The hormone-binding role of 2 cysteines near the C terminus of the mouse glucocorticoid receptor.

Previous biochemical analyses with covalent affinity labels and thiol-blocking reagents suggested possible roles for one methionine residue and multiple cysteine residues in binding of steroid to the 250-amino acid hormone-binding domain at the C-terminal end of mammalian glucocorticoid receptors. To test the functional roles of these residues in the receptor's ability to bind hormone and active transcription of target genes, the mouse glucocorticoid receptor cDNA was specifically mutated to cause single amino acid substitutions for methionine 610 and for each of the 5 cysteines (at positions 628, 644, 649, 671, and 742) in the hormone-binding domain. Among these 6 residues, only mutations in cysteine 671 and cysteine 742 caused substantial reductions in function. In transient transfection assays, the concentration of dexamethasone required for half-maximal activation of a glucocorticoid-responsive reporter gene was increased by 10-40-fold by changing cysteine 742 to serine or cysteine 671 to serine or alanine. At saturating concentrations of dexamethasone, the mutant receptors activated the reporter gene to the same extent as the wild type receptor, indicating that the mutations affected only the hormone-binding function of the receptor and not its ability to bind DNA or activate transcription once the hormone was bound.