Hormone Binding Domain Of Estrogen Receptor Research Articles

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein.

Crystals of an intact GST-estrogen receptor hormone binding domain fusion protein have been grown from solutions of MPD. The crystals grew as clusters of thin plates and needles of maximum dimensions 100 x 20 x 1 micrometer but were unsuitable for X-ray diffraction analysis. However, examination by electron microscopy shows an ordered lattice in which the protein molecules are clearly visible. Image analysis of electron micrographs of the protein crystals revealed electron stain-excluding density which showed a two-domain trimeric structure in projection, with each molecule of dimensions 12.0 x 5.0 nm diameter. The use of GST-fusion proteins in crystallisation are discussed.

Enhancement of Estrogen Receptor Transcriptional Activity by the Coactivator GRIP-1 Highlights the Role of Activation Function 2 in Determining Estrogen Receptor Pharmacology

The human estrogen receptor (ER) contains two major activation functions (AFs) responsible for its transcriptional activity. One of these, activation function 2 (AF-2), located within the hormone-binding domain (HBD), has been shown to mediate the ligand-dependent transcriptional activity of ER as well as other members of the nuclear receptor superfamily. Recently, proteins interacting with the HBD of several nuclear receptors have been cloned. One of these proteins, glucocorticoid receptor interacting protein (GRIP-1), has been shown to interact with ER and was originally hypothesized to mediate its transcriptional activity through AF-2. However, we find in this study that the transcriptional activity of ER, containing mutations in the AF-2 core sequence, can be enhanced by coexpression of the coactivator GRIP-1, suggesting that this protein may not rely solely on the AF-2 domain for interaction. We propose, therefore, that the HBD of ER either contains multiple binding sites that are necessary for association with GRIP-1 or, alternatively, that this coactivator contacts the receptor in an undetermined region within the HBD. Importantly, these studies demonstrate also that mutations or deletion of AF-2 alter the ligand pharmacology of the receptor such that ER loses the ability to discriminate between agonists and antagonists. Interestingly, on these mutant receptors GRIP-1 still functions as a coactivator independent of the nature of the bound ligand. It is likely, therefore, that the C-terminal AF-2 domain may function as a molecular switch allowing the wild-type receptor to discriminate between agonists and antagonists as well as providing a surface with which associated proteins can interact.

Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.

Interference of Myb transactivation activity by a conditional dominant negative protein: functional interference in a cytotoxic T-cell line results in G 1 arrest

The ability to ablate the activity of specific transcription factors in vivo is a potentially important tool to study their roles in cellular processes such as the cell cycle. Previously, production of a dominant interfering c-Myb protein (comprising a fusion of the c-Myb DNA binding domain with the Drosophila Engrailed transrepressor) was found to inhibit the proliferation of immature thymocytes in the developing thymus of transgenic mice. We report here the further development of this stratagem by rendering the c-Myb/Engrailed protein conditionally active by fusion to a modified estrogen receptor hormone binding domain, ER™. Co-transfection experiments in NIH 3T3 fibroblasts showed that the resulting chimeric protein, Myb/En/ER™, repressed transactivation of a c-Myb-responsive reporter only in the presence of the synthetic steroid, 4-hydroxytamoxifen (OHT). Additionally, we found that Myb/En/ER™ could counteract transactivation by C/EBP-β of the mim-1 promoter, which contains juxtaposed Myb and C/EBP binding sites. Cytotoxic T-cells stably producing the inactive Myb/En/ER™ protein were readily obtained by gene transfection. The addition of OHT to these cells resulted in inhibition of proliferation and arrest in G 1. The utility of this experimental system to study Myb and other transcription factors is discussed.

A dominant interfering Myb mutant causes apoptosis in T cells.

The c-Myb transcription factor is required for the production of most hemopoietic lineages, but information is sparse about its mode of action and the key genes it regulates. We have made an inducible dominant interfering Myb protein, by creating a chimera comprising the DNA binding domain of c-Myb, the Drosophila Engrailed repressor domain, and a modified estrogen receptor hormone binding domain. When expressed in the murine thymoma cell line EL4, activation of this mutant results in a significant proportion of the cell population undergoing apoptosis, as assessed by nuclear breakdown and DNA fragmentation, but has no apparent effect on cell-cycle progression. The apoptotic phenotype is mirrored during thymopoiesis in transgenic mice expressing dominant interfering Myb mutants; their T cells are fragile both in vivo and in vitro. Induction of the Myb dominant interfering mutant in EL4 cells correlates with down-regulation of bcl-2, but does not affect transcription of other bcl-2 family members; conversely, overexpression of bcl-2 in the transgenic mouse model rescues thymocytes from death. Analysis of the bcl-2 promoter by run-on transcription, bandshifting, and transient expression assays shows that it is a direct target of Myb. These data suggest a new and important role for Myb proteins as regulators of cell survival during hemopoiesis.

Synthesis, structure and biological properties of Z-17alpha-(2-iodovinyl)-11beta-chloromethyl estradiol-17beta (Z-CMIV), a high affinity ligand for the characterization of estrogen receptor-positive tumors.

