To evaluate the importance of GR, we previously transfected CHO cells with antisense-oriented human GR cDNA and selected stable cell lines. Although the cells showed lower GR activities and increased vulnerability to oxidants than did parental CHO cells, this antisense strategy was not appropriate for the generation of transgenic mice. Because GR is thought to exist as a homodimer in cells and has two active centers, formed by the juxtaposition of Cys58 and Cys63 of one peptide with His467 of the other monomer, we sought to modify cellular GR activities by a dominant negative mutant strategy. To inactivate one or both of the active sites in the dimer, we converted the 59th codon TGC(Cys58) of the human GR cDNA to AGC(Ser) or GCC (Ala), and/or changed the 468th codon CAC (His467) to CCC (Pro) or CTC (Leu), and transiently transfected CHO cells with these mutant cDNA's and measured GR activities. The cells transfected with each single mutant cDNA did not change GR activities, but cells transfected with double mutants showed lower activities(Cys58Ser/His467Pro, 21.9±1.1; Cys58Ser/His467Leu, 23.8±1.7; Cys58Ala/His467Pro, 25.4±1.8: Cys58Ala/His467Leu, 20.6±1.1 mU/mg pro) than were seen in CHO cells (30.3±0.9), while CHO cells transfected with authentic hGR cDNA had increases in GR activities of more than 20-fold (733.1±61.2) The cells transfected with mutated DNA's expressed as much hGR crossreactive proteins as did the cells transfected with authentic hGR cDNA in Western blots analysis, demonstrating that the transfection efficiencies were comparable and indicating that 58Cys and 467His are both essential for activities. All of the double mutants markedly attenuated the increase in GR activities in cells cotransfected with authentic hGR cDNA, suggesting the mutant protein might dimerize more efficiently with human GR protein than with the native CHO GR.
