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. 2018 Jun 1;293(22):8495-8507.
doi: 10.1074/jbc.RA117.001212. Epub 2018 Apr 9.

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

Yukitomo Arao  1 , Kenneth S Korach  2
Affiliations

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

Yukitomo Arao et al. J Biol Chem. .

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 in vitro 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.

Keywords: F domain; domain swapping; estradiol; estrogen; estrogen receptor; estrogen signaling; human; ligand-binding protein; mouse; nuclear receptor superfamily; receptor transactivation; selective estrogen receptor modulator (SERM); tamoxifen.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Differential transcriptional activity between human and mouse ERα. Balb3T3A31 cells were cotransfected with the reporter plasmid (3xERE-TATA-luc), reference plasmid (pRL-TK), and expression vectors for full-length human or mouse ERα (hERαWT or mERαWT) and empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm), E2 (A), or 4OHT (B). Treated concentrations are described in the figures. HepG2 cells were cotransfected with the reporter plasmids (3xERE-TATA-luc, C and D; or C3-110tk-luc, E and F), pRL-TK, and expression vectors for full-length ERα or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm), E2 (C and E), or 4OHT (D and F). The luciferase activities for each panel are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERα and hERα activities; b, hERα activity against vehicle; c, mERα activity against vehicle; p < 0.05. G, whole-cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody to demonstrate expression levels of ERα WT. β-Actin was used as a loading control (Actin). Representative Western blotting is shown.
Figure 2.
Figure 2.
The activity of AF-1 is similar between human and mouse ERα. A, ERα consists of six domains named A–F. A/B domain possesses AF-1. E domain possesses ligand-dependent transactivation function (AF-2). mERα339 is the mouse ERα mutant with entire AF-2 truncation; hERα340 is the human ERα mutant with entire AF-2 truncation. B, whole-cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody to demonstrate expression levels of WT and C-terminal end truncated ERα. β-Actin was used as a loading control (Actin). Representative Western blotting is shown. HepG2 cells were cotransfected with the reporter plasmids (3xERE-TATA-luc, C; or C3-110tk-luc, D), pRL-TK, and expression vectors for full-length ERα (hERαWT or mERαWT), C-terminal end truncated ERα (hERα340 or mERα339), or empty expression plasmid (pcDNA3). Cells were treated with vehicle (−) or 10 nm 4OHT (+). The luciferase activities for each panel are represented as relative activity over pcDNA3. The activity is represented as mean ± S.D. One-way ANOVA was performed to indicate significant difference: a, against pcDNA3; b, WT ERα activity against vehicle; p < 0.05.
Figure 3.
Figure 3.
The activity of AF-2 is different between human and mouse ERα. A, HepG2 cells were cotransfected with pG5-luc and the expression vector for the Gal4DBD-fused ERα LBD (pB-mEF, pB-mEΔF, pB-hEF, or pB-hEΔF). Cells were treated with vehicle (0 nm) or E2 (1–100 nm). The luciferase activities are represented as fold activation over vehicle (0 nm). The activity is represented as mean ± S.D. B, HepG2 cells were cotransfected with pG5-luc, pRL-TK, and the expression vectors for the Gal4DBD-fused NCOA NR-box (pM-SRC1-NR, pM-GRIP1-NR, or pM-ACTR-NR) in the presence of expression vectors for VP16AD (pACT) or VP16AD-fused ERα LBD (pA-mEF, pA-mEΔF, pA-hEF or pA-hEΔF). Cells were treated with vehicle (0 nm) or E2 (10 and 100 nm). The luciferase activities are represented as relative activity over pACT in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, against vehicle (0 nm); b, between mEF and mEΔF activities in each concentration; c, between hEF and hEΔF activities in each concentration; p < 0.05.
Figure 4.
Figure 4.