Linkage of a 11beta-chloromethyl group to estradiol-17beta (E2) dramatically increases the binding affinity of the steroid for the estrogen receptor (ER) with the formation of a quasi-irreversible steroid-receptor complex. We have synthesized the two isomers of 11beta-chloromethyl-17alpha-iodovinyl-estradiol (E-CMIV and Z-CMIV) by a novel route. Both derivatives demonstrated high binding affinity and selectivity for ER (RBAs: ER = 820 and 1008; SHBG = 1.2 and 0.25, respectively; E2 = 100). On the basis of X-ray crystallographic data for Z-CMIV and its precursor, we have postulated that Z-CMIV might interact strongly with aromatic amino-acids within a hydrophobic groove of the ER hormone binding domain (HBD) that incorporates pockets corresponding to the 11beta and 17alpha steroid substituents. The binding properties of Z-CMIV labeled with 125I were investigated, especially its ability to detect and quantify altered ER forms with low binding affinity for E2. Sucrose density gradient analysis revealed that Z-CMIV has a higher activation potency than E2 as it converts a higher proportion of non-activated monomers in the cytosol into activated monomers with the potential to dimerize. In in vitro (MCF-7 cells) and in vivo (rat uterus) determinations of estrogenic activity, Z-CMIV was as potent as E2 in increasing progesterone receptor (PgR) concentrations and decreasing ER levels and in stimulating uterine growth. [125I]-Z-CMIV could open the way to new applications in the diagnosis and therapy of ER-positive breast cancers, especially those containing altered (variant) ERs.

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain.

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313-599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [3H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was approximately 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [3H]estradiol was unaffected by Ca2+ and Mg2+ ions up to 1 mM but was significantly inhibited by Zn2+ ions at concentrations above 10 microM, an effect reversed by EDTA.

Scintillating microtitration plates, a platform for hormone and DNA binding assays. Model experiments with the human estrogen receptor

The estrogen receptor is an important drug target and an allosteric binder that binds hormones, DNA and proteins at different sites on its surface. Recombinantly produced human estrogen receptor (hER) and estrogen receptor hormone binding domain (hER-HBD) was used in conjunction with a novel platform for ligand binding studies, scintillating microtitration plates, to obtain quantitative binding data. Radioligand binding was determined in a multi-detector scintillation counter designed for the microtitration plate format. Methodology was developed that allows determination of equilibrium binding constants and of kinetic rate constants for both hormone-and DNA-binding. The results obtained show that the methodology is valid with regard to binding characteristics of the estrogen receptor. The assay format with scintillating microtitration plates has a major advantage over regular liquid scintillation counting or other solid-phase scintillant procedures as the vial itself is the means for sample holding and detection of radioactivity. Thus, the scintillating microtitration plates are especially suited for high capacity equilibrium and kinetic binding assays.

Estrogen receptors: bioactivities and interactions with cell signaling pathways.

Estrogens regulate the growth, differentiation, and functioning of diverse target tissues, both within and outside of the reproductive system. Most of the actions of estrogens appear to be exerted via the estrogen receptor (ER) of target cells, an intracellular receptor that is a member of a large superfamily of proteins that function as ligand-activated transcription factors, regulating the synthesis of specific RNAs and proteins. To understand how the ER discriminates between estrogen ligands, which activate the ER, and antiestrogen ligands, which fail to effectively activate the ER, we have generated and analyzed human estrogen receptors with mutations in the ER hormone binding domain. These studies provide evidence for the promoter-specific and cell-specific actions of the estrogen-occupied and antiestrogen-occupied and antiestrogen-occupied ER, highlight a regional dissociation of the hormone binding and transcription activation functions in domain E of the receptor, and indicate that some of the contact sites of estrogens and antiestrogens in the ER are likely different. In addition, multiple interactions among different cellular signaling pathways are involved in the regulation of gene expression and cell proliferation by the ER. In several cell types, protein kinase activators and some growth factors enhance the transcriptional activity of the ER. Cyclic AMP also alters the agonist/antagonist balance of some antiestrogens. Estrogens and, to a lesser extent, antiestrogens, as well as protein kinase activators and growth factors increase phosphorylation of the ER and possibly other proteins involved in the ER-specific response pathway, suggesting that changes in cellular phosphorylation state will be important in determining the biological activity of the ER and the effectiveness of antiestrogens as estrogen antagonists. The ER also has important interrelationships with the progesterone receptor (PR) system in modulation of biological responses. Liganded PR-A and PR-B can each suppress estradiol-stimulated ER activity, with the magnitude of repression dependent on the PR isoform, progestin ligand, promoter, and cell type. These findings underscore the mounting evidence for the importance of interactions between members of the steroid hormone receptor family.

