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

Estrogen receptor alpha (ER α ) is a major transducer of estrogen-mediated physiological signals. ER α 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 ER α F domain possesses 45 amino acids; however, its function remains unclear. We noticed that the homology of the F domains between mouse and human ER α s is remarkably lower (75.6% similarity) than that between the entire proteins (94.7% similarity). To assess the functionality of the ER α F domains, here we generated chimeric ER α 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 ER α s. We found that the transcriptional activity of the mouse wild-type ER α is more potent (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 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 of hEhF dimerization activity against vehicle (0 nM), a ; hEmF dimerization activity against vehicle (0 nM), b ; between hEhF dimerization and hEmF dimerization activities in each concentration, c ; p < 0.05.


human F domain swapped ERαs was the inverse of the wild-type ERα activities but not estradiol-mediated transcriptional activities.
Further experiments with constructs containing deletion or point mutations of a predicted βstrand 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.
Estrogen regulates multiple physiological functions in various tissues, including reproduction, bone density and metabolic regulation. Estrogen receptors (ERα and ERβ) are ligand dependent transcription regulators, which transmit estrogen signals to estrogen-mediated physiological functions (1). The phenotypes of ER null mutant mice have suggested that ERα plays a primary role in a number of estrogen mediated physiological responses compared to ERβ null mutant mice (2). Many of these phenotypes of 2 ERα activities are also observed in clinical cases of ERα insensitivity (3)(4)(5). Thus, ERα has been a major target for the development of therapeutic reagents, such as selective estrogen receptor modulators (SERM) and selective estrogen receptor degraders which control estrogenmediated physiological responses.
ERα contains highly-conserved domain structures common among the nuclear receptor superfamily, designated A through F domains. The A/B domain, which contains transcription activation function 1 (AF-1), is localized on the Nterminus and the structure varies between the nuclear receptors. On the other hand, the C domain, also known as the DNA binding domain (DBD), and the E domain, also known as the ligand binding domain (LBD) are highly conserved between nuclear receptors. The E domain contains the ligand binding pocket and transcription activation function 2 (AF-2). The F domain is localized on the very end of the Ctermini of some nuclear receptors and is the most variable domain between the family members. Even within orthologues of ERα, the homology of the F domain sequence is significantly lower than the other domains. For instance, the similarity between human and mouse ERα A/B domain, F domain and the whole protein are 90.6%, 75.6% and 94.7% respectively (Table 1).
Concerns have arisen over differential pharmacokinetics of estrogenic compounds across different species, which are relevant to the findings of differential activities of estrogenic compounds seen in experimental animal studies (6). Due to the higher homology of ERα protein among species, it has been thought that the differential metabolism of chemicals may be the cause of differential estrogenic activities rather than species differences of ERα structure. Indeed, the structural difference of ERα between animals has also been considered as a contributing factor to the differential estrogenic activities. However, there is limited evidence for differential functionality of ERα among different animal species (7). In addition, it is totally unknown whether the F domain, which is the least homologous region among the species, is involved in the differential functionality of ERα. The function of human ERα F domain has been analyzed by several groups using different experimental models (8)(9)(10)(11)(12). These reports have suggested that the human ERα F domain is needed for the partial agonist activity of 4hydroxytamoxifen (4OHT) and the efficacy of estradiol (E2)-mediated transcription activity of ERα. However, the mechanism of the precise F domain functionality is still unclear.
The differential estrogenic activity of tamoxifen between human and mouse has been discussed. However, there are still no informative conclusions. We evaluated the transcription activities of human and mouse ERα using a cell based in vitro reporter assay and found differential transcription activities under equivalent conditions in a human hepatoma cell line. To confirm F domain's contribution to this differential activity, we generated chimeric mouse-human F domain swapped ERα expression plasmids. Analyses suggested that the F domain governs the species difference of ERα activity and a specific region of F domain contributes to the differential functionality for E2 and 4OHT mediated transcription. Our strategy of exchanging the mouse-human F domains of ERα presents new insight into evaluating ERα F domain functionality and potentially for explaining differential estrogenic chemical activities among species.