F domain contributes to the differential transcriptional activity between human and mouse ERα. A, schematic diagram of human/mouse F domain–swapped ERα constructs. B, whole-cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody to demonstrate expression levels of WT and F domain–swapped ERα. β-Actin was used as a loading control (Actin). Representative Western blotting is shown. HepG2 cells were cotransfected with the reporter plasmid (3xERE-TATA-luc, C and D; or C3-110tk-luc, E and F), pRL-TK, and expression vectors for ERα mutants (mERα-mF, mERα-hF, hERα-hF, or hERα-mF) or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm), E2 (C and E), or 4OHT (D and F). Treated concentrations are described in figures. The luciferase activities are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERα-mF and mERα-hF in each concentration; b, between hERα-hF and hERα-mF in each concentration; p < 0.05.
Figure 5.
Figure 5.
F domain is involved in the E2-dependent coactivator recruitment to the LBD. A, HepG2 cells were cotransfected with pG5-luc and the expression vector for the Gal4DBD-fused ERα LBD (pB-mEmF, pB-mEhF, pB-hEhF, or pB-hEmF). Cells were treated with vehicle (0 nm), E2 (1–100 nm), or 4OHT (1–100 nm). The luciferase activities are represented as fold activation over vehicle (0 nm). The activity is represented as mean ± S.D. B, HepG2 cells were cotransfected with pG5-luc, pRL-TK, and the expression vectors for the Gal4DBD-fused NCOA NR-box (pM-SRC1-NR, pM-GRIP1-NR, or pM-ACTR-NR) in the presence of expression vectors for VP16AD (pACT) or VP16AD-fused ERα LBD (pA-mEmF, pA-mEhF, pA-hEhF, or pA-hEmF). Cells were treated with vehicle (0 nm) or E2 (10 and 100 nm). The luciferase activities are represented as relative activity over pACT in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, against vehicle (0 nm); b, between mEmF and mEhF activities in each concentration; c, between hEhF and hEmF activities in each concentration; p < 0.05.
Figure 6.
Figure 6.
F domain is involved in the 4OHT-dependent LBD dimerization. A, HepG2 cells were cotransfected with pG5-luc and expression vector for Gal4DBD-fused mERα LBD in the presence of expression vector for VP16AD (pB-mEmF+pA or pB-mEhF+pA) or VP16AD-fused mERα LBD (pB-mEmF+pA-mEmF or pB-mEhF+pA-mEhF). Cells were treated with either vehicle (0 nm) or 4OHT (0.1–10 nm). The luciferase activity is represented as relative activity that is set as 1 in the vehicle (0 nm)-treated pB-mEmF+pA- or pB-mEhF+pA-transfected cells for pB-mEmF+pA-mEmF or pB-mEhF+pA-mEhF, respectively. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, mEmF dimerization activity against vehicle (0 nm); b, mEhF dimerization activity against vehicle (0 nm); c, between mEmF dimerization and mEhF dimerization activities in each concentration; p < 0.05. B, HepG2 cells were cotransfected with pG5-luc and expression vector for Gal4DBD-fused hERα LBD in the presence of expression vector for VP16AD (pB-hEhF+pA or pB-hEmF+pA) or VP16AD-fused hERα LBD (pB-hEhF+pA-hEhF or pB-hEmF+pA-hEmF). Cells were treated with either vehicle (0 nm) or 4OHT (0.1–10 nm). The luciferase activity is represented as relative activity that is set as 1 in the vehicle (0 nm)-treated pB-hEhF+pA- or pB-hEmF+pA-transfected cells for pB-hEhF+pA-hEhF or pB-hEmF+pA-hEmF, respectively. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, hEhF dimerization activity against vehicle (0 nm); b, hEmF dimerization activity against vehicle (0 nm); c, between hEhF dimerization and hEmF dimerization activities in each concentration; p < 0.05.
Figure 7.
Figure 7.
Mouse-specific residues of F domain contribute to the potency of 4OHT-mediated ERα activation. A, amino acid sequence of mouse and human F domain. Deleted tyrosine-tyrosine-isoleucine residues (YYI) are denoted as red letters. B, whole-cell lysates extracted from the plasmid-transfected HepG2 cells were analyzed by immunoblotting with anti-ERα antibody to demonstrate expression levels of WT and YYI deleted ERα. β-Actin was used as a loading control (Actin). Representative Western blotting is shown. HepG2 cells were cotransfected with the reporter plasmid (3xERE-TATA-luc), pRL-TK, and the expression vector for WT or YYI-deleted ERα (mERαΔYYI and hERαΔYYI) or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm), E2 (C), or 4OHT (D). Treated concentrations are described in the figures. The luciferase activities for each panel