This project proposes to characterize the structure and activities of an endogenous growth inhibitor that has been purified from medium """"""""conditioned"""""""" by exposure to density-inhibited mouse 3T3 fibroblasts. This factor (Mr 13,000) is designated FGR-s (13 K), which stands for Fibroblast Growth Regulator that is shed or secreted into the medium in a soluble form. We have generated a monoclonal antibody that binds this Mr = 13,000 polypeptide and that neutralizes its growth inhibitory activity. Partial amino acid sequence results indicate that FGR-s (13 K) is homologous to a family of thiol proteinase inhibitors called the Cystatins. The specific objectives of the proposed research are: (1) to identify a cDNA for FGR-s (13 K) and to determine the nucleotide sequence of the coding region in the cDNA; (2) to express the recombinant FGR-s (13 K) polypeptide in E. coli and to purify the active protein form the enriched source; (3) to analyze the components of the target cell with which FGR- s (13 K) interacts; (4) to analyze the production and binding of FGR-s (13 K) in normal versus transformed cells, fibroblasts versus other cell types, cells at different densities and at different densities and at different stages of the cell cycle; (5) to probe the cellular sites of action of FGR-s (13 K) and to study the biochemcial mechanism of growth control. The successful attainment of the initial goals would provide a good source of FGR-s (13 K) for the analysis of its mechanism of action. These latter studies should, in turn, contribute to our understanding of the general biological significance of endogenous growth inhibitors in cellular homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027203-09
Application #
3274599
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-02-01
Project End
1992-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Osteopathy
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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Cowles, E A; Agrwal, N; Anderson, R L et al. (1990) Carbohydrate-binding protein 35. Isoelectric points of the polypeptide and a phosphorylated derivative. J Biol Chem 265:17706-12
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Cowles, E A; Moutsatsos, I K; Wang, J L et al. (1989) Expression of carbohydrate binding protein 35 in human fibroblasts: comparisons between cells with different proliferative capacities. Exp Gerontol 24:577-85
Agrwal, N; Wang, J L; Voss, P G (1989) Carbohydrate-binding protein 35. Levels of transcription and mRNA accumulation in quiescent and proliferating cells. J Biol Chem 264:17236-42
Laing, J G; Robertson, M W; Gritzmacher, C A et al. (1989) Biochemical and immunological comparisons of carbohydrate-binding protein 35 and an IgE-binding protein. J Biol Chem 264:1097-10

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