Our studies on the mechanism by which thrombin initiates division of cultured fibroblasts have shown that action at the cell surface is sufficient for cell activation. This prompted studies on the cell surface for interactions and events necessary for the stimulation of cell division. These studies led to the identification of protease nexin, a cell-secreted protein which mediates much of the specific binding of thrombin to the cell surface. Protease nexin is released by cells into the culture medium where it forms a covalent linkage with thrombin. The thrombin-protease nexin complexes then specifically bind to cells and are internalized and degraded. Our studies have shown that linkage of thrombin to protease nexin inactivates the thrombin. Moreover, protease nexin inhibits the stimulation by thrombin, and thus it represents a mechanism by which cells modulate their mitogenic response to thrombin. We have also demonstrated a cell surface binding site for unlinked thrombin and have shown that binding of thrombin to this site is not necessary for the stimulation of cell division. Past studies have shown that the proteolytic activity of thrombin is necessary for cell activation. In fact, all of our results point to a requirement for cleavage of one or more cell surface proteins by thrombin for the stimulation. At this point we have shown that several cell surface proteins are cleaved by thrombin. One of these has been identified as fibronectin. In addition, cell surface proteins of about 140 kilodaltons and 55 kilodaltons were thrombin-sensitive, but they have not yet been identified. Studies are underway to better resolve membrane proteins so we will be able to identify additional ones that might be thrombin-sensitive. We will study which of these cleavages are necessary for cell activation by determining whether they occur in a large series of cloned cell populations that are either responsive or unresponsive to the mitogenic action of thrombin. (A)

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA012306-15
Application #
3163622
Study Section
Cognition and Perception Study Section (CP)
Project Start
1979-06-01
Project End
1989-05-31
Budget Start
1985-06-01
Budget End
1986-05-31
Support Year
15
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Gurwitz, D; Cunningham, D D (1990) Neurite outgrowth activity of protease nexin-1 on neuroblastoma cells requires thrombin inhibition. J Cell Physiol 142:155-62
Rao, J S; Kahler, C B; Baker, J B et al. (1989) Protease nexin I, a serpin, inhibits plasminogen-dependent degradation of muscle extracellular matrix. Muscle Nerve 12:640-6
Cunningham, D D; Gurwitz, D (1989) Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1. J Cell Biochem 39:55-64
Gurwitz, D; Cunningham, D D (1988) Thrombin modulates and reverses neuroblastoma neurite outgrowth. Proc Natl Acad Sci U S A 85:3440-4
Thompson, J A; Lau, A L; Cunningham, D D (1987) Selective radiolabeling of cell surface proteins to a high specific activity. Biochemistry 26:743-50
Raben, D M; Yasuda, K M; Cunningham, D D (1987) Relationship of thrombin-stimulated arachidonic acid release and metabolism to mitogenesis and phosphatidylinositol synthesis. J Cell Physiol 130:466-73
Thompson, J A; Cunningham, D D (1987) Identification of cell surface proteins sensitive to proteolysis by thrombin. Methods Enzymol 147:157-64
Raben, D M; Yasuda, K; Cunningham, D D (1987) Modulation of thrombin-stimulated lipid responses in cultured fibroblasts. Evidence for two coupling mechanisms. Biochemistry 26:2759-65
Bergman, B L; Scott, R W; Bajpai, A et al. (1986) Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I. Proc Natl Acad Sci U S A 83:996-1000
Cunningham, D D; Farrell, D H (1986) Thrombin interactions with cultured fibroblasts: relationship to mitogenic stimulation. Ann N Y Acad Sci 485:240-8

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