Hormone binding and transcription activation by estrogen receptors: analyses using mammalian and yeast systems.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the human estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labeling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. C530 mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed mutagenesis of selected residues in the ER and region-specific chemical mutagenesis of the ER hormone binding domain with initial phenotypic screening in yeast have enabled the identification of a region near C530 important in discrimination between estrogens and antiestrogens and of other residues important in hormone-dependent transcriptional activation. Some ER mutants with alterations in the carboxy-terminal portion of the hormone binding domain are transcriptionally inactive yet bind hormone and also function as potent dominant negative ERs, suppressing the activity of wild-type ER at low concentrations. These studies reveal a separation of the hormone binding and transcription activation functions of the ER. They are also beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer. In addition, some dominant negative receptors may prove useful in examining ER mechanisms of action and in suppressing the estrogen-dependent growth of breast cancer cells.

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 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.

Powerful dominant negative mutants of the human estrogen receptor.

We have identified and characterized three human estrogen receptor (ER) mutants, which, at low concentrations, are capable of blocking the intracellular activity of wild type ER. The mutants, a truncated ER (ER1-530), a point mutant (L540Q), and a frameshift (S554fs), were generated by random chemical mutagenesis of the ER hormone binding domain and screened first for low activity in a yeast selection system. In transient co-transfection assays using ER-deficient Chinese hamster ovary cells, all three mutants exhibited less than 10% of the transcription activation activity of wild type ER, and when co-expressed with wild type ER, each of the mutants effectively suppressed the ability of wild type ER to activate transcription of an estrogen-regulated reporter plasmid. When equal amounts of plasmid encoding the ER mutants and wild type ER were used, S554fs, ER1-530, and L540Q suppressed the activity of wild type ER by 80, 55, and 75%, respectively. At a ratio of 1 part S554fs to 10 parts wild type ER, transcription was still inhibited by 40%. Western blot analysis showed that all three mutants were expressed at approximately the same level as wild type ER. Suppression of transcription was specific for ER, since the mutants did not inhibit progesterone receptor-mediated transcription. Not all mutations leading to inactive ER confer the dominant negative phenotype, as five ER mutants rendered transcriptionally inactive by point mutations between residues 516 and 524 of the ER hormone binding domain were poor inhibitors of wild type ER activity. Binding studies showed that the L540Q and S554fs dominant negative mutants bound 17 beta-estradiol with wild type affinity (Kd = 0.3-0.5 nM), whereas ER1-530 exhibited a 15-fold reduction in affinity for estradiol. The three dominant negative ERs showed significant ability to interact with the estrogen response element (ERE) in promoter interference assays, but ER1-530 and S554fs displayed little or no binding to the ERE in gel mobility shift assays where higher affinity for the DNA may be required for the receptor-ERE complex to remain associated during the electrophoresis. These data support the idea that, in all three mutants, it is loss of function of the COOH-terminal transactivation domain which leads to the dominant negative phenotype. S554fs, a powerful dominant negative mutant, is a good candidate for further studies aimed at suppressing the estrogen-dependent growth of human breast cancer cells.

William L. McGuire Memorial Symposium. Estrogen receptors: ligand discrimination and antiestrogen action.

We have used affinity labeling, site-directed mutagenesis and regional chemical mutagenesis in order to determine regions of the estrogen receptor (ER) important in hormone binding, ligand discrimination between estrogens and antiestrogens, and transcriptional activation. Affinity labelling studies with the antiestrogen, tamoxifen aziridine and the estrogen, ketononestrol aziridine have identified cysteine 530 in the ER hormone binding domain as the primary site of labeling. In the absence of a cysteine at 530 (i.e. Cys530A1a mutant), C381 becomes the site of estrogen-compatible tamoxifen aziridine labeling. Hence these two residues, although far apart in the primary linear sequence of the ER protein, must be close in the three-dimensional structure of the protein, in the ER ligand binding pocket, so that the ligand can reach either site. Site-directed and region-specific chemical mutagenesis have identified a region around C530 important in discrimination between estrogens and antiestrogens, and other mutants have allowed identification of residues important in hormone-dependent transcriptional activation. Some transcriptionally inactive ER mutants also function as potent dominant negative ERs, suppressing the activity of wild-type ERs at low concentrations. These studies are beginning to provide a more detailed picture of the ER hormone binding domain and amino acids important in ligand binding and discrimination between different categories of agonist and antagonist ligands. Such information will be important in the design of maximally effective antiestrogens. In addition, since there is now substantial evidence for a mixture of wild-type and variant ERs in breast cancers, our studies should provide insight about the bioactivities of these variant receptors and their roles in modulating the activity of wild type ER, and should lead to a better understanding of the possible role of variant receptors in altered response or resistance to antiestrogen and endocrine therapy in breast cancer.

Cross-linking of estrogen receptor to chromatin in intact MCF-7 human breast cancer cells: optimization and effect of ligand.

To investigate the effect of ligand (be it hormone, antihormone, or no hormone) on the interaction between estrogen receptor (ER) and chromatin, we have used formaldehyde as a cross-linking agent in intact MCF-7 human breast cancer cells. After a 1- to 2-h hormone treatment, the cells are exposed for 8 min to formaldehyde, which is added directly to their culture medium to minimize environmental perturbation. Nuclei are prepared from formaldehyde-treated cells and their contents are fractionated on CsCl density gradients to separate DNA-protein complexes from free protein. Peak gradient fractions are assayed for the presence of specific proteins by immunoblot of sodium dodecyl sulfate-polyacrylamide gel patterns. Using this approach, we find that 0.15% formaldehyde is optimal for cross-linking ER to chromatin. We detect ER and the large subunit of RNA polymerase II with DNA from formaldehyde-treated, but not from untreated cells. On the other hand, actin (a cytoplasmic protein) and small nuclear ribonucleoprotein particle proteins (nuclear RNA binding proteins) are not cross-linked to DNA. Therefore, cross-linking appears to be selective and fractionation is efficient. Interestingly, we detect similar levels of ER (as well as RNA polymerase II) with DNA from formaldehyde-treated cells, regardless of whether the cells are preexposed to estrogen (17 beta-estradiol at 10(-8) M), antiestrogen (ICI 164,384 at 10(-7) or 10(-6) M), or no hormone. These results, using covalent cross-linking in intact cells, indicate that both ligand-occupied and unoccupied ER are associated with chromatin.