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Title: The interaction between AF-1 and AF-2 in the two human estrogen receptors
Author: Hassanin, Kamel Mohamed AbdAlla
ISNI:       0000 0001 3545 8626
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2004
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Estrogen receptors, ERα and ERβ, are members of the large nuclear receptor superfamily of ligand-activated transcription factors. They stimulate the transcription of their target genes by two different activation functions (AF-1 and AF-2). These two activation functions exert their effects independently but can interact with each other in a synergistic manner. The contributions of AF-1 and AF-2 to overall transcriptional activation vary in a cell- and gene-specific manner. The interaction between AF-1 and AF-2 has therefore been compared in hERα and hERβ. Equivalent point mutations and truncation mutations were created in hERα and hERβ. The hERα or hERβ constructs were co-transfected together with a series of ERE-based reporter genes into Cos-1, HeLa and MCF10A cell lines. A point mutation (L540Q) had previously been reported to abolish the AF-2 activity of hERα without affecting the receptor's ligand-binding properties. We created this mutant and the hERβ equivalent (L491Q) in both full-length ER and in N-terminal truncated receptors. Hormone binding analyses revealed that the two mutants had similar affinities for estradiol to those of their respective wild type receptors. Both reporter-specific and cell type specific differences were observed in the responses of the mutant receptors to estradiol and to the anti-estrogens, transhydroxytamoxifen and ICI 182780. Differences were observed between the ERα and ERβ mutants but their general behaviour was similar. The remaining estradiol-induced transcriptional activity of the two AF-2 mutants appeared to be due to AF-1 activity. hERβ does not possess a strong AF-1, unlike ERα, and was not able to support an agonistic response to transhydroxytamoxifen. However, hERβ did show EGF-induced ligand-independent activation similar to hERα. Findings with truncated ERβ indicated that EGF mediates its effect on ERβ through AF-1 but not AF-2. Similar to hERα, simultaneous treatment of ERβ with EGF and estradiol showed an additive effect indicating that hERβ has two distinct mechanisms for ligand-independent and ligand-dependent activation. Unexpectedly, the AF-2 mutation, L540Q, abolished the ERα EGF response but this was restored after the further addition of either estradiol or ICI 182780. The equivalent AF-2 mutation (L491Q) in hERβ either reduced or abolished EGF-induced response, depending on the promoter and cell contexts. These findings suggest that EGF-activated AF-1 of hERβ and of hERα requires a functioning AF-2 in the receptor with which to interact to stimulate transcription. Point mutants were created in which phosphorylatable serines in the AF-1 of hERβ (Ser 87 & Ser 105) and in the AF-1 of ERα (Ser 118) were substituted by unphosphorylatable alanine residues. The serine 118 of ERα was found to be essential for its EGF response but had no significant role in its estradiol response. Serine 105 in hERβ is involved in both estradiol and EGF responses. Serine 87 of hERβ is important for its EGF response but has no role in its hormone response. These studies have produced further evidence for the interaction between the AF-1 and AF-2 transcriptional activation functions of human estrogen receptors. They have demonstrated significant differences between hERα and hERβ and have identified key amino acids in the two receptors that are involved in the stimulation of the transcription of estrogen-responsive genes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral