The F domain of estrogen receptor α is involved in species-specific, tamoxifen-mediated transactivation

Abstract

Estrogen receptor α (ERα) is a major transducer of estrogen-mediated physiological signals. ERα is a member of the nuclear receptor superfamily, which encompasses ligand-dependent transcription factors. The C terminus of nuclear receptors, termed the F domain, is the least homologous region among the members of this family. The ERα F domain possesses 45 amino acids; however, its function remains unclear. We noticed that the homology of the F domains between mouse and human ERαs is remarkably lower (75.6% similarity) than that between the entire proteins (94.7% similarity). To assess the functionality of the ERα F domains, here we generated chimeric ERα expression constructs with mouse-human-exchanged F domains. Using cell-based assays, we analyzed the transcriptional coactivator interaction and ligand-binding domain dimerization activities of these mouse-human F domain-swapped ERαs. We found that the transcriptional activity of the mouse WT ERα is more potent than that of the human WT ERα in the human hepatoma cell line HepG2. 4-Hydroxytamoxifen (4OHT)-mediated transcriptional activity of mouse-human F domain-swapped ERαs was the inverse of the WT ERα activities but not estradiol-mediated transcriptional activities. Further experiments with constructs containing deletion or point mutations of a predicted β-strand region within the F domain suggested that this region governs the species-specific 4OHT-mediated transcriptional activity of ERα. We conclude that the ERα F domain has a species-specific function in 4OHT-mediated receptor transactivation and that mouse-human F domain-swapped ERα mutants enable key insights into ERα F domain structure and function.

The genomic regulatory elements for estrogen receptor alpha transactivation‐function‐1 regulated genes

“…In contrast, the 4OHT‐bound ERα appears to recognize the HSs differently, because the 4OHT‐mediated Klk1b21p_0.4k transcriptional activity was retained with any combination of the HS mutations. We have previously reported that the differential dimer formation of the ligand‐binding domain (LBD) between SERMs (eg, 4OHT, raloxifene) and E2 bound ERα which is linked to the SERMs‐specific AF‐1–mediated transcriptional activity 15 . The differential 4OHT‐mediated ERα dimer formation might be involved in and could explain the differential recognition of the DRs in the utilization of the EREs compared to that of the E2‐mediated dimer.…”

“…The following plasmids have been described previously, 11 pcDNA3‐mERa; pcDNA3 plasmid contains full‐length mouse ERα (mERα1‐599), pcDNA3‐121‐mERa; pcDNA3 plasmid contains N‐terminal 120 amino acids (AA) truncated mouse ERα (mERα121‐599), pcDNA3‐AF2ER; pcDNA3 plasmid contains L543A, L544A mutated full‐length mouse ERα (mERα1‐599, L543A, L544A), pcDNA3‐mERa339; pcDNA3 plasmid contains 1 to 339 AA of mouse ERα. The protein expression level of these plasmids has been analyzed and reported previously 12,15 . The mouse ERα expression plasmids containing the mutations of N‐terminal serine residues (S108, S110, and S122 of mouse ERα) were generated by PCR‐based site‐directed mutagenesis.…”

“…The protein expression level of these plasmids has been analyzed and reported previously. 12,15 The mouse ERα expression plasmids containing the mutations of N-terminal serine residues (S108, S110, and S122 of mouse ERα) were generated by PCR-based site-directed mutagenesis. The reaction mixture contains the Pfu Turbo DNA polymerase (Agilent Technologies), a pair of sense and anti-sense oligo DNAs, and template DNA.…”

“…We have previously reported that the differential dimer formation of the ligand-binding domain (LBD) between SERMs (eg, 4OHT, raloxifene) and E2 bound ERα which is linked to the SERMs-specific AF-1-mediated transcriptional activity. 15 The differential 4OHT-mediated ERα dimer formation might Note: The peak counts of mERα ChIP-Seq containing the perfect palindromic ERE (5'-GGTCAnnnTGACC-3'), the direct repeat (DR; 5'-AGGTCA-n-AGGTCA-3') and both. The peak counts of DR only and both were subcategorized to the spacing 10-40 nucleotides (DR10-40), 41-70 nucleotides (DR41-70), 71-100 nucleotides (DR71-100), 101-130 nucleotides (DR101-130), and 131-160 nucleotides (DR131-160).…”

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay

“…The F domain is localized immediately adjacent to the E domain and is the most variable domain among the NRs. Even between human and mouse ERα, the homology of the F domain is significantly lower than that of the other domains 1 . The ligand-bound LBD of ERα enhances the homodimerization of the ERα protein to bind the specific estrogen-responsive DNA element directly for regulating the ligand-dependent gene transcription (classical action of ERα).…”