RESULTS
Differential transcription activity between mouse and human ERα in the human hepatoma cell line -At first, we analyzed the estrogen responsive element (ERE)-dependent transcription activity of mouse ERα (mERα) and human ERα (hERα) using ERE-fused luciferase (luc) reporters (3xERE-TATA-luc and C3-110tk-luc). We chose both a mouse and a human cell line to account for both species, mouse fibroblast cell line (Balb3T3A31) and human hepatoma cell line (HepG2) were transfected with the reporter plasmid and the receptor expression plasmid (pcDNA3-mERa or pcDNA3-hERa) or the empty plasmid (pcDNA3). Transactivation levels were expressed as relative activity compared to the level of pcDNA3. The basal activity of mERα without 3 ligands was higher than hERα in the Balb3T3A31 cells but there was no difference in the maximum level of E2-mediated transactivation (Fig. 1A). In the Balb3T3A31 cells, the activity of mERα was blocked by 4OHT. On the other hand, the activity of hERα was not stimulated or blocked by 4OHT (Fig. 1B). In the HepG2 cells, E2 ( Fig. 1C and 1E) and 4OHT ( Fig. 1D and 1F) mediated transcription activity of mERα was significantly higher than hERα. Although the transcription activities of mERα and hERα were different in the HepG2 cells, there was no difference in the receptor protein expression levels (Fig. 1G).
AF-1 activity was similar between mouse and human ERα in the HepG2 cells -To understand the contribution of transactivation function domains (AF-1 and AF-2) in differential activity of mERα and hERα in the HepG2 cells, we analyzed AF-1 activities using the AF-2 truncated ERα mutants (mERα339 and hERα340) ( Fig. 2A  and 2B), which exhibits only a constitutive AF-1 mediated transcription activity (13, 14). There was no difference between mERα339 and hERα340 in ERE-luc reporter activation function ( Fig. 2C and 2D), suggesting that the AF-1 activity of mouse and human ERα is indistinguishable in the HepG2 cells.
F domain contributes to the differential transcription activity of mouse and human ERα -The results of the F domain truncated mutant suggested that the F domain is highly associated with the AF-2 activity of mERα and hERα. To assess the role of the F domain in AF-2 activity, we generated ERα mutants which contain mouse and human exchanged F domains ( Fig. 4A and 4B). HepG2 cells were transfected with an EREluc reporter plasmid and the expression plasmid for the F domain swapped receptor (mERα-hF, hERα-mF) or the control receptor (mERα-mF, hERα-hF). The E2-mediated activity of F domain swapped receptors showed in-between levels compare to the levels of control receptors ( Fig. 4C and 4E). In response to 4OHT activities of mERα and hERα were totally inverted by F domain swapping ( Fig. 4D and 4F). These results suggest that the F domain influences the differential activities of mERα and hERα to 4OHT.
F domain is involved in the 4OHT-dependent LBD dimerization activity -Because we have reported previously that the efficacy of LBD dimerization associates with the partial agonist activity of 4OHT (15), 4OHT-dependent LBD dimerization activity was analyzed by using the mammalian two-hybrid assay. HepG2 cells were cotransfected with the G5-luc reporter and plasmids for expressing Gal4DBD-fused mEmF, mEhF, hEhF or hEmF (pBIND-EF), in the presence of plasmids for expressing VP16AD alone (pACT) or VP16AD-fused mEmF, mEhF, hEhF or hEmF (pACT-EF). Cells were treated with 0.1-10 nM 4OHT or vehicle. Since the 4OHT-mediated dimerization activity of mERα LBD was significantly higher than hERα LBD, the results are shown separately for mERα and hERα ( Fig. 6A and 6B). 4OHT-mediated dimerization activity of the mERα LBD (mEmF) was significantly reduced by the swapping to the human F domain (mEhF) (Fig. 6A). In contrast, the lower 4OHT-mediated dimerization activity of hERα LBD (hEhF) was increased by the swapping and incorporation of the mouse F domain (hEmF) (Fig. 6B). These results suggest that the 4OHTmediated dimerization activities of mouse and human ERα LBD are governed by the F domain. These results coincide with the 4OHT-mediated ERE-dependent transcription activity of F domain swapped ERα mutants ( Fig. 4D and 4F).
