Transcriptional activation by the estrogen receptor requires a conformational change in the ligand binding domain.

402

337

The human estrogen receptor ␣-isoform (ER␣) is a nuclear transcription factor that displays a complex pharmacology. In addition to classical agonists and antagonists, the transcriptional activity of ER␣ 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 ER␣ results in the formation of unique ER␣-ligand conformations. These conformations have been shown to influence ER␣-cofactor binding and, therefore, have a profound impact on ER␣ pharmacology. In this study, we demonstrate that the nature of the bound ligand also influences the stability of ER␣, revealing an additional mechanism by which the pharmacological activity of a compound is determined. Of note we found that although all ER␣-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 ER␣, 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 ER␣. We conclude that the different conformational states adopted by ER␣ in the presence of different ligands influence transcriptional activity directly by regulating cofactor binding and indirectly by modulating receptor stability. ER␣1 resides within the nuclei of target cells in an inactive form in the absence of hormone. Upon binding its cognate ligand estradiol, the receptor undergoes an activating conformational change permitting it to interact with specific cofactors and bind DNA response elements within target gene promoters (1, 2). The DNA-bound receptor-ligand complex is then capable of either activating or repressing target gene transcription, depending on both the cell and the promoter context. The classical model of ER␣ action suggests that the role of agonists, such as estradiol, is that of a switch converting the receptor from an inactive to an active form. It now appears that ER␣ pharmacology is more complex, since it has been observed that different ER␣-ligands induce different changes in receptor conformation and that target cells can distinguish between these complexes (3-5). For instance, the anti-estrogen tamoxifen opposes estrogen action in the breast, whereas it manifests estrogenic activities in bone, the cardiovascular system, and the uterus. Reflecting its complex pharmacology, tamoxifen has recently been reclassified as a selective estrogen receptor modulator (SERM). Additional SERMs have been identified, such as raloxifene, GW5638, TSE424, lasofoxifene, and arzoxifene, each of which has distinct agonist/antagonist profiles (6). The challenge, therefore, has been to understand the mechanism(s) underlying SERM-mediated action an...

Section: Er␣mentioning

confidence: 99%

Section: The Estrogen Receptor Is a Short Lived Protein Whose Stabilimentioning

confidence: 99%

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

Wijayaratne

McDonnell

2001

402

337

“…Tryptic digestion of nuclear receptors has resulted in specific, ligand-dependent proteolytic patterns indicative of different receptor conformations (39,40). That these conformational differences exist with various ligands have been substantiated particularly well for the estrogen receptor in x-ray diffractions studies (41,42).…”

Section: Fig 7 2md Is a Potent Inducer Of Vdr/rxr Interaction A Bmentioning

confidence: 99%

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 Potently Stimulates Gene-specific DNA Binding of the Vitamin D Receptor in Osteoblasts

Yamamoto¹,

Shevde

Warrier

et al. 2003

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitaminD 3 (2MD) is a highly potent analog of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) whose actions are mediated through the vitamin D receptor (VDR). In this report, we have replicated this increased potency of 2MD in vitro using osteoblastic cells and explored its underlying molecular mechanism. 2MD stimulates the expression of several vitamin D-sensitive genes including 25-hydroxyvitamin D 3 -24 hydroxylase (Cyp24), osteopontin and receptor activator of NFB ligand and suppresses osteoprotegerin at concentrations two logs lower than that for 1,25(OH) 2 D 3 . 2MD is also more potent in stimulating transfected chimeric reporter genes under either Cyp24 or the osteocalcin promoter control. Enhanced potency is retained regardless of medium serum content. Interestingly, the uptake of both 1,25(OH) 2 D 3 and 2MD into cells is similar, as is their rapid association with the VDR. This indicates that comparable levels of occupied VDR do not elicit equivalent levels of transactivation. Using chromatin immunoprecipitation (ChIP), however, we observed a strong correlation between DNA-bound receptor and the level of induced transcription suggesting a 2MD-induced increase in affinity of the VDR for DNA. Additional studies using a mammalian two-hybrid system and ChIP indicate that 2MD is also more potent in promoting interaction with RXR and the coactivators SRC-1 and DRIP205. Finally, protease digestion studies revealed a unique VDR conformation in the presence of 2MD. These studies suggest that the molecular mechanism of 2MD potency is due to its ability to promote enhanced levels of specific DNA binding by the VDR and could suggest possible explanations for the tissue-and gene-selective actions of 2MD.

“…In the absence of hormone, ER resides in a latent form in target cell nuclei associated within a large macromolecular complex comprising heat shock protein 90 (hsp90), hsp70, p59, and other proteins (6). Upon ligand binding, the receptor undergoes a dramatic conformational change (7,8), initiating a cascade of events leading ultimately to the association of an ER dimer with specific estrogen response elements (EREs) within the regulatory regions of target genes (9). The mechanism by which the bound receptor modulates gene transcription is unknown.…”

mentioning

confidence: 99%

Identification of a New Subclass of Alu DNA Repeats Which Can Function as Estrogen Receptor-dependent Transcriptional Enhancers

Norris¹,

Fan²,

Alemán

et al. 1995

210

139

We have utilized a genetic selection system in yeast to identify novel estrogen-responsive genes within the human genome and to define the sequences in the BRCA-1 gene responsible for its estrogen responsiveness. This approach led to the identification of a new subclass within the Alu family of DNA repeats which have diverged from known Alu sequences and have acquired the ability to function as estrogen receptor-dependent enhancers. Importantly, these new elements confer receptor-dependent estrogen responsiveness to a heterologous promoter when assayed in mammalian cells. This transcriptional activity can be attenuated by the addition of either of three different classes of estrogen receptor antagonists, indicating that these elements function as classical estrogen receptor-dependent enhancers. Furthermore, this enhancer activity is restricted to a specific subset of DNA repeats because consensus Alu elements of four major subfamilies do not respond to the estrogen receptor. Previously, most Alu sequences have been considered to be functionally inert. However, this work provides strong evidence that a significant subset can confer estrogen responsiveness upon a promoter within which they are located. Clearly, Alu sequences must now be considered as important contributors to the regulation of gene transcription in estrogen receptor-containing cells.The steroid hormone estrogen is a key intracellular modulator of the processes involved in establishment and maintenance of female reproductive function (1). In addition, its actions play an important role in maintaining female cardiovascular tone and regulating bone cell differentiation (2). In pathological states, the mitogenic activity of estrogen facilitates progression of breast cancers (3) and is implicated in the abnormalities of uterine function observed in endometriosis and possibly uterine fibroids (4). These actions of estrogen all appear to be mediated through specific high affinity receptors located within target cell nuclei (1). Molecular cloning has revealed that the estrogen receptor (ER) 1 is a member of a superfamily of receptors which mediate the nuclear actions of the sex steroids, retinoic acid, vitamin D 3 , and thyroid hormones (5). In the absence of hormone, ER resides in a latent form in target cell nuclei associated within a large macromolecular complex comprising heat shock protein 90 (hsp90), hsp70, p59, and other proteins (6). Upon ligand binding, the receptor undergoes a dramatic conformational change (7,8), initiating a cascade of events leading ultimately to the association of an ER dimer with specific estrogen response elements (EREs) within the regulatory regions of target genes (9). The mechanism by which the bound receptor modulates gene transcription is unknown. Because of its diverse biological functions and the implied complexity of its targets, there has been a keen interest in defining the genes which are regulated by estrogen. To date, a relatively small number of genes have been identified in humans which are modulated direc...