Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells

Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor alpha (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-tagged lac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integrated lac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP-SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP-SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP-SRC-1, while antagonist additions diminish YFP-SRC-1-CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER-SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP-SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.     

Identification of novel estrogen receptor alpha antagonists

We have identified novel estrogen receptor alpha (ERalpha) antagonists using both cell-based and computer-based virtual screening strategies. A mammalian two-hybrid screen was used to select compounds that disrupt the interaction between the ERalpha ligand binding domain (LBD) and the coactivator SRC-3. A virtual screen was designed to select compounds that fit onto the LxxLL peptide-binding surface of the receptor, based on the X-ray crystal structure of the ERalpha LBD complexed with a LxxLL peptide. All selected compounds effectively inhibited 17-beta-estradiol induced coactivator recruitment with potency ranging from nano-molar to micromolar. However, in contrast to classical ER antagonists, these novel inhibitors poorly displace estradiol in the ER-ligand competition assay. Nuclear magnetic resonance (NMR) suggested direct binding of these compounds to the receptors pre-complexed with estradiol and further demonstrated that no estradiol displacement occurred. Partial proteolytic enzyme digestion revealed that, when compared with 17-beta-estradiol- and 4 hydroxy-tamoxifen (4-OHT) bound receptors, at least one of these compounds might induce a unique receptor conformation. These small molecules may represent new classes of ER antagonists, and may have the potential to provide an alternative for the current anti-estrogen therapy.     

Design and synthesis of peptide-based chimeric molecules to induce degradation of the estrogen and androgen receptors

Peptide-based inducers of estrogen receptor (ER) α and androgen receptor (AR) degradations via the ubiquitin–proteasome system (UPS) were developed. The designated inducers were composed of two biologically active scaffolds: the helical peptide PERM3, which is an LXXLL-like mimic of the coactivator SRC-1, and various small molecules (MV1, LCL161, VH032, and POM) that bind to E3 ligases (IAPs, VHL, and cereblon, respectively), to induce ubiquitylation of nuclear receptors that bind to SRC-1. All of the synthesized chimeric E3 ligand-containing molecules induced the UPS-mediated degradation of ERα and AR. The PERM3 peptide was applicable for the development of the ERα and AR degraders using these E3 ligands.     

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres

BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.     

Rifampicin-independent interactions between the pregnane X receptor ligand binding domain and peptide fragments of coactivator and corepressor proteins

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s(-1), respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.