Open Journal of Endocrine and Metabolic Diseases, 2013, 3, 12-19 Published Online August 2013 (
The Transactivating Function 2 (AF-2) of Estrogen
Receptor (ER) α Is Indispensable for ERα-Mediated
Physiological Responses and AF-1 Activity
Yukitomo Arao, Katherine J. Hamilton, Kenneth S. Korach*
Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology,
National Institute of Environmental Health Sciences/NIH, Research Triangle Park, North Carolina, USA
Email: *
Received May 11, 2013; revised June 11, 2013; accepted July 11, 2013
Copyright © 2013 Yukitomo Arao et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Estrogen has various ph ysiological functions and the estrogen receptor (ER) is a key regulator of those functions. ERα
is a ligand-dependent transcription factor and that activity is mediated by the transactivating function-1 (AF-1) in the
N-terminal domain and transactivating function-2 (AF-2) in the C-terminal ligand-binding domain. The functions of
ERα AF-1 and AF-2 have been characterized by various in vitro experiments, however, there is still less information
about the in vivo physiological functions of ERα AF-1 and AF-2. Recently, we established a genetically mutated ERα
AF-2 knock-in mouse (AF2ERKI) that possessed L543A, L544A mutated-ERα. This AF-2 core mutation disrupted
AF-2 function and resulted in ERα null phenotypes. This mouse model revealed that proper AF-2 core structure and
function were indispensable for ERα-mediated physiological responses and AF-1 functionality. AF2ER mutation re-
verses the ERα antagonists to agonists and that activity is mediated by AF-1 solely. The pure antagonist,
ICI182780/fulvestrant, activated several estrogen-mediated physiological responses in the AF2ERKI mouse. The
AF2ERKI mouse model will be useful to discern estrogen physiological func tions which involve AF-1.
Keywords: Knock-In Mouse; SERMs; Antagonist Reversal
1. Introduction
1.1. Transcriptional Activation Domains in
Estrogen Receptor
Estrogen receptor (ER) is a member of the nuclear re-
ceptor superfamily which share highly conserved domain
structures, including the DNA binding domain (DBD)
and ligand binding domain (LBD) [1,2]. The DBD is
localized in the middle of the protein and the carboxyl-
terminal (C-terminal) end of the protein possesses the
LBD. In contrast, the stru cture of the amino-terminal (N-
terminal) end is varied between the nuclear receptors [3].
The transcriptional activation function (AF) domains of
ERα are distributed in the N-terminal (known as AF-1)
and C-terminal (known as AF-2) of the ER protein. AF-2
is localized in the LBD on helix 12, an element in the
LBD that is defined as the core of the AF-2 region [4,5].
The configuration of helix 12 is changed by the ligand
binding to the LBD which induces a transcriptionally
active or inactive form of the receptor. When agonists
(transcription activating chemicals) bind to the LBD,
helix 12 makes a co-activator binding surface and re-
cruits transcriptional co-activators, such as SRC1/p160,
SRC2/GRIP1and SRC3/AIB1 to the LBD [6]. On the
other hand, when antagonists (transcription inactivating
chemicals) bind to the LBD, helix 12 is relocated to pr e-
vent the co-activator binding and recruits transcriptional
co-repressors to the LBD [7,8]. Thus the AF-2 is desig-
nated as a “ligand-dependent” transcription regulation
domain. The N-terminal of ERα possesses the constitu-
tively active transcriptional domain (AF-1), because of
the fact that the deletion of the LBD from the ERα pro-
tein induces high basal activ ity [9,10]. This basal activ ity
of ERα AF-1 is dependent upon the gene promoter con-
text and cell types [10]. Several phosphorylation sites
have been identified in the N-terminal end of the ERα
protein, such as serines 104/106 [11], serine 118 [12,13]
and serine 168 [14] on human ERα and serine 122 on
mouse ERα (identical to serine 118 of human ERα; [15]).
