scholarly journals AF-2 activity and recruitment of steroid receptor coactivator 1 to the estrogen receptor depend on a lysine residue conserved in nuclear receptors.

1997 ◽  
Vol 17 (4) ◽  
pp. 1832-1839 ◽  
Author(s):  
P M Henttu ◽  
E Kalkhoven ◽  
M G Parker
Keyword(s):  
Estrogen Receptor ◽  
Nuclear Receptors ◽  
Transcriptional Activation ◽  
Alpha Helix ◽  
Steroid Receptor ◽  
Activation Function ◽  
Binding Activity ◽  
Lysine Residue ◽  
Steroid Receptor Coactivator ◽  
Helix 12

Hormone-dependent transcriptional activation by nuclear receptors depends on the presence of a conserved C-terminal amphipathic alpha-helix (helix 12) in the ligand-binding domain. Here we show that a lysine residue, which is conserved in most nuclear receptors in the predicted helix 3, is also required for estrogen-dependent transactivation. The replacement of lysine 366 with alanine appreciably reduced activation function 2 (AF-2) activity without affecting steroid- or DNA-binding activity in the mouse estrogen receptor. The mutation dramatically reduced the ability of the receptor to bind steroid receptor coactivator 1 (SRC-1) but had no effect on receptor-interacting protein 140 (RIP-140) binding, indicating that while their sites of interaction overlap, they are not entirely consistent and in keeping with the proposal that the recruitment of coactivators, such as SRC-1, is required for AF-2 activity. Although the function of RIP-140 remains to be established, RIP-140 appears to be capable of recruiting the basal transcription machinery, since overexpression of the protein markedly increased the transcriptional activity of the mutant receptor. Since the lysine residue is conserved, we propose that it is required, together with residues in helix 12, to form the surface by which members of the nuclear receptor family interact with coactivators.

scholarly journals Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

2003 ◽  
Vol 23 (1) ◽  
pp. 335-348 ◽  
Author(s):  
Mari Luz Acevedo ◽  
W. Lee Kraus
Keyword(s):  
Estrogen Receptor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Preinitiation Complex ◽  
Transcription System ◽  
Steroid Receptor Coactivator ◽  
Transcription Reinitiation

ABSTRACT Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromatin assembly and transcription system, to examine the functional role for Mediator in the transcriptional activity of estrogen receptor α (ERα) with chromatin templates, as well as functional interplay between Mediator and p300/CBP during ERα-dependent transcription. Using three different approaches to functionally inactivate Mediator (immunoneutralization, immunodepletion, and inhibitory polypeptides), we find that Mediator is required for maximal transcriptional activation by ligand-activated ERα. In addition, we demonstrate synergism between Mediator and p300/CBP-SRC during ERα-dependent transcription with chromatin templates, but not with naked DNA. This synergism is important for promoting the formation of a stable transcription preinitiation complex leading to the initiation of transcription. Interestingly, we find that Mediator has an additional distinct role during ERα-dependent transcription not shared by p300/CBP-SRC: namely, to promote preinitiation complex formation for subsequent rounds of transcription reinitiation. These results suggest that one functional consequence of Mediator-ERα interactions is the stimulation of multiple cycles of transcription reinitiation. Collectively, our results indicate an important role for Mediator, as well as its functional interplay with p300/CBP-SRC, in the enhancement of ERα-dependent transcription with chromatin templates.

scholarly journals Transcriptional activation by estrogen receptor (ERalpha) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells

2003 ◽  
Vol 30 (3) ◽  
pp. 411-422 ◽  
Author(s):  
HM Sheppard ◽  
S Matsuda ◽  
JC Harries ◽  
KB Kindle ◽  
DM Heery
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Transcriptional Activity ◽  
Steroid Receptors ◽  
Steroid Receptor ◽  
Yeast Cells ◽  
Creb Binding Protein ◽  
Subtle Difference ◽  
Steroid Receptor Coactivator ◽  
Helix 12

Steroid receptors activate transcription in yeast cells via interactions with endogenous coactivators and/or basal factors. We examined the effects of mutations in the ligand binding domain on the transcriptional activity of ERalpha in yeast. Our results show that mutations in Helix 3 (K366A) and Helix 12 (M547A, L548A) disrupt transcriptional activity of ERalpha in yeast, as previously observed in mammalian cells. However, replacement of a conserved tyrosine residue in Helix 12 with alanine or aspartate (Y541A and Y541D), which renders ERalpha constitutively active in mammalian cells, had only a weak stimulatory effect on ligand-independent reporter activation by ERalpha in yeast. Two-hybrid interaction experiments revealed that a Y541A mutant expressed in yeast was capable of ligand-independent binding to a mammalian coactivator, suggesting that there is a subtle difference in how this mutant interacts with mammalian and yeast cofactors. We also show that the ligand-dependent activities of ERalpha and progesterone receptor (PR) in yeast cells were strongly enhanced by the human p160 protein steroid receptor coactivator (SRC1), but not by CREB-Binding Protein (CBP) or the p300/CBP associated factor (P/CAF). Although the SRC1 activation domains AD1 and AD2 are functional in yeast, deletion of these sequences only partially impaired SRC1 coactivator function in this organism; this is in contrast to similar experiments in mammalian cells. Thus SRC1 sequences involved in recruitment of CBP/p300 and Co-Activator-Associated Arginine Methyltransferase (CARM-1) in mammalian cells are not essential for its function in yeast, suggesting that SRC1 operates via distinct mechanisms in yeast and mammalian cells.

