INHIBITION OF MINK LUNG EPITHELIAL-CELL PROLIFERATION BY TRANSFORMING GROWTH-FACTOR-BETA IS COUPLED THROUGH A PERTUSSIS-TOXIN-SENSITIVE SUBSTRATE

Transforming growth factor β1 (TGFβ1) inhibits the proliferative response of mink lung epithelial cells (CCL64) to serum and to epidermal growth factor (EGF). This response to TGFβ1 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 TGFβ1-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 TGFβ1. Whereas in the parental CCL64 cells TGFβ1 stimulates both GTP[35S] (guanosine 5'-[γ-[35S]thio]triphosphate) binding and GTPase activity, in the CCL64-D1 variants TGFβ1 is without effect. Quantitative immunoblotting with antisera for G-protein α- and β-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 TGFβ-resistant clone, MLE-M, which we show lacks detectable type I receptor protein, the CCL64-D1 cells retain all three TGFβ cell-surface binding proteins. On the basis of these studies, we propose that a necessary component of TGFβ1-mediated growth inhibition in CCL64 epithelial cells is the coupling of TGFβ1 receptor binding to G-protein activation.