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 concentrations of glucocorticoid agonists, was used as a convenient genetic system for isolating sublines containing mutant glucocorticoid receptors (GR) with hormone-binding deficiencies. Partially hormone-resistant cell clones were derived from chemically mutagenized cell populations by selecting for growth in moderate concentrations of dexamethasone (Dex) and then screening for failure to grow in high Dex concentrations. Such clones are likely to have mutant GR. In GR cDNA clones from the partially resistant cell sublines, three different functionally significant mutations in the hormone-binding domain were identified: Leu-569 changed to Phe (L569F), Leu-670 to Phe (L670F), and Met-672 to Ile (M672I). Dose-response analyses with Dex and affinity labeling studies with dexamethasone 21-mesylate in transiently transfected cells indicated that all three mutant GR species had hormone-binding deficiencies. However, at saturating Dex concentra...