It has been reported that the phospho-status of the N-
*Corresponding a uthor.
opyright © 2013 SciRes. OJEMD
terminal modulates the transcription activity of ERα
independently of estrogen ligand [11,16,17]. Thus, the
AF-1 is designated as a “ligand-independent” transcrip-
tion regulati on domain.
1.2. Estrogenic Compounds
Various estrogenic-active compounds (agonists) have
been identified using the binding assay with ERα protein
[18-20], or using the reporter assay with a full-length
ERα protein expressed yeast [21] or with ERα positive
mammalian cells [22]. These compounds include both
man-made chemicals and the natural compounds (known
as xenoestrogens) which have various chemical struc-
tures. Various estrogen receptor antagonists and selective
estrogen receptor modulators (SERMs) have been de-
veloped in pharmaceutical research by analyzing deriva-
tives of certain estrogenic compounds [23-25]. Tamoxi-
fen (4-hydoxytamoxifen; 4OHT) is one of the most well-
characterized SERMs. 4OHT displays both agonistic and
antagonistic effects in a tissue specific manner, namely,
4OHT is an agonist in uterine tissue but works as an an-
tagonist in the mammary gland [26]. A well- known pure
antagonist, fulvestrant/ICI182780 (ICI) has been classi-
fied as a selective estrogen receptor down-regulator
(SERD), which induces ERα protein degradation [27-29].
Thus, the binding affinity to ERα is not simply correlated
to the transcriptional activity of estrogenic chemicals. It
has been reported that the partial agonist activity of
4OHT is only derived from ERα AF-1 activity and that is
a major cause of tissue selective action of 4OHT [10]. It
is important to determine if estrogenic action of chemi-
cals involves a preference of AF-1 in order to understand
the tissue specific action of estrogenic responsiveness.
However, to date there is little information about the AF-
1 depen den c y of estrogenic chemicals.
2. AF-2 Inactivated ERα Mouse Model
2.1. Generation of AF2ERKI Mouse Model
The various physiological roles of ERα have been previ-
ously demonstrated by the ERα knock-out (αERKO)
mouse model [30]. The αERKO does not express func-
tional ERα protein as a result of a genetic modification of
the Esr1 allele [31,32]. Thus this model cannot discern
the selective function ality of ERα AF-1 or AF-2 in ERα-
mediated physiological responses in vivo. To assess the
effect of loss of AF-2 function in vivo, we generated the
AF-2 mutated knock-in (KI) mice (AF2ERKI) through
the modification of the Esr1 allele [33,34]. AF2ERKI
possesses the following mutations: the leucines 543 and
544 of mouse ERα were mutated to alanines and a 6x
His-tag was added to the C-terminal end of the ERα pro-
tein (Figure 1). Leucines 543 and 544 are components of
helix 12 and the mutation of those residues causes dis-
ruption of E2-mediated transcription without the reduc-
tion of ligand binding activity [35].
2.2. AF2ERKI Homozygote Female
Continuous breeding studies indicated that AF2ERKI
homozygote females were infertile. The AF2ERKI fe-
male does not display signs of a normal estrous cycle and
vaginal smears display a pattern of persistent diestrus.
The serum LH level, which is an end point linked to re-
productive cyclicity and responsiveness, was elevated in
AF2ERKI 4-fold over the wild-type (WT) female. The
serum E2 level of AF2ERKI was 2-fold higher than the
WT female [33]. These hormone levels in the AF2ERKI
female were similar to the αERKO female. Such elevated
LH levels suggest that the negative feedback of the hy-
pothalamic-pituitary gonadal axis in the AF2ERKI fe-
male is disrupted as has been reported in the αERKO
animals [30], consistent with the infertility. The uterine
tissues of AF2ERKI possess luminal epithelium but few-
er glandular structures compared to WT uteri (Figure
2(a)). Foxa2, which is implicated in uterine gland devel-
opment, was significantly lower in the AF2ERKI uterus
than WT which is consistent with less glandular mor-
phology [33]. Though uterine tissue is the most sensitive
organ of E2-responsiveness, the AF2ERKI uterus is ire-
sponsive to not only endogenous E2 but also exogenous
pharmacological levels of E2 (2 mg/kg) treatment to the
ovariectomized animal [33]. This observation clearly
suggests that the AF-2 of ERα is indispensable for estra-
Figure 1. (a) Schematic diagram of WT and AF2ER-mutant
ERα; (b) Representative results of Western blots probed for
the ERα (ERα66 and ERα46), His-tagged ERα (His-Tag)
and β-Tubulin in the 8-week-old WT and AF2ERKI homo-
zygote individual mouse uterus and pituitary are shown.
