Intermolecular interactions identify ligand-selective activity of estrogen receptor alpha/beta dimers

Abstract

Estrogen receptor (ER) dimerization is prerequisite for its activation of target gene transcription. Because the two forms of ER, ERalpha and ERbeta, exhibit opposing functions in cell proliferation, the ability of ligands to induce ERalpha/beta heterodimers vs. their respective homodimers is expected to have profound impacts on transcriptional outcomes and cellular growth. However, there is a lack of direct methods to monitor the formation of ERalpha/beta heterodimers in vivo and to distinguish the ability of estrogenic ligands to promote ER homo- vs. heterodimerization. Here, we describe bioluminescence resonance energy transfer (BRET) assays for monitoring the formation of ERalpha/beta heterodimers and their respective homodimers in live cells. We demonstrate that although both partners contribute to heterodimerization, ligand-bound ERalpha plays a dominant role. Furthermore, a bioactive component was found to induce ERbeta/beta homodimers, and ERalpha/beta heterodimers but had minimal activity on ERalpha/alpha homodimers, posing a model that compounds promoting ERalpha/beta heterodimer formation might have therapeutic value. Thus, ER homodimer and heterodimer BRET assays are applicable to drug screening for dimer-selective selective ER modulators. Furthermore, this strategy can be used to study other nuclear receptor dimers.

Fig. 1.

ER within BRET fusion proteins is functional. (A) Western blotting reveals protein expression. (B) Coimmunoprecipitation with an antibody vs. ERα followed by Western blotting using an ERβ antibody (Upper) or an ERα antibody (Lower). (C) Whole-cell ligand-binding assays on transfected cells. (D) Electrophoretic mobility shift assays using nuclear extracts prepared from transfected cells. Arrows indicate supershifted bands in the presence of antibody. Error bars represent SEM. (E) Luciferase units of cells transfected with the indicated constructs and an ERE-reporter were measured and normalized to β-gal. RLU, relative luciferase units.

Fig. 2.

Optimization of BRET assays. (A) Altering the ratio of RLuc-YFP fusions determines efficiency of resonance energy transfer. (B) E₂ dose–response curves showing ligand-dependent dimerization of all 3 ER pairs. (C) Bystander BRET assays reveal that the background BRET signals for ERβ homodimers are caused by ligand-independent dimerization, whereas the background BRET signals for the ERα homodimer and ERα/β heterodimer conditions are caused by random collisions between the RLuc and YFP fusions. (D) Dimer formation over time in the presence of 100 nM E₂ occurs quickly for all 3 ER combinations. Error bars represent SEM.

Fig. 3.

ERα is the dominant partner within an ERα/β heterodimer. (A) Low concentrations of PPT lead to heterodimerization via binding to ERα, but low concentrations of DPN do not lead to heterodimerization via binding to ERβ. PPT and DPN are specific to their respective subtypes at this low concentration. (B) Ligand binding to ERα may induce dimerization in the absence of ligand-bound ERβ, although the maximum dimerization potential is not achieved unless the LBD of ERβ is also wild type. Error bars represent SEM.

Fig. 4.

Various ER agonists and antagonists induce different conformations in the 3 dimer pairs. (A) Agonist ligands induce similar conformations among dimer pairs according to ligand affinity. Antagonist ligands induce varying conformations (compare each gray bar with each black bar) and may also be contrasted from each other (compare among the 3 gray bars). These antagonists also induce diverse conformations among the 3 dimer pairs (e.g., raloxifene). (B) Western blotting reveals that 1-h ligand treatment does not lead to degradation of the transiently transfected fusion proteins. Error bars represent SEM.

Fig. 5.

Naturally occurring estrogenic ligands exhibit dimer pair selectivity. (A) Genistein induces ERβ homodimerization at 10 nM, whereas ERα homodimerization and ERα/β heterodimerization are significantly induced at a lower limit of 100 nM. (B) Liquiritigenin induces ERβ homodimers and ERα/β heterodimers at 1 μM, but ERα homodimers are not induced at this concentration (Upper). Liquiritigenin induces heterodimerization by binding to ERα (Lower) despite the finding that ERα homodimers are not induced at this concentration. Error bars represent SEM.