Mutations in the Estrogen Receptor Ligand Binding Domain Discriminate between Hormone-dependent Transactivation and Transrepression

Abstract

The estrogen receptor (ER) suppresses transcriptional activity of the RelA subunit of nuclear factor-B in a hormone-dependent manner by a mechanism involving both the receptor DNA binding domain and ligand binding domain (LBD). In this study we examine the role of the ER LBD in mediating ligand-dependent RelA transrepression. Both ER␣ and ER␤ inhibit RelA in response to 17␤-estradiol but not in the presence of antihormones. We have identified residues within the ER␣ LBD that are responsible for receptor dimerization and show that dimerization is necessary for transactivation and transrepression. Moreover we have generated mutant receptors that have lost their ability to inhibit RelA but retain their capacity to stimulate transcription and conversely mutants that are transcriptionally defective but capable of antagonizing RelA. Overexpression of p160 and cAMP-response element-binding protein-binding protein/p300 co-activators failed to relieve repression of RelA, which is consistent with the demonstration that RelA inhibition can occur independently of these co-activators. These findings suggest it is unlikely that sequestration of these cofactors required for ER transcriptional activation can account for hormone-dependent antagonism of RelA. The identification of ER mutants that discriminate between transactivation and transrepression implies that distinct surfaces within the LBD are involved in mediating these two receptor functions.The pleiotropic effects of estrogens are mediated by estrogen receptors (ERs), 1 which function as hormone-activated transcription factors regulating the expression of a variety of estrogen-responsive genes. In common with other members of the nuclear receptor (NR) superfamily, ER contains three functional domains, an N-terminal region with a hormone-independent activation function (AF1), a conserved central DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD), which is responsible for high affinity ligand binding, dimerization, and hormone-dependent activation (AF2) (1-3). There are two forms of ER, ER␣, and ER␤, which share a high degree of homology in their DBD and LBD but contain divergent N-terminal domains (4, 5). In response to hormone binding, ER homodimers contact estrogen response elements (ERE) located within the regulatory sequences of target genes, resulting in the recruitment of co-activator proteins and consequent initiation of gene transcription (6, 7). Elucidation of the crystal structure of ER␣ and ER␤ LBD has revealed that they share a common modular structure with the LBD of related NRs, which can be divided into 12 discrete helices (see Refs. 8 -11, and the references therein). Comparison of these unliganded and liganded crystal structures suggests that upon ligand binding the LBD undergoes a conformational change whereby the C-terminal helix (H12 in ER) is realigned over the ligand binding pocket and in ER is packed against H3, H5/6, and H11 (8 -11). The activity of ER␣ AF2 is dependent on the integrity of a hydrophobic interaction surface gen...

Characterization of a human REL-estrogen receptor fusion protein with a reverse conditional transforming activity in chicken spleen cells

“…Taken together, these results indicate that the ability of ERa to inhibit REL-directed transactivation is enhanced by estrogen. Similar to our results, estrogen has previously been shown to enhance the ability of ERa to repress RelA-dependent transactivation (Valentine et al, 2000).…”

“…Several previous reports have demonstrated that fulllength ERa can inhibit NF-kB-induced transactivation in an estrogen-dependent manner (Ray et al, 1994;Stein and Yang, 1995;Ray et al, 1997;Cerillo et al, 1998;Valentine et al, 2000;Evans et al, 2001;Sharma et al, 2001;Tzagarakis-Foster et al, 2002), but the mechanism by which this inhibition occurs is unclear. However, two studies have reported that ERa can bind preferentially to REL, as compared to other NF-kB subunits.…”

Identification of pathway-selective estrogen receptor ligands that inhibit NF-κB transcriptional activity

“…The inhibition of NF-B transcriptional activity by E2 bound to ER␣ or ER␤ has been well documented (6,27). Here, we demonstrate that WAY-169916 retains the ability to inhibit NF-B activity through both human ER␣ and ER␤ in vitro with little or no induction of CK activity, suggesting that WAY-169916 pharmacologically dissociates the NF-B-suppressive activity of ER from its transcriptional activity.…”

“…The ability to dissociate these two activities has been previously demonstrated by using site-directed mutagenesis on ER␣. Mutations within both helix 12 and helix 3 of ER␣ were identified that impaired ER transcriptional activity but retained the ability to antagonize NF-B transcriptional activity in a ligand-dependent fashion (27). It will be important to determine whether these residues also contribute in the selectivity observed with WAY-169916.…”

“…ER␣ expression predominates in the uterus, pituitary, kidney, and adrenal gland, whereas ER␤ expression is highest in the prostate, ovary, bladder, and lung. The two receptors have nearly identical DNA-binding domains and can activate transcription through binding to identical ER response elements (25,26), and both can antagonize NF-B functional activity (6,27).…”