Roger White

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.

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.

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.

Identification of phosphorylation sites in the mouse oestrogen receptor

Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17 beta-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radio-labelled with [32P]orthophosphate and a number of 3H- or 14C-labelled amino acids, immunopurified and then tryptic peptides were separated by thin layer chromatography or high performance liquid chromatography. Amino acid sequence analysis indicated that Ser-122, Ser-156, Ser-158 and Ser-298 were phosphorylated. The substitution of Ser-122 and Ser-298 with alanine had a negligible effect on the transcriptional activity of the receptor in transfected cells. However, a reduction of transcriptional activity was observed when Ser-122 was mutated in the context of mutations in a putative amphipathic alpha-helix involved in AF-2 activity. Thus a region of AF-1 that encompasses Ser-122 appears to interact with AF-2 in the full-length receptor.

Aromatase activity and CYP19 gene expression in breast cancers

The aromatase enzyme complex is responsible for the conversion of C19 androgens to oestrogens. Aromatase expression in oestrogen-responsive breast cancers may be an important mechanism of autocrine regulation in tumour growth. To evaluate whether aromatase cytochrome P450 (P450arom) transcript levels within breast tumours were correlated to the enzyme activity, a specific competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed. In this reaction, a 32 base-deleted complementary RNA was used as internal standard. In vitro aromatase activity was measured by either the tritium release assay or characterization of oestrogen fractions. Results indicate that there is a positive correlation between P450arom transcript levels and enzyme activity, but the relationship does not reach statistical significance. Therefore, whereas aromatase mRNA quantification may be an option by which to monitor the potential of tumour to synthesize oestrogens, it will not accurately reflect enzyme activity in a minority of tumours. Preliminary evidence was obtained in a tumour with low enzyme activity and a high P450arom transcript level for the presence of an endogenous aromatase inhibitor. This study highlights the necessity to characterize factors involved in the regulation of aromatase activity in such tumours.

Analysis of oestrogen receptor dimerisation using chimeric proteins

Sequences essential for dimerisation have been identified in the hormone binding domain of the mouse oestrogen receptor by insertional and point mutagenesis and sequence comparisons reveal that equivalent residues may be conserved in other members of the nuclear hormone receptor superfamily. To assess functional compatibility of this region between members of the receptor superfamily, peptide sequences corresponding to the equivalent regions of the human androgen receptor and retinoic acid receptor have been substituted for the dimerisation domain of the mouse oestrogen receptor. The resulting chimeric proteins were analysed for high affinity DNA binding using a gel retardation assay and shown to bind with reduced affinity compared to the wild type oestrogen receptor. The reduction in DNA binding observed may result from the intramolecular incompatibility of functional elements within the hormone binding domain of nuclear hormone receptors.

Analysis of regulatory sequences in androgen-responsive genes☆

We have analysed the effect of androgens on the activity of promoters from MMTV, and the rat prostate C3(1) and mouse secretory protease inhibitor genes. MMTV promoter activity was stimulated by testosterone as well as progesterone and dexamethasone but not by oestradiol. Deletion analysis indicated that the three steroids acted through DNA sequences between nucleotides -201 and -69 upstream of the MMTV cap site. In contrast, the promoters for the C3(1) gene and the protease inhibitor gene were unaffected by testosterone in a number of cell types, including prostate cells, despite the fact that the MMTV promoter was stimulated in such cells.