Structure-function analysis of the hormone binding domain of the human estrogen receptor by region-specific mutagenesis and phenotypic screening in yeast

To investigate the structural requirements for recognition and response to ligands by the human estrogen receptor (hER), a series of point mutations were generated in the hormone binding domain (HBD) of the receptor using a limited formic acid treatment of its cDNA. Receptors having a reduced sensitivity to the estrogen, 17 β-estradiol (E₂), or to the antiestrogen, trans-hydroxytamoxifen, were selected from a library of intact hER cDNAs containing these mutant HBDs by expression and phenotypic screening in yeast (Saccharomyces cerevisiae). Several were sequenced, and the encoded receptors were characterized in both yeast and mammalian (Chinese hamster ovary) cells using hormone-binding and transactivation assays. In general, parallel phenotypes were observed in yeast and in Chinese hamster ovary cells following estrogen exposure. We report on 15 receptors having point mutations located at various positions throughout the HBD. Four categories of mutants were identified: 1) those showing no change from wild type in their response to E₂; 2) those showing a greatly reduced transactivation response over the range of ligand concentrations tested; 3) those requiring much higher concentrations of E₂ for maximal transactivation, indicating a reduced sensitivity to ligand; and 4) those showing reduced response to E₂, but little change in response to trans-hydroxytamoxifen in yeast. Two mutations in the carboxyl terminus of the HBD eliminated hormone-dependent transactivation despite the continued ability to bind E₂ with high affinity. Therefore, our results show a separation of the transactivation and hormone-binding functions of the hER, and indicate that the integrity of many regions throughout the large, approximately 250-amino acid HBD is important for these functions. Our studies also demonstrate the advantages of using regional mutagenesis combined with phenotypic screening in yeast to complement sitedirected mutagenesis when investigating a large, functionally complex region.