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 iaminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed a and 13, 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 a and 13, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the ct 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 a and 13 subunits of the class I and II antigens by peptide mapping indicated that the 13 subunits were identical while the ct 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 I13 and 1113 subunits were structurally related to the 13 subunit of the fibronectin receptor described by others. However, none of these receptors shared the same ct 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 or 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 13 subunit and a unique a subunit. The class II receptor appears to be a primary mediator of specific cell adhesion to collagen. Th...
Laminin 5 regulates anchorage and motility of epithelial cells through integrins α6β4 and α3β1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the α3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all α3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin α6β4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin α3β1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin α3β1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin α6β4, suggesting that signaling through β1 or β4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.
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