β-Tubulin was used as a loading control. +/+; WT, KI/KI;
AF2ERKI homozygote. (Reproduced from [33]).
Copyright © 2013 SciRes. OJEMD
Figure 2. Histology of 8-week-old representative AF2ERKI
female mice. (a) Uterine and (b) ovarian tissue H&E stain-
ing from WT (left), AF2ERKI homozygote (middle) and
αERKO (left) mice. Scale bar = 100 μm; (c) Mammary
gland whole mount Carmine Alum staining from 8-week-
old representative mice. Scale bar = 1 cm. (Reproduced
from [33]).
diol responsiveness in uterine responses.
Growth factors, such as insulin like growth factor 1
(Igf1) and EGF are able to stimulate uterine cell prolif-
eration without estrogen in ovariectomized WT mice but
not in the αERKO uteri [36]. This observation suggests
that the grow th factor dependent phosphorylation signals
activate “ligand-independent” transcription through ERα
in vivo [37]. However, Igf1 or EGF did not induce the
uterine cell proliferation in AF2ERKI [33], suggesting
that the AF-1 activ ity of ERα is regulated by the “ligand-
dependent” function al domain AF-2.
The ovaries in the AF2ERKI female mice show cystic
and hemorrhagic follicles, reminiscent of the αERKO
ovarian phenotype. A few primary follicles can be seen,
but no corpora lutea were observed in AF2ERKI ovaries
(Figure 2(b)). The hemorrhagic follicles develop after
the prepubertal period. The gene expression profile of
steroidogenic enzymes in the AF2ERKI ovary is also
similar to the αERKO ovary [unpublished observation by
YA, KJH and K SK].
In adult WT mice, the mammary ducts elongate to the
end of the mammary fat pad and side branches from the
primary ducts fill the fat pad. On the other hand,
AF2ERKI mammary glands never develop beyond a ru-
dimentary epithelial ductal tree similar to αERKO fe-
males (Figure 2(c)). Taken together, these results sug-
gest that estrogen-dependent AF-2 mediated transactiva-
tion of ERα is essential for developing and maintaining
female reproductive tissues.
2.3. AF2ERKI Homozygote Male Reproduction
AF2ERKI homozygote males sired no offspring during a
continuous breeding study. The sperm count and motility
were significantly lower in adult AF2ERKI male mice
than WT. The morphological features of the AF2ERKI
testis are characterized by dilated seminiferous tubules
and rete testis (Figure 3). This phenotype is similar to
what was seen previously in αERKO male mice [38,39].
The dilation of the seminiferous tubule was observed in
20-day-old AF2ERKI males and in adults the tubule dila-
tion was of similar severity as the αERKO male mice. It
has been reported that the repression of certain mem-
brane proteins in the efferent duct of the αERKO is re-
lated to seminiferous tubule dilation and male infertility
[40]. The expression level of the sodium/hydrogen ex-
changer 3 (Slc9a3), aquaporin 9 (Aqp9), carbonic anhy-
drase 2 (Car2) and Aqp1, all related to testicular fluid
reabsorption in efferent ducts, were significantly de-
creased in the AF2ERKI efferent duct as seen in αERKO
males [34]. These results suggest that the AF-2 function
is also indispensable for normal male reproduction. Se-
rum hormone levels are disrupted in AF2ERKI male
mice [34]. The AF2ERKI male has a 10 times higher le-
vel of testosterone than WT and that level was similar to
the αERKO male. However, the serum LH level in the
AF2ERKI male was lower than the αERKO and was si-
milar to WT [34]. This might suggest differential usage
of ERα AF-1 in negative feedback of the hypothalamic-
pituitary gonadal axis in male versus female mice.
