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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037785-07
Application #
3175609
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-01-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1992-06-30
Support Year
7
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Albany Medical College
Department
Type
Schools of Medicine
DUNS #
City
Albany
State
NY
Country
United States
Zip Code
12208
Panetti, T S; McKeown-Longo, P J (1993) The alpha v beta 5 integrin receptor regulates receptor-mediated endocytosis of vitronectin. J Biol Chem 268:11492-5
Panetti, T S; McKeown-Longo, P J (1993) Receptor-mediated endocytosis of vitronectin is regulated by its conformational state. J Biol Chem 268:11988-93
Ciambrone, G J; McKeown-Longo, P J (1992) Vitronectin regulates the synthesis and localization of urokinase-type plasminogen activator in HT-1080 cells. J Biol Chem 267:13617-22
Kowalczyk, A P; McKeown-Longo, P J (1992) Basolateral distribution of fibronectin matrix assembly sites on vascular endothelial monolayers is regulated by substratum fibronectin. J Cell Physiol 152:126-34
Ciambrone, G J; McKeown-Longo, P J (1990) Plasminogen activator inhibitor type I stabilizes vitronectin-dependent adhesions in HT-1080 cells. J Cell Biol 111:2183-95
Kowalczyk, A P; Tulloh, R H; McKeown-Longo, P J (1990) Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation. Blood 75:2335-42
Allio, A E; McKeown-Longo, P J (1988) Extracellular matrix assembly of cell-derived and plasma-derived fibronectins by substrate-attached fibroblasts. J Cell Physiol 135:459-66
Mann, D M; McKeown-Longo, P J; Millis, A J (1988) Binding of soluble fibronectin and its subsequent incorporation into the extracellular matrix by early and late passage human skin fibroblasts. J Biol Chem 263:2756-60
McKeown-Longo, P J; Etzler, C A (1987) Induction of fibronectin matrix assembly in human fibrosarcoma cells by dexamethasone. J Cell Biol 104:601-10