (original) (raw)
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Monoclonal antibody to fibronectin which inhibits extracellular matrix assembly
FEBS Letters, 1987
A monoclonal antibody L8 specific to fibronectin was shown to inhibit fibronectin incorporation into the fibroblast extracellular matrix. Antibody L8 could not interact with fibronectin complexed with gelatin. The results suggest the existence of a specific site on the fibronectin molecule playing a critical role in the assembly of the fibronectin extracellular matrix. This site is located near the collagen-binding domain.
Journal of Cell Biology, 1987
Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent m...
Recent advances in research on fibronectin and other cell attachment proteins
Journal of Cellular Biochemistry, 1985
Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [I-lo]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. We shall also present various popular or provocative hypotheses and speculations about future work in the field.
Cell Biology International Reports, 1988
Monoclonal and polyclonal antibodies were raised against a placenta plasma membrane protein preparation, which was obtained by fractionation on Blue B dye matrix and by HPLC-anionexchange, and which was shown to contain fibronectin receptors. lmmunochemical and functional evidence showed that monoclonal antibody DH12 recognized the 8 subunit of the human fibronectin receptor on fibroblasts. This monoclonal antibody reacted with two proteins in Western blots and in double immune precipitations of whole cell preparations. Only the higher Mr protein became labeled by surface iodination of intact fibroblasts. The lower Mr protein is thought to be an intracellular precursor of the B subunit of the fibronectin receptor.
Journal of Cellular Biochemistry, 1985
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 l), 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 l), 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.
Focal adhesion sites and the removal of substratum-bound fibronectin
The Journal of Cell Biology, 1986
Fibronectin was not removed from the substratum beneath tbcal adhesion sites when fibroblasts spread in serum-free medium on adsorbed fibronectin substrata, or when fibroblasts spread in serumcontaining medium on covalently cross-linked fibronectin substrata. Under these conditions, there was colocalization between 140-kD fibroneetin receptors and focal adhesion sites. It was concluded that removal of adsorbed fibronectin from beneath focal adhesion sites was a mechanical process that required serum. The effect of serum was nonspecific since serum could be replaced by equivalent concentrations of serum albumin, ovalbumin, or gamma globulins. Quantitative measurements indicated that the presence
Cell biology international reports, 1982
Distribution of fibronectin-containing structures on the surface of different cell parts of substrate-spread normal mouse fibroblasts was examined by indirect immunofluorescence. Relatively large fibronectin fibrils were preferentially located on the surface of central cell part (endoplasm) while peripheral cell part (?ame??op?asm) was characterized by the presence of smaller structures ("spots" and "streaks"). Colcemid-treated fibroblasts had similar distribution of fibronectin; cytochalasin B-treated cells had mostly fibronectin spots on a?? parts of the surface. It is suggested that centripetal movement of receptor-attached fibronectin molecules on the cell surface may promote the formation of fibronectin fibrils.
Experimental Cell Research, 1997
and participate in the pathology of various inflamma-Fibronectin, a large dimeric glycoprotein synthe-tory conditions [1]. PMN responses are largely regusized and secreted by several cell types, mediates cell lated by adhesion to other cell types and extracellular adherence to surfaces. In infections and inflammatory matrix proteins [2, 3]. Migration, chemotaxis, cytotoxresponses, blood polymorphonuclear leukocytes (PMNs) icity, and phagocytosis of large particles are examples adhere to cells and matrix proteins during extravasaof adhesion-dependent PMN activities that involve intion and accumulation at inflammatory sites. The prestegrins as cell-surface adhesive receptors [4-6]. To acence of fibronectin in blood PMNs has been poorly cumulate at inflammatory loci, blood PMNs adhere studied, and the characteristics and subcellular localfirst to vascular endothelial cells, cross the vessel wall, ization of this endogenous adhesive molecule are pracand thereafter migrate in the connective tissue, entically unknown. By immunofluorescence flow cytomecountering fibronectin and other matrix proteins [2, 3]. try, purified rabbit antibodies and a monoclonal anti-Indeed, PMNs have been found to adhere to plasma body to plasma fibronectin reacted with isolated blood fibronectin by using integrin receptors [7]. PMNs also PMNs, only after permeabilization of the cells. By adhere readily to glass, nylon, or plastic surfaces in the Western blot analysis, the antibodies recognized, unabsence of serum or other exogenous proteins, although der reducing conditions, a protein with an apparent adherence may be modulated by the presence of serum molecular mass of 230 kDa in the cell lysate. Eleven or plasma [4, 6, 8]. Major advances on the molecular monoclonal antibodies to common frame fibronectin basis of leukocyte adhesion have been achieved during epitopes, including the RGD-containing cell-binding the last decade, but several determinants of PMN adhedomain, also reacted with PMN fibronectin by Western sion still remain unknown. blotting. In contrast, two antibodies to ED-A, the alter-Fibronectin is a high-molecular-weight glycoprotein natively spliced region characteristic of ''cellular'' fibronectin, were unreactive, but recognized platelet found in many extracellular matrices and in blood fibronectin. On average, 1 million PMNs contained 6.8 plasma [reviewed in 9]. It promotes cell adhesion and ng { 1.4 (SD) of fibronectin, as measured by sandwich affects cell morphology, migration, and differentiation. ELISA. Immunogold labeling and electron microscopy This molecule is composed of two similar subunits studies indicated localization of most fibronectin in bound by disulfide bridges. Each subunit is composed PMN granules. Moreover, double-immunofluorescence of three different types of internal repeats, designated and digital image analysis demonstrated colocaliza-I, II, and III, which in turn form structural and function of fibronectin with lactoferrin, a marker of spetional domains specialized for binding to cell surface cific (secondary) granules. The results indicate that integrin receptors or other extracellular matrix moleblood PMNs contain approximately 8000 molecules per cules. Different cell-type-specific fibronectin isoforms cell of intact ED-A-negative fibronectin localized arise by alternative splicing of the transcript of a single mainly in their specific granules.
International Journal of Cancer, 1989
We studied the function and localization of the fibronectin receptor complex in cultured normal and SV40-transformed human fibroblasts and in human fibrosarcoma cells by using monoclonal antibodies (MAbs) against the f.3 sub-unit of the receptor. Immunoprecipitation, fibronectin fragment affinity chromatography and immunoblotting results suggested that all the cells studied had similar amounts of the receptor. In normal fibroblasts MAbs additionally immunoprecipitated a smaller polypeptide, revealed as the precursor for the f. 3 subunit and another polypeptide shown to be the Q sub-unit of the VIA-I complex. The emergence of vinculin-positive focal adhesion