15 O - Isolation of an estrogen receptor variant with increased activity from premalignant breast lesions

Recent molecular evidence suggests that at least half of the premalignant proliferative lesions of the breast have already suffered genetic alterations, suggesting that many of these lesions are in fact neoplasms with an increased propensity to evolve to malignancy. A growing body of literature also suggests that a high percentage of proliferative hyperplastic breast lesions, in contrast to nonnal breast epithelium, express high levels of the estrogen receptor (ER). Thus we hypothesize that either inappropriate overexpression of nonnal ER, or expression of certain variant forms of the ER, may drive abnormal proliferation in breast epithelium, providing a favorable environment for the genetic alterations. We have recently isolated an ER variant from breast hyperplasias with a point mutation within exon 4, which leads to a change of lysine to arginine at the beginning of the ER hormone binding domain. This variant is expressed in more than 30% of proliferative hyperplasias as assessed by single strand conformation polymorphism (SSCP). The ER variant displays increased agonistic properties in transactivations assays in MDA-MB-231 or HeLa cells. It exhibits significantly higher activity levels as compared to WT ER on a vitellogenin ERE reporter, and displays a 200-fold increase in estrogen sensitivity. Furthermore, when the variant receptor is stably transfected into the ER-positive MCF-7 breast cancer cell line, these cells now proliferate in response to very low levels of estradiol.

The human estrogen receptor hormone binding domain dimerizes independently of ligand activation

High level expression of biochemically active human estrogen receptor hormone binding domain (hER-HBD) was achieved using a Saccharomyces cerevisae expression system. Using dissociation kinetic analysis, density gradient centrifugation and cross-linking studies, a spontaneous dimerization activity of hER-HBD independent of the presence of the DNA binding domain, ligand, and of elevated temperature is demonstrated.

Hexestrol diazirine photoaffinity labeling reagent for the estrogen receptor.

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65,000, the same species that is labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.

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. C530A mutant), C381 becomes the site of estrogen-competible 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.

Modulation of cell proliferation and gene expression by a p53-estrogen receptor hybrid protein.

We report that p53her, a chimeric protein consisting of the complete human wild-type p53 and the human estrogen receptor hormone-binding domain, strongly suppresses proliferation and induces characteristic morphological changes in Saos-2 human osteosarcoma cells when induced by 17 beta-estradiol. In contrast, p53her constitutively transactivates a p53-responsive promoter in transfection assays, so that transactivation is not regulated by estradiol. However, coexpression of p53her and oncoprotein MDM-2, which associates with and presumably inactivates p53, results in suppression of p53her-mediated transactivation in the absence, but not the presence, of estradiol. Similarly, p53her induces expression of an endogenous MDM-2 transcript only in the presence of estradiol. These results suggest a correlation between the growth suppressor function of p53her and release of a transactivation block mediated by MDM-2.

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.

Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast.

We demonstrate that the hormone-binding domain (HBD) of the human estrogen receptor (ER) can function as an autonomous regulatory domain in the budding yeast, Saccharomyces cerevisiae. As in mammalian cells, the HBD can subject the activity of a heterologous protein, which is fused to it, to hormonal control. Thus, a chimeric transcriptional activator consisting of (i) the DNA-binding domain of GAL4, (ii) the ER HBD, and (iii) the activation domain of viral protein 16 (VP16) stimulates both episomal and integrated reporter genes exclusively in the presence of steroid hormone. Steroids being gratuitous signals for yeast, this fusion protein is a convenient tool for highly regulated production of proteins of interest. Notably, it can be exploited to activate the commonly used galactose-inducible expression vectors without switching the carbon source.

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.

Comparison of the 90-kilodalton heat shock protein interaction with in vitro translated glucocorticoid and estrogen receptors.

The rat glucocorticoid receptor is a 795-amino acid protein with the hormone binding domain located in the C-terminal portion of the molecule. In the absence of hormone, this domain displays a protein inactivation activity that represses the nuclear localization, DNA binding, and transcriptional regulatory activities of the receptor. This inactivation activity, which appears to be mediated by the 90-kilodalton heat shock protein (HSP90), is stronger in the glucocorticoid receptor than the corresponding activity of the estrogen receptor hormone binding domain. In order to analyze these differences, we have directly compared in vitro translated glucocorticoid and estrogen receptors in terms of their interaction with HSP90 by a coimmunoprecipitation assay employing two monoclonal antibodies, AC88 and 8D3, which react with different regions of the HSP90 molecule. Intact forms of both the glucocorticoid receptor and the estrogen receptor coimmunoprecipitated with endogenous HSP90 in reticulocyte lysates, indicating that both receptors were capable of binding to HSP90 when translated in vitro. By assaying a series of receptor deletion mutants, we found that the sequences required for HSP90 binding mapped to a similar region within the hormone binding domain of both receptors. While the hormone binding domain was found to be the only structural requirement for HSP90 binding to the glucocorticoid receptor, additional sequences N-terminal to the hormone binding domain were shown to be required for HSP90 binding to the estrogen receptor. These results are consistent with a postulate that differences in the protein inactivation activities of the glucocorticoid and estrogen receptor hormone binding domains may be secondary to differences in the interactions of these domains with HSP90.