Abstract
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 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 site-directed mutagenesis when investigating a large, functionally complex region.
Highlights
While OHT was anagonist of reporter gene activity in yeast, this ligand was a pure Ezantagonist in ouCr HO cell system (Fig. 5,panels D-F),and did not show any of the agonistic activity seen in yeast.despite the fact that the mutants displayed widely different levels of transactivation in response E2 needed foractivity of the wild type ER in yeast uersus CHO
Sincewe found that theKd of wild type and mutantERs
Ez-stimulated CAT activity byOHTfollowed similar curves for estradiol was the same in yeast and CHOcell extracts (Fig. 5, panels D-F).This result indicates that thesensitivities (Table I, Footnote b), it is possible that thehigher ligand conof the mutants to Ez and OHT are altered coordinately
Summary
Dept.of Physiology and Biophysics, Universityof Illinois, 524Bum Hall, 407 S. Droxytamoxifen; CHO, Chinese hamster ovary; CAT, chloramphenicol acetyltransferase;wt, wild type; ERE, estrogen response element; kb, kilobase pairk.);bp, base paids); X-gal, 5-bromo-4-chloro-3-indoyl0-0galactoside; mER, mouse estrogen receptor. To complement and extend our site-directed analysis of the ing uracilbefore transformation, and was plated onto the same medium hER HBD, we report our useof regional mutagenesis to create lacking both uracil and tryptophan afterwards. These were inverteodver a glass disohf chloroform for 15 min to located throughout the HBD, that alter sensitivity to one or both of these ligands, with or without altering hormone binding. The plates were incubated at 30 "C for 1-3 h until blue color development
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