Section 10
Chapter 9,068

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

Neff, S.; Sadowski, C.; Miksicek, R.J.

Molecular Endocrinology 8(9): 1215-1223


ISSN/ISBN: 0888-8809
PMID: 7838154
DOI: 10.1210/mend.8.9.7838154
Accession: 009067440

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To further our understanding of the role played by cysteine residues in ligand recognition by the human estrogen receptor (hER), we have individually mutated residues 381, 417, 447, and 530 within the hormone-binding domain from cysteine to serine. We have also examined 10 additional nonconservative amino acid changes at C530. Four mutants were identified with defects in their ability to stimulate transcription in response to hormone: C447S, C530P, C530W, and C530G. Accumulation of the mutant proteins to comparable steady state levels after transient expression in COS-7 cells leads us to conclude that none of these substitutions results in increased turnover of receptor protein. None of these mutations had a reduced affinity for estradiol when compared with wild-type hER [dissociation constant (Kd) = 0.29 nM]. Although C447 and C530 reside outside of motifs previously defined as being necessary for DNA binding or receptor dimerization, all four of these mutants unexpectedly displayed altered DNA-binding properties when analyzed using a gel mobility shift assay. Their loss of DNA binding could be overcome, at least in part, by hormone treatment or by the addition of antibody. Additionally, mutations C447S, C530S, and C530P displayed patterns 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 ability of this receptor to bind estradiol. These mutations, however, can effect DNA-binding activity and the distribution of hER within cells.

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