MUTATIONAL ANALYSIS OF CYSTEINE RESIDUES WITHIN THE HORMONE-BINDING DOMAIN OF THE HUMAN ESTROGEN-RECEPTOR IDENTIFIES MUTANTS THAT ARE DEFECTIVE IN BOTH DNA-BINDING AND SUBCELLULAR-DISTRIBUTION

To further our understanding of the role played by cysteine residues i n ligand recognition by the human estrogen receptor (hER), we have ind ividually mutated residues 381, 417, 447, and 530 within the hormone-b inding domain from cysteine to serine. We have also examined 10 additi onal nonconservative amino acid changes at C530. Four mutants were ide ntified with defects in their ability to stimulate transcription in re sponse to hormone: C447S, C530P, C530W, and C530G. Accumulation of the mutant proteins to comparable steady state levels after transient exp ression in COS-7 cells leads us to conclude that none of these substit utions results in increased turnover of receptor protein. None of thes e mutations had a reduced affinity for estradiol when compared with wi ld-type hER [dissociation constant (K-d) = 0.29 nM]. Although C447 and C530 reside outside of motifs previously defined as being necessary f or DNA binding or receptor dimerization, all four of these mutants une xpectedly displayed altered DNA-binding properties when analyzed using a gel mobility shift assay. Their loss of DNA binding could be overco me, at least in part, by hormone treatment or by the addition of antib ody. Additionally, mutations C447S, C530S, and C530P displayed pattern s of subcellular localization in the absence of hormone that differed from wild-type hER. These results argue that cysteine residues 447 and 530 within the hormone-binding domain play no direct role in the abil ity of this receptor to bind estradiol. These mutations, however, can effect DNA-binding activity and the distribution of hER within cells.