PROTEIN-PROTEIN INTERACTIONS ARE IMPLIED IN GLUCOCORTICOID RECEPTOR MUTANT 465-ASTERISK-MEDIATED CELL-DEATH

Previously we have shown that ICR-27, a clone of glucocorticoid-resist ant human leukemic T cells, showed rapid cell loss upon transient tran sfection with plas mids expressing certain fragments of the human gluc ocorticoid receptor lacking the ligand binding domain, An extreme exam ple was the frameshift GR mutant 465, mutated after amino acid 465. T his generated a novel al-amino acid ''tail,'' beginning within the sec ond zinc finger of the human glucocorticoid receptor DNA binding domai n, a region required for ICR-27 cell death caused by hologlucocorticoi d receptor plus hormone, The cell loss mediated by 465 was faster but quantitatively equivalent to that caused by hologlucocorticoid recept or plus hormone, We are therefore investigating the mechanism of actio n of 465. We overexpressed 465* with or without a glutathione S-trans ferase tag fused to its N terminus and tested its ability to affect gl ucocorticoid response element (GRE)-driven reactions in vitro, Partial ly purified 465 showed little binding to a consensus GRE, caused virt ually no stimulation of transcription from a GRE, and failed to inhibi t GR-driven transcription, However, GST-465 ''trapped'' several prote ins from ICR-27 cell extracts, including c-Jun; recombinant c-Jun also was bound in vitro, In co-transfection assays of CV-1 cells, 465 exp ression reduced phorbol ester (12-O-tetradecanoylphorbol-13-acetate) t ranscriptional activation from a promoter containing multiple AP-1 sit es. Further studies proved the repressive activity of 465 was c-Jun-s pecific and not due to squelching artifacts, The data suggest that int eraction of 465 with other proteins, such as c-Jun, might be responsi ble for its cell killing function.