Specific region of F domain potentiates 4OHT-mediated mouse ERα activity -The function of possible structural regions of human ERα F domain have been reported, including a predictive β-strand in the C-terminal of the F domain, however actual conformation is still uncertain (10,11). We analyzed the transcription activity of several ERα mutants focused on the element near a suggested β-strand in the F domain to confirm the functional relevance of this region. Three amino acids (tyrosine-tyrosine-isoleucine, YYI) were deleted from the F domain to disrupt the predicted β-strand structure (mERαΔYYI and hERαΔYYI) (Fig. 7A). The expression level of mutant receptor protein was lower than WT in both species (Fig. 7B). Although the protein level was lower than WT, mouse and human ERαΔYYI exhibited E2-mediated transcription activity similar to WT (Fig. 7C). Interestingly, 4OHTmediated transcription activity of mERαΔYYI was significantly decreased but not hERαΔYYI (Fig.  7D). Furthermore, we generated a mutant hERα protein expression plasmid which replaced the three amino acids on the flanking of YYI to the mouse corresponding residues. Namely, the lysine (K581) on the N-terminal flanking of YYI sequence was replaced to threonine (T) and the threonine-glycine (T585, G586) residues on the Cterminal flanking of YYI sequence were replaced to prolines (PP) (hERαK581T,T585P,G586P; denoted as hERαTyyiPP) (Fig. 7E). The E2mediated transcription activity of hERαTyyiPP was identical to hERα-hF (Fig. 7F). On the other hand, 4OHT-mediated transcription activity of hERαTyyiPP was significantly higher than hERα-hF and similar to mERα-mF (Fig. 7G). Furthermore, we analyzed the LBD dimerization activity of K581T,T585P,G586P mutated human ERα (hEF-TyyiPP) using the mammalian twohybrid assay. 4OHT-mediated dimerization activity of hEF-TyyiPP was significantly higher than WT human ERα LBD (hEF) (Fig. 7H). These results suggest that the mouse specific residues around the predictive β-strand in the F domain are involved in the differential activities of 4OHTmediated mERα and hERα transcriptional regulation.
Mouse ERα is more potent than human ERα for estrogen responsive gene activation -Lastly, we examined the efficacy of E2-or 4OHTmediated endogenous gene activation through mouse and human ERα using mERα or hERα transfected HeLa cells (Fig. 8). Total RNA was extracted from the cells which were treated with 10 nM E2 or 4OHT for 24 h. As examples, the expression levels of SERPINB9 (PI9) and TFF1 (pS2) genes were analyzed. These genes are known estrogen responsive genes in several cell lines (16)(17)(18)(19). The mRNA level of ERα regulated genes was normalized by the transcript of neomycin resistant gene (Neo), which is expressed from the pcDNA3 plasmid for normalizing the transfection efficiency. PI9 and pS2 genes were 5 activated by E2 in an ERα dependent manner ( Fig.  8A and 8C). E2-induced mRNA levels of both genes were slightly higher in the mERα transfected cells than hERα transfected cells. PI9 gene was activated by 4OHT in an ERα dependent manner ( Fig. 8B) but pS2 gene was not activated by 4OHT (Fig. 8D). Importantly, the 4OHTinduced PI9 mRNA level was significantly higher in the mERα transfected cells than hERα transfected cells (Fig. 8B), which is consistent with the in vitro observations in this report.

DISCUSSION
The differential estrogenic activity of tamoxifen in experimental animals has been reported (6). These findings, led to a question of the differential estrogenic activity of tamoxifen between the human and mouse species. However, the conclusion to explain these differential activities is still unclear. In particular, there is no findings associating the structural difference between human and mouse ERα with the differential estrogenic activity of tamoxifen. The high homology between human and mouse ERα (94.7% similarity) has led to the thought that the function of these receptors is identical. However, we found that the efficacy of transcription activity of mERα is more potent than hERα. Specifically, the results of F domain swapped ERα mutants suggested that the F domain contributes to the tamoxifen-mediated differential transcriptional activity of mERα and hERα.
