Our goal is to elucidate mechanisms by which regulatory serine proteases are controlled at and near the cell surface by three protease nexins (PN-1, PN-2 and PN-3) which we identified in earlier studies. The PNs are protein protease inhibitors that are synthesized and released by a variety of cultured cells. They form covalent complexes with certain proteases in the extracellular environment; the complexes bind back to the cells and are rapidly internalized and degraded. With purified PN-1 and seven regulatory proteases it inactivates, we will measure second order association rate constants to provide a quantitative foundation for our studies. Then, based on our finding that the surface of fixed fibroblasts accelerates the reaction between PN-1 and thrombin, we will determine if the reactions between PN-1 and the other proteases are similarly accelerated. Some of the acceleration of the reaction between PN-1 and thrombin appears to involve cell surface/extracellular matrix glycosaminoglycans. We will check this by treating fibroblasts with glycosaminoglycan lyases, fixing the cells, and examining the effect of this on the ability of the cells to accelerate the reactions. In complimentary studies we will examine the effects of purified glycosaminoglycans, in the absence of cells, on the reactions between PN-1 and the proteases. About 50% of the acceleration of the PN-1 and thrombin reactions appears to involve the Mr=150,000 cell surface binding sites for thrombin. We will study the basis of this and determine if there are cell surface binding sites for the six other regulatory proteases that might similarly participate in their control. With PN-2 that we recently purified to homogeneity, we will screen for serine proteases it effectively inactivates, measure second order association rate constants with them, and begin studies on PN-2 along the lines of the above studies for PN-1. Finally, we will purify PN-3 from serum-free culture medium conditioned by fibroblasts.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM031609-04
Application #
3279747
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1983-07-01
Project End
1991-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
4
Fiscal Year
1986
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
Kim, T; Choi, B H; Choe, W et al. (1992) Expression of protease nexin-II in human dorsal root ganglia. A correlative immunocytochemical and in situ hybridization study. Mol Chem Neuropathol 16:225-39
Cunningham, D D; Wagner, S L; Farrell, D H (1992) Regulation of protease nexin-1 activity by heparin and heparan sulfate. Adv Exp Med Biol 313:297-306
Cunningham, D D (1992) Regulation of neuronal cells and astrocytes by protease nexin-1 and thrombin. Ann N Y Acad Sci 674:228-36
Van Nostrand, W E; Wagner, S L; Shankle, W R et al. (1992) Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients. Proc Natl Acad Sci U S A 89:2551-5
Van Nostrand, W E; Farrow, J S; Wagner, S L et al. (1991) The predominant form of the amyloid beta-protein precursor in human brain is protease nexin 2. Proc Natl Acad Sci U S A 88:10302-6
Van Nostrand, W E; Schmaier, A H; Farrow, J S et al. (1991) Protease nexin-2/amyloid beta-protein precursor in blood is a platelet-specific protein. Biochem Biophys Res Commun 175:15-21
Choi, B H; Suzuki, M; Kim, T et al. (1990) Protease nexin-1. Localization in the human brain suggests a protective role against extravasated serine proteases. Am J Pathol 137:741-7
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
Cavanaugh, K P; Gurwitz, D; Cunningham, D D et al. (1990) Reciprocal modulation of astrocyte stellation by thrombin and protease nexin-1. J Neurochem 54:1735-43
Van Nostrand, W E; Wagner, S L; Farrow, J S et al. (1990) Immunopurification and protease inhibitory properties of protease nexin-2/amyloid beta-protein precursor. J Biol Chem 265:9591-4

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