Using a model system to study fibronectin fibrillogenesis, we have proposed that the incorporation of soluble fibronectin into the extracellular matrix involves the binding of fibronectin to specific cell surface receptors which recognize a cell binding site in the amino-terminus of the fibronectin molecule. We have recently identified an 18K protein on the surface of human embryonic skin fibroblasts which binds to the amino terminal 70K fragment of fibronectin (Appendix). The purpose of this proposal is to understand the function of the 18K protein and to test the hypothesis that the 18K protein is the previously described matrix assembly receptor. The 18K protein will be purified by affinity chromatography on Affi-gel coupled to 70K fragment. Monoclonal antibodies prepared against this protein will be tested for the ability to disrupt cell mediated fibronectin fibrillogenesis and fibronectin mediated cell attachment and spreading. The 18K protein will be further purified by immuno-affinity chromatography, incorporated into liposomes and tested for the ability to bind purified fibronectin and fibronectin fragments. Purified 18K will be sequenced and examined for homology with other known fibronectin receptors. The possible regulation of the 18K protein by growth factors and hormones will be studied in normal fibroblasts and fibrosarcoma (HT-1080) cells. Regulation of 18K protein by changes in phosphorylation, actin association and surface distribution will be investigated. The susceptibility of the 18K protein to proteolysis by urokinase and plasmin will be tested to understand whether the dexamethasone induced increase in fibronectin binding sites is related to increases in the level of plasminogen activator inhibitor. Experiments will be done to identify whether the model system which we have developed for soluble fibronectin is distinct from the system which has been described by others using substrate absorbed fibronectin. The regulation of 18K protein function may have important implications in morphogenesis (e.g. cell migration and differentiation) and pathological processes such as fibrosis and tumor cell metastasis.