Our results suggested that the N-terminal transactivation function (AF-1) is the primary element for the 4OHT-dependent transcription activity of full-length ERα, following the previous reports (13 , 14) and that the F domain plays a role in this regulation. We have previously reported that the SERM mediated partial agonist activity of ERα is correlated with the efficacy of LBD (EF domain) dimerization cooperating with ERE binding (15). As we demonstrate in this report, 4OHT-dependent dimerization of mouse ERα LBD is more potent than human ERα LBD, which is consistent with the 4OHT-dependent full-length ERα transcription activities. The species difference of 4OHT-mediated LBD dimerization efficiency was totally changed by the F domain swapping and that level reflects the full-length mutant ERα transcription activities. These results clearly suggest that the F domain structure is contributing to an important role in 4OHTdependent LBD dimerization and partial agonist activity of ERα.
As we showed here, the PI9 gene was activated by 4OHT in the ERα-transfected HeLa cells but not pS2. This observation agrees with the promoter context dependent tamoxifen activity as previously reported (13). 4OHT-dependent PI9 gene expression was higher in the mERα than hERα transfected cells, suggesting that the agonist activity of 4OHT through mERα was more potent than hERα in the same promoter context. 4OHT activity via mERα was higher than hERα with each of the different promoters that we analyzed (3xERE-TATA, C3-110tk and SERPINB9). This may suggest that the species differences of 4OHTmediated ERα activity is independent of the promoter context. In other words, structural differences between human and mouse ERα could cause the species differences of agonist activity of 4OHT.
F domain replacement did not affect E2mediated full-length ERα transactivation strikingly, which was different from 4OHTmediated regulation. However, the efficacy of E2mediated NCOA interaction to the AF-2 was reversed by F domain swapping, and it was consistent with the E2-dependent AF-2 activity of F domain swapped ERα mutants. These results suggest that the F domain contributes to the modulation of the E2-dependent coactivator interacting surface of LBD, but it provides a limited contribution to the E2-dependent fulllength ERα transactivation. The differential activities of F domain swapped ERα on the C3-110tk and 3xERE-TATA reporters imply that the E2-activated full-length ERα is functioning in a different manner on each responsive element. To verify this hypothesis, we performed further analysis using the reporters which contain 1, 2 or 3 consensuses ERE motifs fused with a thymidine kinase (tk) promoter ( Fig. 9). E2-mediated fulllength mouse ERα transcription activity was higher than human ERα for those reporters in 6 HepG2 cells. Interestingly, the profile of transcription activities of F domain swapped ERα mutants for 1xERE-tk reporter was similar to the profile of C3-110tk reporter. On the other hand, the profiles of 2xERE-tk and 3xERE-tk reporters were similar to 3xERE-TATA-luc. Importantly, sole activity of AF-1 and AF-2 was not observed on the 1xERE-tk reporter different from 2xERE-tk and 3xERE-tk reporters. These results suggested that the monomeric or multimeric binding of E2mediated ERα dimer on the ERE regulates transcription activity in a different manner. Namely, the AF-2 (transactivation function of EF domain) works cooperatively with AF-1 when ERα is bound on a single ERE. On the other hand, AF-2 and AF-1 work additively expressing E2mediated transcription activity when ERα is bound on multiple EREs. The characteristics of the F domain may be emphasized when ERα dimer is monomerically bound on the ERE.
Schwartz et al. have suggested that the residues of QKYYIT (hERα 580 to 585) in the human ERα F domain contribute to a possible βstrand structure (10). However, the function of this specific region is not well known. The computational prediction analysis for the protein secondary structure suggested that a β-strand is likely to exist in this region of mouse ERα F domain similar to human ERα. The results of the YYI sequence deleted mutant ERα suggested that this region is strongly correlated with the species difference of 4OHT-mediated ERα transcription activity. We recognized that the flanking amino acids of YYI are varied between species; the lysine (K), which is a 5'-flanking residue, is highly conserved among primates but most other mammals have threonine (T). On the other hand, the threonine (T), which is a 3'-flanking residue, is conserved among mammals except rodents; rodents have proline (P) instead of T. Further analyses using a human ERα mutant with the three-point mutation swapped to the mouse ERα residues in the predicted β-strand region (hERαTyyiPP) suggested that the characteristics of the amino acids flanking the YYI residues are important for the species-specific 4OHT-mediated transactivation function of ERα. Our strategy of exchanging the mouse-human F domain sequence may be useful to examine the structural features of the ERα F domain, since there is no published crystallographic structure of the F domain. Even though our study demonstrates a functional difference.
