Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique α and common β subunits

Abstract

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed α and β, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed α and β, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the α subunits. Pulsechase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the α and β subunits of the class I and II antigens by peptide mapping indicated that the β subunits were identical while the α subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class Iβ and IIβ subunits were structurally realted to the β subunit of the fibronectin receptor described by others. However, none of these receptors shared the same α subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment of spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates. These results suggest that the class I and II receptors are two new members of a family of cell surface receptors for the extracellular matrix involved in mediating cell adhesion and shall be referred to as ECMRI and II. Each member of this family possesses a common β subunit and a unique α subunit. The class II receptor appears to be a primary mediator of specific cell adhesion to collagen. The promiscuous class I receptor also mediates cell adhesion to collagen but appears to interact with fibronectin and laminin as well. The class III receptor is not a member of this family of extracellular matrix adhesion receptors but does bind specifically to native collagens.