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

作者：Carol K. Wrenn, Benita S. Katzenellenbogen · 发表于：Journal of Biological Chemistry · 年份：1993 · DOI：10.1016/s0021-9258(20)80497-9 · 被引用次数：187 · 研究领域：Estrogen and related hormone effects、Steroid Chemistry and Biochemistry、Biotin and Related Studies

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 beta-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...