Previous reports using the yeast two-hybrid assay have suggested that the F domain prevents the E2-mediated SRC1 (NCOA1) RID interaction to the human ERα LBD (11,12). Those results are inconsistent with the transcription activity of F domain truncated ERα in mammalian cells (8,11). We showed that the truncation of F domain clearly reduced E2-mediated NCOA RID interaction and AF-2 activity of mouse and human ERα in HepG2 cells. Our results suggested that the F domain facilitates the E2-mediated NCOA interaction to the AF-2 surface rather than preventing interaction. The contradiction between our results and previous reports which used a yeast system suggests that the function of F domain is cell type dependent. We showed here the differential activity of hERα and mERα in HepG2 cells (human hepatoma cell). We observed the same trends of ERα dependent gene regulation in a human cervical cancer cell line, HeLa cells. Thus far, only the mouse fibroblast cell line, Balb3T3A31 cells showed different results in our analyzed cell types. These results imply that species-specific factor(s) may exist for modulating the F domain function.
In summary, we revealed that the speciesspecific amino acids of the predicted β-strand region on the C-terminal of ERα F domain govern the species dependent tamoxifen mediated transcription activity. Our results also suggest that considerations should be made when analyzing SERM activity in mouse versus human samples because of the existence of differential F domain functionality between human and mouse ERα.
Cell culture and transfection condition for luciferase assay -HepG2 cells (human hepatocellular carcinoma) were cultured in phenol red-free α-MEM (Invitrogen) supplemented with 10% FBS (Gemini-Bio) and 1% penicillinstreptomycin solution (Sigma-Aldrich). Balb3T3A31 cells (mouse fibroblast) were cultured in phenol red-free α-MEM supplemented with 10% FBS, 2 mM sodium pyruvate (Sigma-Aldrich) and 1% penicillin-streptomycin solution. For transient transfections, the cells were cultured in phenol red-free medium supplemented with 10% charcoal-stripped FBS (Gemini-Bio) and seeded in 24-well plates at a density of 1.2x10 5 cells/well (HepG2) and 0.8x10 5 cells/well (Balb3T3A31). The cells were transfected with the following DNA mixture for 6 h using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For reporter assay, a DNA mixture containing 50 ng of expression plasmids for WT or mutated ERα, 100 ng of reporter plasmids for 3xERE-TATA-luc or C3-110tk-luc, and 100 ng pRL-TK was transfected in each well. For assessment of AF-2 activity, the 9 DNA mixture contained 50 ng expression plasmids for GAL4 DBD fusion proteins (pBIND) and 100 ng pG5-Luc reporter plasmid was transfected in each well. pBIND plasmid contains renilla luciferase expression unit for transfection normalization.
For assessment of LBD dimerization, the DNA mixture contained 50 ng expression plasmids for GAL4DBD fusion proteins (pBIND), 50 ng expression plasmids for VP16 activation domain (AD) fusion proteins (pACT) and 100 ng pG5-Luc reporter plasmid was transfected in each well. For assessment of coactivator interaction, the DNA mixture contained 100 ng expression plasmids for GAL4DBD fused NCOA NR-box (pM), 50 ng expression plasmids for VP16AD fusion proteins (pACT), 50 ng pRL-TK and 100 ng pG5-Luc reporter plasmid was transfected in each well.
Luciferase assay -The cells were cultured in fresh medium supplemented with the ethanol as a vehicle, E2 (Steraloids) or 4OHT (Sigma Aldrich) 6 h after transfections. Luciferase and renilla luciferase activities were assayed 18 h after treatments using Dual-luciferase Reporter Assay System (Promega). Luciferase activity was normalized for transfection efficiency using renilla luciferase as an internal control. All results are representative of at least two independent experiments and represent the mean ± S.D. of triplicate samples.
