Malcolm G. Parker

Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors.

The oestrogen receptor stimulates transcription by means of at least two distinct transcriptional activation domains, TAF‐1 in the N‐terminal domain and TAF‐2 in the hormone binding domain. Here we show that TAF‐2 activity requires a region in the C‐terminus of the hormone binding domain between residues 538 and 552 in the mouse oestrogen receptor which is conserved among many nuclear hormone receptors. Point mutagenesis of conserved hydrophobic and charged residues significantly reduced ligand dependent transcriptional activation but had no effect on steroid or DNA binding. Mutation of the corresponding residues in the glucocorticoid receptor also abolished transcriptional activation. We therefore propose that the conserved region may be essential for ligand dependent transcriptional activation by other members of the nuclear receptor family.

ESTROGEN RECEPTORS ALPHA AND BETA FORM HETERODIMERS ON DNA

The estrogen receptor (ER) is expressed in two forms, ERα and ERβ. Here we show that ERα and ERβ, expressed both in vitro and in vivo, form heterodimers which bind to DNA with an affinity (K d of approximately 2 nm) similar to that of ERα and greater than that of ERβ homodimers. Mutation analysis of the hormone binding domain of ERα suggests that the dimerization interface required to form heterodimers with ERβ is very similar but not identical to that required for homodimer formation. The heterodimer, like the homodimers, are capable of binding the steroid receptor coactivator-1 when bound to DNA and stimulating transcription of a reporter gene in transfected cells. Given the relative expression of ERα and ERβ in tissues and the difference in DNA binding activity between ERα/ERβ heterodimers and ERβ it seems likely that the heterodimer is functionally active in a subset of target cells.

Characterization and colocalization of steroid binding and dimerization activities in the mouse estrogen receptor

We have identified a region within the steroid binding domain of the mouse estrogen receptor that is required for both receptor dimerization and high affinity DNA binding. Analysis of sequences in this region revealed that a heptad repeat of hydrophobic residues was conserved in all members of the nuclear receptor superfamily. Single amino acid substitutions of residues in the N-terminal half, but not the C-terminal half, of the repeat prevented receptor dimerization. Steroid binding was abolished by point mutations in the center of the conserved region, implying that the steroid binding and dimerization domains overlap. The role of this region in steroid receptor function is discussed in relation to other models of protein dimerization and DNA binding.

Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor

Steroid receptor co‐activator (SRC1) is one of a number of transcriptional co‐activators that are capable of potentiating the activity of nuclear receptors including the oestrogen receptor (ER). Here we report that two isoforms, SRC1a and SRC1e, which diverge at their C‐termini, are functionally distinct as they differ in their abilities to enhance the activity of the ER in intact cells. SRC1e enhanced the ability of the ER to stimulate transcription to a greater extent than SRC1a, which had negligible effects on certain promoters. To elucidate the basis of this functional difference, we compared the nuclear receptor‐binding properties and mapped the transcriptional activation domains in the two SRC1 isoforms. Both isoforms share a triplet of nuclear receptor‐binding motifs (LXXLL motifs) for binding to functional ER dimers, and an activation domain which co‐localizes with the CBP‐binding domain, while SRC1a contains a unique LXXLL motif in its C‐terminus. Although this LXXLL motif increases the affinity for the ER in vitro, it does not appear to be responsible for the functional difference between the two isoforms. This difference is due to a second activation domain that is CBP independent and is suppressed in the SRC1a isoform. Thus, SRC1 exists as functionally distinct isoforms which are likely to play different roles in ER‐mediated transcription.

A comparison of transcriptional activation by ERα and ERβ

Abstract We have compared the ability of ERα and ERβ to stimulate transcription from a number of reporter genes in different cell lines and demonstrate that the activity of AF1 in ERβ is negligible compared with that of ERα on ERE based reporters. The activity of AF2 in ERα and ERβ is similar and this is likely to reflect their similar ability to bind coactivators. As a consequence, when transcription from a gene depends on both AF1 and AF2 the activity of ERα greatly exceeds that of ERβ but when AF1 is not required ERα and ERβ have similar transcriptional activities.

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

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.

Structure and Function of the Estrogen Receptor

The hormone binding domain of the estrogen receptor is required not only for binding estradiol but also to form stable homodimers of the protein and mediate transcriptional activation by the receptor. Residues that are essential for estrogen binding are also involved in dimerization, suggesting that the hormone-binding pocket is at or near the dimer interface. Distinct hydrophobic and charged residues are essential for hormone-dependent transcriptional activation, and these appear to be conserved by other members of the nuclear receptor family. We have found that the pure antiestrogens ICI 164384 and ICI 182780 increase the turnover of the receptor compared with that in the presence of estradiol. Because it is likely that the pure antiestrogens bind to a similar region of the receptor as that of estradiol, we propose that they inhibit receptor dimerization by means of their 7 alpha alkyl-amide extension. It appears that as a consequence nuclear uptake is inhibited and the receptor more rapidly degraded in the cytoplasm.

Effect of androgen on the transcription of rat prostatic binding protein genes

The mechanisms whereby androgens modulate mRNA levels for the C1, C2 and C3 prostatic binding protein genes in the rat ventral prostate have been investigated. Transcription rates for the 3 genes were lowered 2-3-fold upon castration but found to increase again upon re-administration of testosterone. Although this demonstrated a direct effect of androgens at the transcriptional level for these genes, the rates of mRNA synthesis were insufficient to account for previously observed steady-state measurements. This was confirmed by analysis of the nuclear steady-state levels for each mRNA following hormonal manipulation of the rats in vivo. It is concluded that besides direct transcriptional regulation, androgens must also exert a major effect of the half-life of these mRNAs in the rat ventral prostate.

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

The estrogen receptor (ER) suppresses transcriptional activity of the RelA subunit of nuclear factor-κB 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 ERα and ERβ inhibit RelA in response to 17β-estradiol but not in the presence of antihormones. We have identified residues within the ERα 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.

Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif.

Steroid hormones induce the transcriptional activity of their cognate receptors by recruiting a variety of cofactors. One of these, steroid receptor co-activator-1 (SRC-1) interacts with the ligand binding domains of a number of different receptors by means of LXXLL motifs. We have investigated the relative interaction of four such motifs in SRC-1a using a yeast two-hybrid assay. We demonstrate that ERalpha, ERbeta and ERbeta2 preferentially interact with motif 2 while GR, AR, PPARalpha and PPARgamma preferentially interact with motif 4. We show that the interactions depend not only on the LXXLL motif itself but also on residues flanking the motif.