Conformation Of ERalpha Research Articles

Resistance to Antiestrogen Arzoxifene Is Mediated by Overexpression of Cyclin D1

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)alpha-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERalpha-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERalpha transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERalpha in the presence of arzoxifene. In this altered conformation, ERalpha still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERalpha-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERalpha antagonist into an agonist. This can be explained by a stabilization of the ERalpha/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERalpha upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Protein Disulfide Isomerase Serves as a Molecular Chaperone to Maintain Estrogen Receptor α Structure and Function

The effects of the steroid hormone 17beta-estradiol are mediated through its interaction with the nuclear estrogen receptor (ER). Upon binding 17beta-estradiol, the ER initiates changes in gene expression through its interaction with specific DNA sequences, estrogen response elements (EREs), and recruits coregulatory proteins that influence gene expression. To better understand how estrogen-responsive genes are regulated, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus ERE. One of these proteins, protein disulfide isomerase (PDI), has two distinct functions: acting as a molecular chaperone to maintain properly folded proteins and regulating the redox state of proteins by catalyzing the thiol-disulfide exchange reaction through two thioredoxin-like domains. Using a battery of biochemical and molecular techniques, we have demonstrated that PDI colocalizes with ERalpha in MCF-7 nuclei, alters ERalpha conformation, enhances the ERalpha-ERE interaction in the absence and presence of an oxidizing agent, influences the ability of ERalpha to mediate changes in gene expression, and associates with promoter regions of two endogenous estrogen-responsive genes. Our studies suggest that PDI plays a critical role in estrogen responsiveness by functioning as a molecular chaperone and assisting the receptor in differentially regulating target gene expression.

Mutation of Leu-536 in Human Estrogen Receptor-α Alters the Coupling between Ligand Binding, Transcription Activation, and Receptor Conformation

The estrogen receptor (ER), of which there are two forms, ERalpha and ERbeta, is a ligand-modulated transcription factor important in both normal biology and as a target for agents to prevent and treat breast cancer. Crystallographic studies of the ERalpha ligand-binding domain suggest that Leu-536 may be involved in hydrophobic interactions at the start of a helix, "helix 12," that is crucial in the agonist-stimulated activity of ERalpha, as well as in the ability of antagonists to block the activity of ERalpha. We found that certain mutations of Leu-536 increased the ligand-independent activity of ERalpha although greatly reducing or eliminating the agonist activity of 17beta-estradiol (E2) and 4-hydroxytamoxifen (4OHT), on an estrogen response element-driven and an AP-1-driven reporter. The mutations impaired the interaction of the ER ligand-binding domain with the SRC1 receptor-interacting domain in a mammalian two-hybrid system. When tested in the yeast two-hybrid system, mutation of Leu-536 increased the basal reactivity of ERalpha to probes that recognize the agonist-bound conformation but did not significantly alter its reactivity to these probes in the presence of E2. Most interestingly, mutation of Leu-536 reduced the interaction of the 4OHT-bound ERalpha and increased the reactivity of the raloxifene- or ICI 182,780-bound ERalpha, with probes that recognize the 4OHT-bound ERalpha conformation in a yeast two-hybrid system. These results show that Leu-536 is critical in coupling the binding of ligand to the modulation of the conformation and activity of ERalpha.

Simulation of the different biological activities of diethylstilbestrol (DES) on estrogen receptor alpha and estrogen-related receptor gamma.

We describe the application of the molecular dynamics (MD) and molecular mechanics-generalized Born/surface area (MM-GB/SA) approaches to the simulation of the different biological activity of diethylstilbestrol (DES) on two highly homologous nuclear receptors-estrogen receptor alpha (ER-alpha) and estrogen-related receptor gamma (ERR-gamma). DES exerts an agonistic effect against ER-alpha and an antagonistic effect against ERR-gamma. Using the x-ray crystal structures of ER-alpha in the canonical agonist bound form (PDB code: 3ERD) and antagonist bound form (PDB code: 3ERT), ERR-gamma homology models have been constructed for the receptor in two different conformations. MM-GB/SA binding free energy calculations of DES in the ER-alpha and ERR-gamma structures suggest that DES exhibits a greater free energy of binding in the agonist bound conformation of ER-alpha, while the antagonist bound conformation is preferred for ERR-gamma. Further dissection of the free energy contributions coupled with calculation of the ligand binding pocket volume suggests that the van der Waals interactions for DES within the smaller binding pocket volume of ERR-gamma are less favorable and this is the main factor for DES antagonism in ERR-gamma. This approach has potential general applicability to the prediction of the biological activity of nuclear receptor ligands.

Estrogen response element binding induces alterations in estrogen receptor-alpha conformation as revealed by susceptibility to partial proteolysis.

