Coactivator Proteins as Determinants of Estrogen Receptor Structure and Function: Spectroscopic Evidence for a Novel Coactivator-Stabilized Receptor Conformation

Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Molecular Endocrinology (Impact Factor: 4.02). 07/2005; 19(6):1516-28. DOI: 10.1210/me.2004-0458
Source: PubMed


The direct regulation of gene transcription by nuclear receptors, such as the estrogen receptor (ER), involves not just ligand and DNA binding but the recruitment of coregulators. Typically, recruitment of p160 coactivator proteins to agonist-liganded ER is considered to be unidirectional, with ligand binding stabilizing an ER ligand binding domain (LBD) conformation that favors coactivator interaction. Using fluorophore-labeled ERalpha-LBDs, we present evidence for a pronounced stabilization of ER conformation that results from coactivator binding, manifest by decreased ER sensitivity to proteases and reduced conformational dynamics, as well as for the formation of a novel coactivator-stabilized (costabilized) receptor conformation, that can be conveniently monitored by the generation of an excimer emission from pyrene-labeled ERalpha-LBDs. This costabilized conformation may embody features required to support ER transcriptional activity. Different classes of coactivator proteins combine with estrogen agonists of different structure to elicit varying degrees of this receptor stabilization, and antagonists and coactivator binding inhibitors disfavor the costabilized conformation. Remarkably, high concentrations of coactivators engender this conformation even in apo- and antagonist-bound ERs (more so with selective ER modulators than with pure antagonists), providing an in vitro model for the development of resistance to hormone therapy in breast cancer.

Download full-text


Available from: John A Katzenellenbogen, Mar 06, 2014
  • Source
    • "While hydrogen bonding between the hydroxyl of this phenolic ring and Thr-347 (Fig. 1) is of importance for ERα binding, hydrophobic interactions between the C-11 subregion of the steroid core and the LBD are largely dominant [8]. Such differences in binding modes between type I and II ligands have been reported to confer to ERα distinct conformations that should influence its ability to recruit coactivators containing a consensus LxxLL binding motif (L = Leucine, x = any amino acid) [9] [10] [11] [12] [13] such as those of the CBP / p300 or the SRC / p160 families known as acetyltransferase enzymes and / or adaptator proteins for the transcription machinery recruitment (see [14] [15] [16]). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Estrogen receptor alpha (ERalpha) belongs to the superfamily of nuclear receptors and as such acts as a ligand-modulated transcription factor. Ligands elicit in ERalpha conformational changes leading to the recruitment of coactivators required for the transactivation of target genes via cognate response elements. In many cells, activated ERalpha also undergoes downregulation by proteolysis mediated by the ubiquitin/proteasome system. Although these various molecular processes have been well characterized, little is known as to which extent they are interrelated. In the present study, we used a panel of type I (estradiol derivatives and "linear", non-steroidal ligands) and type II ("angular" ligands) estrogens, in order to identify possible relationships between ligand binding affinity, recruitment of LxxLL-containing coactivators, ERalpha downregulation in MCF-7 cells and related transactivation activity of ligand-bound ERalpha. For type I estrogens, there was a clear-cut relationship between ligand binding affinity, hydrophobicity around C-11 of estradiol and ability of ERalpha to associate with LxxLL motifs, both in cell-free condition and in vivo (MCF-7 cells). Moreover, LxxLL motif recruitment by ERalpha seemed to be a prerequisite for the downregulation of the receptor. By contrast, type II ligands, as well as estradiol derivatives bearing a bulky side chain at 11beta, had much less tendency to promote ERalpha-LxxLL interaction or even behaved as antagonists in this respect, in agreement with the well known partial estrogenicity/antiestrogenicity of some of these compounds. Interestingly, some type II ligands which antagonized LxxLL motif recruitment were nonetheless able to enhance ERalpha-mediated gene transactivation.
    Full-text · Article · Oct 2009 · Biochemical pharmacology
  • Source
    • "interaction with the co-regulator proteins and dimerization partners [7] [31]. It was recently revealed that binding of co-activators to estrogen receptor (ER) induces novel markedly stabilized receptor conformation [32]. Furthermore, it was demonstrated that the ligand binding significantly increases ER thermal stability, the helical content of its LBD [33], and directly influences ER dimer affinity and dimer dissociation rates [34]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.
    Full-text · Article · Aug 2008 · Biophysical Chemistry
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1 is a primary transcriptional coregulator for estrogen receptor (ER). Six SRC-3 phosphorylation sites have been identified, and these can be induced by steroids, cytokines, and growth factors, involving multiple kinase signaling pathways. Using phosphospecific antibodies for six phosphorylation sites, we investigated the mechanisms involved in estradiol (E2)-induced SRC-3 phosphorylation and found that this occurs only when either activated estrogen receptor alpha (ERalpha) or activated ERbeta is present. Both the activation function 1 and the ligand binding domains of ERalpha are required for maximal induction. Mutations in the coactivator binding groove of the ERalpha ligand binding domain inhibit E2-stimulated SRC-3 phosphorylation, as do mutations in the nuclear receptor-interacting domain of SRC-3, suggesting that ERalpha must directly contact SRC-3 for this posttranslational modification to take place. A transcriptionally inactive ERalpha mutant which localizes to the cytoplasm supports E2-induced SRC-3 phosphorylation. Mutations of the ERalpha DNA binding domain did not block this rapid E2-dependent SRC-3 phosphorylation. Together these data demonstrate that E2-induced SRC-3 phosphorylation is dependent on a direct interaction between SRC-3 and ERalpha and can occur outside of the nucleus. Our results provide evidence for an early nongenomic action of ER on SRC-3 that supports the well-established downstream genomic roles of estrogen and coactivators.
    Full-text · Article · Oct 2005 · Molecular and Cellular Biology
Show more