are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERαWT and mERαΔYYI activities in each concentration; b, between hERαWT and hERαΔYYI activities in each concentration; p < 0.05. E, diagram of mutated human ERα F domain. HepG2 cells were cotransfected with the reporter plasmid (3xERE-TATA-luc), pRL-TK, and expression vectors for full-length ERα (mERα-mF and hERα-hF), F domain mutated ERα (hERαTyyiPP), or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm), E2 (F), or 4OHT (G). The luciferase activities for each panel are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERα-mF and hERαTyyiPP activities in each concentration; b, between hERα-hF and hERαTyyiPP activities in each concentration; p < 0.05. H, HepG2 cells were cotransfected with pG5-luc and expression vector for Gal4DBD-fused WT or mutant hERα LBD in the presence of expression vector for VP16AD (pB-hEF+pA or pB-hEF-TyyiPP+pA) or VP16AD-fused WT or mutant hERα LBD (pB-hEF+pA-hEF or pB-hEF-TyyiPP+pA-hEF-TyyiPP). Cells were treated with either vehicle (0 nm) or 4OHT (0.1–10 nm). The luciferase activity is represented as relative activity that set as 1 in the vehicle (0 nm)-treated pB-hEF+pA- or pB-hEF-TyyiPP+pA-transfected cells for pB-hEF+pA-hEF or pB-hEF-TyyiPP+pA-hEF-TyyiPP, respectively. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between hEF dimerization and hEF-TyyiPP dimerization activities in each concentration; p < 0.05.
Figure 8.
Figure 8.
Mouse ERα is more potent than human ERα for estrogen-responsive gene activation. HeLa cells were transfected with the expression vectors for WT ERα (hERα or mERα) or empty expression plasmid (pcDNA3). The transfected cells were treated with vehicle, 10 nm E2, or 10 nm 4OHT for 24 h. SERPINB9, TFF1, or Neo mRNA was measured by quantitative PCR. The expression level of SERPINB9 (A and B) and TFF1 (C and D) mRNAs was normalized by the Neo mRNA level. The results of ERα-transfected cells and pcDNA3-transfected cells were shown separately, because the expression level of Neo mRNA was significantly different between these cells. The mRNA level is represented as relative level over vehicle-treated hERα-transfected cells. The mRNA level of pcDNA3 transfected cells is represented as relative level over vehicle. E, Neo mRNA level is shown. Neo mRNA level was normalized by RPLP0 (also known as 36B4). Each data are the average of triplicate determinations represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference, *, p < 0.05; **, p < 0.01; ****, p < 0.0001; ns, non-significant difference.
Figure 9.
Figure 9.
Species specific function of F domain for the E2-dependent transactivation appears on the single ERE. Top panel, HepG2 cells were cotransfected with the reporter plasmids (1xERE-tk-luc, 2xERE-tk-luc, or 3xERE-tk-luc), pRL-TK, and expression vectors for C-terminal end truncated ERα (hERα340 or mERα339) or empty expression plasmid (pcDNA3). The luciferase activity is represented as relative activity over pcDNA3 in each reporter. The activity is represented as mean ± S.D. One-way ANOVA was performed to indicate significant difference: a, against pcDNA3 in each reporter; p < 0.05. Middle panel, HepG2 cells were cotransfected with the reporter plasmids, pRL-TK, and expression vectors for N-terminal end truncated ERα (117-hERα or 121-mERα) or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm) or E2. Treated concentrations are described in the figures. The luciferase activities are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERα and hERα activities; b, hERα activity against vehicle; c, mERα activity against vehicle; c, p < 0.05. Bottom panel, HepG2 cells were cotransfected with the reporter plasmids, pRL-TK, and expression vectors for ERα mutants (mERα-mF, mERα-hF, hERα-hF, or hERα-mF) or empty expression plasmid (pcDNA3). Cells were treated with vehicle (0 nm) or E2. The luciferase activities are represented as relative activity over pcDNA3 in each concentration. The activity is represented as mean ± S.D. Two-way ANOVA was performed to indicate significant difference: a, between mERα-mF and mERα-hF in each concentration; b, between hERα-hF and hERα-mF in each concentration; p < 0.05.
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