Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).
Transforming growth factor beta (TGF beta 1) is a potent regulator of DNA synthesis and cellular proliferation. In this study, we investigated whether the growth stimulatory signal of TGF beta 1 is transduced intracellularly by guanine nucleotide regulatory proteins (G-proteins). In plasma membranes from AKR-2B cells, TGF beta 1 increased binding of the radiolabelled, non-hydrolysable GTP analogue, guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]), in a dose-dependent manner. Maximal effects occurred between 0.4 and 1.0 nM-TGF beta 1. Specific binding of GTP[35S] occurred with a Kd of 3.2 x 10(-8) M which was not affected by addition of TGF beta 1. Instead, TGF beta 1 increased the number of available binding sites for GTP[35S] from 16.2 +/- 1.2 to 21.6 +/- 2.1 pmol/mg of protein. GTP[35S] binding was both nucleotide- and growth-factor-specific. Only guanine nucleotides were able to compete for binding, and of the growth factors tested (epidermal growth factor, platelet-derived growth factor, insulin, TGF beta 1 and TGF beta 2) only TGF beta 1 affected GTP[35S] binding. TGF beta 1 increased GTPase activity, as determined by the release of 32PO4(3-) from GTP gamma[32P], from 116 +/- 5.5 to 175 +/- 4.3 pmol/mg of protein following a 15 min incubation. Pretreatment of the membranes with pertussis toxin inhibited both TGF beta 1-stimulated binding of GTP[35S] as well as TGF beta 1-stimulated GTPase activity. These inhibitory actions of pertussis toxin were associated with toxin-induced ADP-ribosylation of a 41 kDa protein. Furthermore, the stimulatory effects of TGF beta 1 on c-sis mRNA expression were shown to be pertussis-toxin sensitive and could be mimicked by direct activation of G-proteins with AIF4-. These results demonstrate that in AKR-2B cells a pertussis-toxin-sensitive guanine nucleotide regulatory protein(s) is coupled to TGF beta 1 receptor binding.
Howe
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PH
Department of Cell Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232.
Leof
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D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008648:Mesoderm / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D037342:Pertussis Toxin; D015398:Signal Transduction; D015291:Transforming Growth Factors / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D010566:Virulence Factors, Bordetella; D015291:Transforming Growth Factors; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins