This project proposes to characterize the structure and activities of an endogenous growth regulator that may play a role in density dependent inhibition of growth. We have purified this growth inhibitory activity from medium conditioned by exposure to density-inhibited fibroblasts and have identified a Mr=13,000 polypeptide as the molecule responsible for growth inhibition. This purified factor is designated FGR-s (13 K), which stands for Fibroblast Growth Regulator that is shed or released into the medium in soluble form. We have generated a monoclonal antibody that binds this Mr=13,000 polypeptide and that neutralizes its growth inhibitory activity. Finally, we have demonstrated that this monoclonal antibody will reverse the effect of density inhibition, suggestion that FGR-s (13 K) or a cross-reactive precursor may be functioning in the normal mechanism of density-dependent growth control. We wish to continue these studies, with particular emphasis on identifying the membrane bound form of FGR-s (13 K). The specific objectives of the research include: (a) to chemically characterize FGR-s (13 K) in terms of partial amino acid sequence and post-translationally modified residues; (b) to identify the membrane-bound form of GFR-s (13 K) and to analyze the structural relationships between FGR-s (13 K) and the membrane-bound molecule; (c) to identify and characterize the receptors for FGR-s (13 K) and to trace the fate of the ligand-receptor complex; (d) to survey the production and binding of FGR-s (13 K) in normal and transformed cells, fibroblasts and other cell types, cells at different densities and at different stages of the cell cycle; and (e) to probe the cellular sites of action of FGR-s (13 K) and the biochemical mechanisms of growth control. All of these studies should contribute to our understanding of the phenomenon of density-dependent inhibition of growht in cultured cells and establish the general biological significance of endogenous growth inhibitors in cellular homeostasis.
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