Ligand-independent activation of the oestrogen receptor by mutation of a conserved tyrosine

Abstract

The oestrogen receptor is a member of the nuclear receptor family of transcription factors which, on binding the steroid hormone 17β‐oestradiol, interacts with co‐activator proteins and stimulates gene expression. Replacement of a single tyrosine in the hormone‐binding domain generated activated forms of the receptor which stimulated transcription in the absence of hormone. This increased activation is related to a decrease in hydrophobicity and a reduction in size of the side chain of the amino acid with which the tyrosine is replaced. Ligand‐independent, in common with ligand‐dependent transcriptional activation, requires an amphipathic α‐helix at the C‐terminus of the ligand‐binding domain which is essential for the interaction of the receptor with a number of potential co‐activator proteins. In contrast to the wild‐type protein, constitutively active receptors were able to bind both the receptor‐interacting protein RIP‐140 and the steroid receptor co‐activator SRC‐1 in a ligandindependent manner, although in the case of SRC‐1 this was only evident when the receptors were pre‐bound to DNA. We propose, therefore, that this tyrosine is required to maintain the receptor in a transcriptionally inactive state in the absence of hormone. Modification of this residue may generate a conformational change in the ligand‐binding domain of the receptor to form an interacting surface which allows the recruitment of co‐activators independent of hormone binding. This suggests that this tyrosine may be a target for a different signalling pathway which forms an alternative mechanism of activating oestrogen receptor‐mediated transcription.

Tyrosine phosphorylation of estradiol receptor by Src regulates its hormone-dependent nuclear export and cell cycle progression in breast cancer cells

“…Expectedly, 40 the ER537F mutant retains most of its ability to activate ERE-dependent transcription. Previous papers reported that different substitutions in this residue differently affect ER transcriptional activity.…”

Loss of ERβ expression as a common step in estrogen-dependent tumor progression

“…The N-terminal domain of nuclear receptors encodes a ligand-independent activation function (AF-1) (Tora et al 1989, Berry et al 1990, a region of the receptor involved in proteinprotein interactions (Onate et al 1998), and transcriptional stimulation of target gene expression. The activation function-2 (AF-2) domain, located in the LBD (Tora et al 1989), is responsible for hormonedependent activation through recruitment of coactivator proteins (Tremblay et al 1997, White et al 1997. There is very little conservation in the N-terminal AF-1 domain, a fact which could explain why different sets of proteins in the transcription complexes may interact with ERa and ERb and direct them to specific targets.…”

Concerted expression of BK virus large T- and small t-antigens strongly enhances oestrogen receptor-mediated transcription.

“…The plasmid pEREvit-TK-LUC contains the consensus ERE of the vitellogenin gene of Xenopus laevis linked to the minimal thymidine kinase (TK) promoter of herpes simplex virus type 1. The plasmids pRcCMV and pRcCMV-CBP were kindly provided by R. Goodman (Lundblad et al, 1995), while pMT2.MOR1-599 Y541-A was a kind gift from M. Parker (White et al, 1997). Plasmid pO-LUC was generously made available to us by F. Argenton (Argenton et al, 1996).…”

“…Cells received a luciferase reporter plasmid containing either the wild-type BKV Dunlop promoter (plasmid pDun-LUC) or an ERE-mutated BKV Dunlop promoter [pDun(mERE)-LUC] and were co-transfected with an empty expression vector or pMT2.MOR1-599 Y541-A. The latter encodes a constitutive active ER (White et al, 1997). The use of such a constitutive activated receptor allows us to perform experiments in the absence of oestrogens, which have been shown to alter gene expression independent of their receptor.…”