Genes whose expression is highly induced by estradiol (E(2)) contain multiple estrogen response elements (EREs) in their promoters. Previously we reported that estrogen receptor-alpha (ERalpha) binds cooperatively to and E(2) synergistically activates reporter gene expression from three or four tandem copies of a consensus ERE (EREc38). Here we evaluated how ERalpha binding to one, two, three or four tandem copies of EREc38 affects ERalpha conformation as detected by altered ERalpha trypsin digestion patterns in Western blots. E(2)- or 4hydroxytamoxifen (4-OHT)-occupied ERalpha bound to the pS2 ERE or to a single copy of EREc38 showed enhanced susceptibility to trypsin digestion compared to E(2)- or 4-OHT-ERalpha incubated with DNA lacking an ERE. ERalpha binding to multiple tandem copies of EREc38 further increased sensitivity to trypsin digestion. These results correlate with synergistic transcription and cooperativity of ERalpha binding to multiple tandem copies of EREc38. These observations suggest that EREc38 binding alters the overall conformation of ERalpha and that multiple tandem copies of EREc38 enhance these conformational changes. We hypothesize that ERE-induced alterations in ERalpha conformation modulate interaction with coregulatory proteins, resulting in synergistic transcriptional activation.

The Human Estrogen Receptor-α Is a Ubiquitinated Protein Whose Stability Is Affected Differentially by Agonists, Antagonists, and Selective Estrogen Receptor Modulators

The human estrogen receptor alpha-isoform (ERalpha) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ERalpha can be regulated by selective estrogen receptor modulators, a new class of drugs whose relative agonist/antagonist activity is determined by cell context. It has been demonstrated that the binding of different ligands to ERalpha results in the formation of unique ERalpha-ligand conformations. These conformations have been shown to influence ERalpha-cofactor binding and, therefore, have a profound impact on ERalpha pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ERalpha, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ERalpha-ligand complexes can be ubiquitinated and degraded by the 26 S proteasome in vivo, the mechanisms by which they are targeted for proteolysis appear to be different. Specifically, for agonist-activated ERalpha, an inverse relationship between transcriptional activity and receptor stability was observed. This relationship does not extend to selective estrogen receptor modulators and pure antagonists. Instead, it appears that with these compounds, the determinant of receptor stability is the ligand-induced conformation of ERalpha. We conclude that the different conformational states adopted by ERalpha in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability.

Rsk2 allosterically activates estrogen receptor alpha by docking to the hormone-binding domain.

We describe a novel mechanism for transcriptional regulation, in which docking of p90 ribosomal S6 kinase 2 (Rsk2) to the hormone-binding domain (HBD) of estrogen receptor alpha (ERalpha) induces a conformational change that enhances the transcriptional activation function contained in the HBD. A constitutively active mutant of Rsk2 specifically enhances ERalpha-mediated transcription by phosphorylation of Ser167 in ERalpha and by physically associating with residues 326-394 of the ERalpha HBD. The anti-estrogen 4-hydroxytamoxifen blocks Rsk2-mediated activation of ERalpha, by inducing a conformation of ERalpha in which the Rsk2 docking site is masked. Transcriptional activation and docking are specific for ERalpha and do not occur with the related isoform, ERbeta. ERalpha phosphorylation, docking and transcriptional activation are regulated by the Rsk2 N-terminal kinase domain. The allosteric regulation of a target protein, independent of phosphorylation, may be paradigmatic of a general function for protein kinase docking sites.

Aspartate 351 of Estrogen Receptor α Is Not Crucial for the Antagonist Activity of Antiestrogens

The antagonist activity of antiestrogens is due to the presence of a long carbon side chain at positions 7alpha or 11beta or equivalent on their steroid or steroid-like skeletons. These side chains establish hydrophobic interactions with amino acids of the estrogen receptor alpha (ERalpha) ligand binding domain. In addition, a hydrogen bond formed between amino acid Asp-351 and the tertiary amine present at the end of the side chain of partial antiestrogens is considered to be crucial for their antiestrogenicity. Here, we have investigated the role of Asp-351 in antiestrogen action in transiently transfected HeLa and MDA-MB-231 cells. Our results indicate that disruption of the negative charge at position 351 does not increase the agonist activity of partial antiestrogens and thus that the hydrogen bond with the antiestrogen side chain is not determinant in positioning the side chain in an antagonist position. The negative charge at position 351 was not required for transcriptional activity in the presence of hormone, but its presence was necessary for basal activity of the wild-type receptor and constitutive activities of mutants L536P and Y537A, suggesting a role of Asp-351 in stabilizing the active conformation of ERalpha. This stabilizing role of Asp-351 could be due to interaction of Asp-351 with the amide group of the peptide bond between Leu-539 and Leu-540 in helix 12 observed in the active conformation of the ERalpha ligand binding domain.