Dg. Chen et Mr. Stallcup, THE HORMONE-BINDING ROLE OF 2 CYSTEINES NEAR THE C-TERMINUS OF THE MOUSE GLUCOCORTICOID RECEPTOR, The Journal of biological chemistry, 269(11), 1994, pp. 7914-7918
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 glucoco
rticoid receptors. To test the functional roles of these residues in t
he receptor's ability to bind hormone and activate transcription of ta
rget genes, the mouse glucocorticoid receptor cDNA was specifically mu
tated 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 fun
ction. In transient transfection assays, the concentration of dexameth
asone required for half maximal activation of a glucocorticoid-respons
ive reporter gene was increased by 10-40-fold by changing cysteine 742
to serine or cysteine 671 to serine or alanine. At saturating concent
rations 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 hor
mone was bound.