Western blot assay -The transfected cells on 24-well plate were washed with PBS and 100 µl of 2x Laemmli sample buffer (Bio-Rad) near 100 °C was added to the wells. The cells were pipetted vigorously and then into a 1.5-ml centrifuge tube. The tubes were heated at 100 °C for 10 min, cooled on ice, and stored at −80 °C until samples were analyzed on SDS-PAGE. Proteins were resolved by SDS-PAGE with 4-12% gradient gel (Invitrogen) and subsequently transferred to nitrocellulose membrane. Blots were incubated overnight in 4˚C with primary antibody for ERα (1:600; H-184, Santa Cruz Biotechnology) or βactin (1:2000; AC-74, Sigma). The blots were washed then incubated with IRDye 800CWconjugated anti-rabbit antibody (LI-COR Biosciences) for ERα or with IRDye 680RDconjugated anti-mouse antibody (LI-COR Bioscience) for β-actin. The signals were visualized by Odyssey infrared imaging system (LI-COR Biosciences). BenchMark protein ladder (Invitrogen) was used for evaluating the molecular weight.
Condition for examining the ERα dependent endogenous gene expression -HeLa cells (human cervical cancer cell) were cultured in phenol red free α-MEM supplemented with 10% FBS, 2 mM sodium pyruvate and 1% penicillin-streptomycin solution. When the cells were transfected with DNAs, the cells were cultured in phenol red-free medium supplemented with 10% charcoal-stripped FBS and seeded in 6-well plates at a density of 4.5x10 5 cells/well. The cells were transfected with the 2 µg of pcDNA3-mERa, pcDNA3-hERa or pcDNA3 for 6 h using Lipofectamine 2000. The cells were cultured with flesh phenol red-free medium supplemented with 10% charcoal-stripped FBS and ligand for 24 h. The cells were homogenized with TRIzol reagent (Invitrogen) for extracting RNA. Total RNA was treated with TURBO DNase (Invitrogen) then reversetranscribed by Superscript II (Invitrogen) to generate the cDNA following the manufacture's instruction. The quantitative PCR was perfumed by using the Fast CYBR Green Master Mix  (22). The level of Neo mRNA, which is expressed from the pcDNA3 plasmid, was analyzed for normalizing the transfection efficiency. The data was obtained from three independent experiments.

10
Statistical analysis -ANOVA with Tukey's multiple comparison test was performed by GraphPad Prism (GraphPad software). p < 0.05 was considered statistically significant.     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 against vehicle (0 nM), a; between mEF and mEΔF activities in each concentration, b; between hEF and hEΔF activities in each concentration, c; p < 0.05.

FIGURE 4.
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 blot 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 between mERα-mF and mERα-hF in each concentration, a; between hERα-hF and hERα-mF in each concentration, b; p < 0.05. 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 against vehicle (0 nM), a; between mEmF and mEhF activities in each concentration, b; between hEhF and hEmF activities in each concentration, c; p < 0.05.

FIGURE 7.
A, the amino acid sequence of mouse and human F domain. Deleted tyrosine-tyrosineisoleucine residues (YYI) are denoted as red letters. B, whole cell lysates extracted from the plasmidtransfected 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 blot 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, 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.. Twoway ANOVA was performed to indicate significant difference between mERαWT and mERαΔYYI activities in each concentration, a; between hERαWT and hERαΔYYI activities in each concentration, b; 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, 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 between mERα-mF and hERαTyyiPP activities in each concentration, a; between hERα-hF and hERαTyyiPP activities in each concentration, b; 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 between hEF dimerization and hEF-TyyiPP dimerization activities in each concentration, a; p < 0.05. . Two-way ANOVA was performed to indicate significant difference, *; P < 0.05, **; P < 0.01, ****; P < 0.0001.

FIGURE 9.
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 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 against pcDNA3 in each reporter, a; p < 0.05. Middle panel, HepG2 cells were cotransfected with the reporter plasmids, pRL-TK and expression vectors for Nterminal 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 between mERα and hERα activities, a; hERα activity against vehicle, b; 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.. Twoway ANOVA was performed to indicate significant difference between mERα-mF and mERα-hF in each concentration, a; between hERα-hF and hERα-mF in each concentration, b; p < 0.05.