Capacity of type I and II ligands to confer to estrogen receptor alpha an appropriate conformation for the recruitment of coactivators containing a LxxLL motif—Relationship with the regulation of receptor level and ERE-dependent transcription in MCF-7 cells

Abstract

Estrogen receptor α (ERα) belongs to the superfamily of nuclear receptors and as such acts as a ligand-modulated transcription factor. Ligands elicit in ERα conformational changes leading to the recruitment of coactivators required for the transactivation of target genes via cognate response elements. In many cells, activated ERα 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, ERα downregulation in MCF-7 cells and related transactivation activity of ligand-bound ERα. For type I estrogens, there was a clear-cut relationship between ligand binding affinity, hydrophobicity around C-11 of estradiol and ability of ERα to associate with LxxLL motifs, both in cell-free condition and in vivo (MCF-7 cells). Moreover, LxxLL motif recruitment by ERα 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 11β, had much less tendency to promote ERα–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 ERα-mediated gene transactivation.

Introduction

Estrogen receptor alpha (ERα) is a member of the nuclear receptor superfamily, the members of which function as ligand-regulated transcription factors [1]. Estrogens are essential for the development and the maintenance of the female reproductive system. Besides, ERα is also known to play a pivotal role in the etiology of hormone-dependent forms of breast cancer [2]. Hence, for the last 30 years or so there has been an intensive search for agents capable of modulating and/or inhibiting ERα-mediated cell proliferation. This has led to the identification of two main classes of ligands both containing two hydroxyl functions that contribute to their binding to the receptor (Table 1, Table 2) [3]. Type I ligands include estrogenic steroids and diphenolic structural analogs that similarly stimulate target tissues (trans stilbene derivatives such as diethylstilbestrol, isoflavones such as genistein, coumestanes such as coumestrol). These planar/linear agonists differentiate from angular weak agonists (type II) for which two subsets have been described: cis stilbene-like and geminal structures. Such angular ligands can in some cases antagonize the effect of strong type I estrogens [4]. Insofar as their pharmacological profile varies among different tissues, they are currently referred to as SERMs (Selective Estrogen Receptor Modulators).

All investigated type I ligands have been reported to fit within a cleft of the hormone binding domain (HBD) in such a way that their hydroxyl groups can form hydrogen bonds with a few amino acids (for E₂, 3-OH: Glu-353 and Arg-394; 17β-OH: His-524; Fig. 1) [5], [6]. Although this property does not hold for type II ligands because of inappropriate orientation of their hydroxyl groups, interactions with Glu-353 and Arg-394 are conserved and concur to produce non-covalent ligand–receptor complexes. Stability of these complexes results from additional electrostatic interactions between the second phenolic ring of these ligands and Thr-347, a residue located in a subregion of the ligand binding domain (LBD) known to attract substituents in C-11 of estradiol (E₂) [6], [7]. 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 acetyl-transferase enzymes and/or adaptator proteins for the transcription machinery recruitment (see [14], [15], [16]).

Even though there is a general consensus that the ability to recruit such coactivators determines the capacity of ERα to induce gene transactivation, no structure/activity study substantiating this concept has been reported so far. Similarly, there are no systematic data concerning a potential relationship between coactivator recruitment (or the absence of recruitment) and the capacity of a ligand to modulate ERα turnover, a factor known to influence receptor-mediated gene transactivation [17], [18], [19]. This gap in our knowledge has led us to conduct such a comparative study based on a panel of type I and type II estrogens. Of note, a part of our investigations focuses on several E₂ derivatives substituted in C-3, C-11 and C-17 since, as discussed above, these three reactive positions of the steroid core are known to play a prominent role in ligand binding and to influence the receptor conformations leading to an estrogenic response. Our experimental approach for this program relied on an ELISA-based assay allowing the quantitative measurements of ligand-activated ERα associated with immobilized LxxLL motif-containing peptide. In previous studies, the specificity of the assay was established by competition experiments showing that a peptide derived from the SRC-1 coactivator effectively suppresses receptor binding to LxxLL-coated plates through a complexation process [20].