Plasma fibronectin has previously been shown to bind to cell surface receptors which may mediate the assembly of soluble fibronectin into the extracellular matrix. Preliminary data suggest that the mechanism for incorporation of soluble fibronectin into the extracellular matrix is not functional in the transformed cell line, HT-1080. However, the mechanism can be induced when HT-1080 cells are treated with the glucocorticoid dexamethasone. The hypothesis that will be tested in these studies is that the elaboration of a fibronectin matrix requires the participation of specific receptors and that these receptors are no longer functional in transformed and differentiating cells which do not incorporate fibronectin into the extracellular matrix. Identification of the fibronectin receptor will be made using monoclonal antibodies which have been prepared against fibronectin-binding proteins present in detergent extracts of human fibroblasts. After identification of receptor(s), experiments will be done to determine whether this molecule(s) functions in the assembly of endogenous (cell synthesized) fibronectin in the extracellular matrix or in the attachment of cells to fibronectin matrices. Immunofluorescent colocalization studies will be done on both normal and transformed cell lines to analyze the distribution of fibronectin and its receptor in the cell layer. Fibronectin receptor expression will be studied in two transformed cell lines: a human fibrosarcoma, HT-1080, and a rat cell line, 2-R, which contain a temperature-sensitive transforming virus. Receptor expression also will be monitored during in vitro chondrogenesis of limb mesenchymal cells. HT-1080 cells will be used to study the potential role of the fibronectin receptor during dexamethasone induction of fibronectin matrix deposition. The 2-R cells will be used to determine whether the loss of a functional fibronectin receptor can be correlated with the loss of the pericellular matrix during transformation at the permissive temperature. Differentiating chondrocytes will be used to correlate fibronectin receptor loss with overt chondrogenesis and to determine whether the modulation of chondrocyte phenotype by Vitamin A is mediated by the fibronectin receptor. (A)