Mechanisms of androgen receptor signalling via steroid receptor coactivator-1 in prostate.

2004 ◽  
Vol 11 (1) ◽  
pp. 117-130 ◽  
Author(s):  
S M Powell ◽  
V Christiaens ◽  
D Voulgaraki ◽  
J Waxman ◽  
F Claessens ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Point Mutations ◽  
Steroid Receptor ◽  
Activation Function ◽  
Steroid Receptor Coactivator ◽  
Basal Transcriptional Machinery ◽  
Independent States

The androgen receptor (AR) is a member of the nuclear receptor superfamily. These ligand-activated transcription factors usually contain two activation functions, a ligand-independent activation function 1(AF1) in the divergent N-terminal domain and a ligand-dependent AF2 in the more conserved C-terminal ligand-binding domain. To promote transcription from target promoters, DNA-bound nuclear receptors recruit coactivator proteins that promote transcription by modifying histones within nucleosomes, resulting in altered topology of chromatin to allow access of the basal transcriptional machinery, or stabilising the pre-initiation complex. It is well known that most coactivators interact with AF2 of many nuclear receptors via conserved, helical LxxLL motifs (where L is leucine and x is any amino acid). The AF2 of the AR is very weak, but we were able to demonstrate that its intrinsic ligand-dependent activity is potentiated by steroid receptor coactivator-1 (SRC1) and that this region interacts with coactivators via LxxLL motifs. However, a mutant SRC1 coactivator with no functional LxxLL motifs was still able to potentiate AR activity. We found that SRC1 can also be recruited to (and increase activity of) AF1 of the AR via a conserved, glutamine-rich region. Point mutations within this region abolish SRC1 interaction with AF1 and also abolish or severely impair its ability to potentiate AR activity on all promoters tested. Thus the AR interacts with SRC1 via two different regions and the AF1 interaction is functionally the more important, although the contribution of the two interactions varies in a promoter-dependent fashion. SRC1 then potentiates receptor activity via recruitment of CBP/p300, a histone acetyltranferase. This is important in the context of prostate cancer as SRC1 and other coactivators including CBP are coexpressed with AR in the luminal epithelial cells of the prostate, where over 90% of prostate tumours arise. There is a need for effective second-line prostate cancer therapy aimed at blocking the AR pathway when anti-androgen therapy has failed. Since there is growing evidence that nuclear receptor cofactors may be implicated in the progression of hormone-dependent tumours to hormone-independent states, novel targets could include the interaction of AR with coactivator proteins. We suggest that the N-terminal interaction would be a more specific and effective target in the case of prostate cancer than the LxxLL/AF2 interaction.

Abstract 3104: Stapled peptide inhibitors of the estrogen receptor/steroid receptor coactivator interaction

2016 ◽  
Author(s):  
Terry W. Moore ◽  
Thomas E. Speltz ◽  
Sean W. Fanning ◽  
Christopher G. Mayne ◽  
Jonna Frasor ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Steroid Receptor ◽  
Peptide Inhibitors ◽  
Stapled Peptide ◽  
Steroid Receptor Coactivator

scholarly journals Ligands Differentially Modify the Nuclear Mobility of Estrogen Receptors α and β

Endocrinology ◽  
2007 ◽  
Vol 149 (1) ◽  
pp. 339-345 ◽  
Author(s):  
Anastasios E. Damdimopoulos ◽  
Giannis Spyrou ◽  
Jan-Åke Gustafsson
Keyword(s):  
Estrogen Receptor ◽  
Comparative Analysis ◽  
Nuclear Receptors ◽  
Estrogen Receptor Α ◽  
Estrogen Response Element ◽  
Subnuclear Localization ◽  
Estrogen Response ◽  
Subtype Specificity ◽  
Helix 12 ◽  
Moderate Effect

Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor α (ERα) and ERβ mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERα, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERα. Interestingly, we found that for ERβ such a classification was not possible because ERβ ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERα, whereas ERβ retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERβ retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.

scholarly journals Identification of Regions within the F Domain of the Human Estrogen Receptor α that Are Important for Modulating Transactivation and Protein-Protein Interactions

2007 ◽  
Vol 21 (4) ◽  
pp. 829-842 ◽  
Author(s):  
Akiko Koide ◽  
Changqing Zhao ◽  
Misuzu Naganuma ◽  
Judith Abrams ◽  
Sarah Deighton-Collins ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Ligand Binding ◽  
Protein Interactions ◽  
Mammalian Cells ◽  
Steroid Receptor ◽  
Estrogen Receptor Α ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Steroid Receptor Coactivator ◽  
Human Estrogen Receptor

Abstract The estrogen receptor (ER)α is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ERα modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ERα (hERα) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins (“monobodies”) that specifically bind to ERα/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579–584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a β-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hERα ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hERα selectively modulate its activity and its interactions with other proteins.

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