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Inhibition of mink lung epithelial cell proliferation by transforming growth factor-beta is coupled through a
P H Howe1, M R Cunningham, E B Leof
1Department of Cell Biology, School of Medicine, Vanderbilt University, Nashville, TN 37232.
Abstract:
Transforming growth factor beta 1 (TGF beta 1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGF beta 1 can be inhibited by prior exposure of the cells to nanogram concentrations of pertussis toxin (PT), suggesting the involvement of a guanine-nucleotide-binding regulatory protein (G-protein) in mediating TGF beta 1-induced growth inhibition. To characterize further this G-protein dependence, we have isolated, by chemical mutagenesis, a CCL64 variant (CCL64-D1) that is resistant to TGF beta 1. Whereas in the parental CCL64 cells TGF beta 1 stimulates both GTP[35S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGF beta 1 is without effect. Quantitative immunoblotting with antisera for G-protein alpha- and beta-subunits, as well as PT-catalysed ADP-ribosylation analyses, revealed no appreciable changes in the level of G-protein expression in the CCL64-D1 variants compared with parental cells. In contrast with another TGF beta-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGF beta cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGF beta 1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGF beta 1 receptor binding to G-protein activation.
Insights
Transforming growth factor beta 1 (TGF beta 1) growth inhibition in mink lung cells involves a G-protein. A TGF beta 1-resistant variant shows impaired G-protein activation, indicating its crucial role in TGF beta 1 signaling.
Area of Science:
- Cell biology
- Molecular signaling
- G-protein coupled receptors
Background:
- Transforming growth factor beta 1 (TGF beta 1) is a key regulator of cell proliferation.
- TGF beta 1's inhibitory effects on mink lung epithelial cells (CCL64) are mediated by G-proteins.
- Pertussis toxin (PT) sensitivity suggests G-protein involvement in TGF beta 1-induced growth inhibition.
Purpose of the Study:
- To investigate the role of G-proteins in TGF beta 1-mediated growth inhibition.
- To characterize a TGF beta 1-resistant cell line (CCL64-D1) and its signaling pathways.
- To elucidate the mechanism of TGF beta 1 signal transduction in CCL64 cells.
Main Methods:
- Chemical mutagenesis to isolate TGF beta 1-resistant CCL64-D1 variant.
- Assays for GTP[35S] binding and GTPase activity.
- Quantitative immunoblotting and PT-catalyzed ADP-ribosylation for G-protein analysis.
- Characterization of TGF beta cell-surface binding proteins.
Main Results:
- CCL64-D1 cells are resistant to TGF beta 1-induced growth inhibition.
- TGF beta 1 fails to stimulate GTP binding or GTPase activity in CCL64-D1 cells.
- G-protein expression levels are unchanged in CCL64-D1 variants.
- CCL64-D1 cells retain TGF beta cell-surface binding proteins, unlike other resistant clones.
Conclusions:
- G-protein activation is essential for TGF beta 1-mediated growth inhibition in CCL64 cells.
- The defect in CCL64-D1 cells lies downstream of TGF beta receptor binding, affecting G-protein coupling.
- This study highlights the critical role of G-protein signaling in mediating TGF beta 1's cellular responses.