The cell substrate attachment (CSAT) antigen is an integral membrane glycoprotein complex that participates in the adhesion of cells to extracellular molecules. The CSAT monoclonal antibody, directed against this complex, inhibited adhesion of cardiac and tendon fibroblasts and ske[etat myoblasts to both laminin and fibronectin, thus implicating the CSAT antigen in adhesion to these extracellular molecules.Equilibrium gel filtration was used to explore the hypothesis that the CSAT antigen functions as a cell surface receptor for both laminin and fibronectin. In this technique, designed for rapidly exchanging equilibria, the gel filtration column is pre-equilibrated with extracellular ligand to ensure receptor occupancy during its journey through the column. Both laminin and fibronectin formed complexes with the CSAT antigen. The association with laminin was inhibited by the CSAT monoclonal antibody; the associations with both fibronectin and laminin were inhibited by synthetic peptides containing the fibronectin cell-binding sequence. Estimates of the dissociation constants by equilibrium gel filtration agree well with those available from other measurements. This suggests that these associations are biologically significant. SDS PAGE showed that all three glycoproteins comprising the CSAT antigen were present in the antigen-ligand complexes. Gel filtration and velocity sedimentation were used to show that the three bands comprise an oligomeric complex, which provides an explanation for their functional association. The inhibition of adhesion by the CSAT monoclonal antibody and the association of the purified antigen with extracellular ligands are interpreted as strongly implicating the CSAT antigen as a receptor for both fibronectin and laminin and perhaps for other extracellular molecules as well.The interaction of cells with components of the extracellular matrix plays a major role in determining cell morphology, cell migration, and tissue maintenance (1). Putative sites of cell-matrix contact have been identified and studied using various microscopic techniques. In fibroblasts they occur in regions where the cellular cytoskeleton and associated components interact with the cell surface (2-4). Integral membrane proteins are hypothesized to be present in these sites and to serve as transmembrane links connecting cytoskeletal and the extracellular matrix components. These transmembrane proteins would then serve as dual receptors for cytoskeletal and extracellular matrix components.We have identified a candidate for such a cell surface molecule using an adhesion and morphology perturbing cell 2134 substrate attachment (CSAT) ~ monoclonal antibody (5). This monoclonal antibody alters the adhesion of several different cell types in culture. The nature and degree of perturbation by the CSAT monoclonal antibody is characteristic for different cell types (6). The antigen to which the antibody is directed has been purified by immunoaffinity chromatography and partially characterized (7). It is a complex of th...

We have described a monoclonat antibody that rounds and detaches chick skeletal myoblasts and myotubes from extracellular substrata. The antibody also inhibits the attachment of myogenic cells to a gelatin-coated substratum but has no detectable effect on myoblast fusion. The cellular response to antibody treatment varies wffh differentiation and cell type. Young myoblasts and myotubes are rapidly rounded and detached by the antibody. Older myotubes require longer incubation times or higher antibody titers for rounding and detachment. Chick embryo fibroblasts, cardiac cells, and neurons are not similarly rounded and remain attached. Since the antibody also detaches cells from embryonic muscle tissue explants, the cell-substratum interaction perturbed by the antibody appears relevant to the in vivo interaction of myogenic cells with their extracellular matrices. Binding studies using iodinated antibody revealed 2-4 x 105 sites per myoblast with an apparent Kd in the range of 2-5 x 10 -9 molar. Embryo fibroblasts bind antibody as well and display approximately twice the number of binding sites per cell. The fluorescence distribution of antigen on myoblasts and myotubes is somewhat punctate and particularly bright along the edge of the myotube. The distribution on fibroblasts was also punctate and was particularly bright along the cell periphery and portions of stress fibers. For both cell types the binding was distinctly different than that reported for collagen, fibronectin, and other extracellular molecules. The antigen, as isolated by antibody affinity chromatography, inhibits antibody-induced rounding. SDS PAGE reveals two unique polypeptides migrating in the region of ~120 and 160 kilodaltons (kd). The most straightforward mechanism for the antibody-induced rounding and detachment is the perturbation of a membrane molecule involved in adhesion. The hypothesized transmembrane link between extracellular macromolecules and the cytoskeleton provides an ovbious candidate.

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Neff et al. (1982, J. Cell Biol., 95:654-666) have described a monoclonal antibody, CSAT, directed against a cell surface antigen that participates in the adhesion of skeletal muscle to extracellular matrices. We used the same antibody to compare and parse the determinants of adhesion and morphology on myogenic and fibrogenic cells. We report here that the antigen is present on skeletal and cardiac muscle and on tendon, skeletal, dermal, and cardiac fibroblasts; however, its contribution to their morphology and adhesion is different. The antibody produces large alterations in the morphology and adhesion of skeletal myoblasts and tendon fibroblasts; in contrast, its effects on the cardiac fibroblasts are not readily detected. The effects of CSAT on the other cell types, i.e., dermal and skeletal fibroblasts, cardiac muscle, 5-bromodeoxyuridine-treated skeletal muscle, lie between these extremes. The effects of CSAT on the skeletal myoblasts depends on the calcium concentration in the growth medium and on the culture age. We interpret these differential responses to CSAT as revealing differences in the adhesion of the various cells to extracel[ular matrices. This interpretation is supported by parallel studies using quantitative assays of cell-matrix adhesion. The likely origin of these adhesive differences is the progressive display of different kinds of adhesion-related molecules and their organizational complexes on increasingly adhesive cells. The antigen to which CSAT is directed is present on all of the above cells and thus appears to be a lowest common denominator of their adhesion to extracellular matrices.Adhesive differences among embryonic cells are thought to play a prominent role in directing morphogenesis (2, 32). Numerous examples of such differential adhesions have been described and evidence has been presented that relates them to morphogenic phenomena. The histotypic sorting out of embryonic cells is rationalized as reflecting the graded, differential adhesive interactions of the constituent cells (24,26,30,31). In the nervous system, the high specificity of retinotectal interactions appears to reflect the adhesive gradients generated by the retinal and tectal cells (12,24). The migration of neuronal processes to their peripheral targets is also highly specific and is hypothesized to reflect the differential affinities of the processes for extracellular substrates (7,23).Recently membrane proteins have been identified and isolated using adhesion-perturbing monoclonal antibodies whose antigens localize in the region of cell-substratum adhesions (13, 27, 28). One of these antibodies, called CSAT, is directed against an antigen that participates in the adhesion of embryonic skeletal muscle to extracellular matrices (17,18,27). T The active determinant appears to be part of an integral membrane protein complex located in the vicinity of transmembrane assemblies involved in adhesion. The identification of such adhesion-related molecules allows an investigation of their role in regulating the...

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