A SOMATIC-CELL GENETIC METHOD FOR IDENTIFICATION OF UNTARGETED MUTATIONS IN THE GLUCOCORTICOID RECEPTOR THAT CAUSE HORMONE-BINDING DEFICIENCIES

Mouse T lymphoma cell line W7MG1,which is killed by physiological conc entrations of glucocorticoid agonists, was used as a convenient geneti c system for isolating sublines containing mutant glucocorticoid recep tors (GR) with hormone-binding deficiencies. Partially hormone-resista nt cell clones were derived from chemically mutagenized cell populatio ns by selecting for growth in moderate concentrations of dexamethasone (Dex) and then screening for failure to grow in high Dex concentratio ns. Such clones are likely to have mutant GR. In GR cDNA clones from t he partially resistant cell sublines, three different functionally sig nificant mutations in the hormone-binding domain were identified: Leu- 569 changed to Phe (L569F), Leu-670 to Phe (L670F), and Met-672 to lie (M672I). Dose-response analyses with Dex and affinity labeling studie s with dexamethasone PI-mesylate in transiently transfected cells indi cated that all three mutant GR species had hormone-binding deficiencie s. However, at saturating Dex concentrations the mutant and wild type GR activated a hormone-inducible reporter gene to the same extent; thu s, these three mutations did not affect the ability of GR to activate transcription of the reporter gene after hormone was bound. In dose-re sponse curves conducted with several glucocorticoid agonists, mutation s L670F and M672I caused no change in ligand-binding specificities, wh ile mutation L569F caused a modest change in specificity. Quantitative hormone-binding studies conducted with mutant GR synthesized in cell- free reactions showed that mutant GR species L569F and M672I had reduc ed Dex-binding affinities both at 0 C and at 22 C in the presence of m olybdate. In contrast, for the L670F mutant, which exhibited the sever est deficiency in vivo, the hormone-binding deficiency in the cell-fre e system was evident only at 26-37 C and primarily in the absence of m olybdate. We propose that the L670F On is an activation-labile type of mutant, which binds hormone normally in the presence of heat shock pr otein 90 but loses hormone rapidly after dissociation from heat shock protein 90. These three mutations define two new subregions of the GR polypeptide that are important for hormone binding.