2.4. AF2ERKI Body Mass and Bone Density
Estrogen is important in regulating body fat as evidenced
by studies indicating ovariectomy leads to increased
body weight in rodents [41] and further supported by the
obesity of αERKO [42] and Cyp19 (aromatase) KO fe-
males [43]. The body weights of 3-week-old male and
female AF2ERKI mice were not different from WT. At 9
weeks, the body weights of AF2ERKI females were sig-
nificantly higher than WT females and that level was
comparable to the same age male mice, however the
body weights of the AF2ERKI males were not different
from WT (Figure 4). The body fat percentages of the
AF2ERKI females were significantly higher than WT,
Figure 3. Representative longitudinal sections of adult WT,
AF2ERKI homozygote and αERKO testes and epididymis.
Arrow indicates efferent duct, T indicates testis, RT indi-
cates rete testis. The bar in the figure indicates 3 mm. (Re-
produced from [34]).
Copyright © 2013 SciRes. OJEMD
Figure 4. Body weight (a and b) and fat content (c and d) of
3-week and 9-week-old WT (+/+) and AF2ERKI homozy-
gote (KI/KI) male (M) and female (F) mice. Fat content was
analyzed by DEXA (n = 6). Data are expressed as mean ±
S.D. Statistical analysis was performed by unpaired t-test. **
indicates p < 0.01, * indicates p < 0.05.
which may contribute to the increased body weights.
Interestingly, the body fat percentages in 9-week-old
AF2ERKI males were significantly higher than WT in
spite of the fact that the body weights were not different
between the genotypes (Figure 4).
Clinical as well as experimental data imply a role of
estrogen in osteoprotective action. For example, bone
mineral density (BMD) is lower in postmenopausal wo-
men, and in the castrated rodents. [44]. It has been re-
ported that the BMD in the αERKO female is relatively
normal. On the other hand, BMD in the male αERKO is
significantly lower than WT [45]. There was no signifi-
cant difference of BMD of the female femur between
AF2ERKI and WT mice. In contrast, BMD of the male
femur was significantly lower in 12-week-old AF2ERKI
mice compared to WT mice and that level was similar to
the female femur (Figure 5). The skeletal phenotypes of
AF2ERKI mice are similar to those of the αERKO mice.
3. Antagonist Reversal Activity of AF2ER
3.1. ERα Antagonists Induce Estrogenic
Responses in AF2ERKI Mouse Uterus
As we described above, the AF2ER mutation diminished
estradiol (E2) mediated physiological responses in mice.
Interestingly, the AF2ER mutation reversed ERα antago-
nists, such as ICI or tamoxifen (TAM), into agonists.
Ovariectomized female mice were injected in a 3-day
bioassay with vehicle, ICI, TAM or E2. The treatment of
E2 and TAM significantly induced the uterine weight of
WT mice and ICI treatment was ineffective. In contrast,
ICI and TAM treatment increased the AF2ERKI uterine
weight but E2 treatment did not (Figure 6). The prolif-
eration of endometrial epithelial cells in the AF2ERKI
uterus was induced by ICI and TAM [33]. Estrogen re-
sponsive uterine genes in the WT female such as Igf1 and
lactotransferrin were regulated similarly by ICI and TAM
Figure 5. Bone mineral densities (BMD) of the femurs in
12-week-old WT (+/+) and AF2ERKI homozygote (KI/KI)
male (M) and female (F) were analyzed by DEXA (n = 6).
Data are expressed as mean ± S.D.. Statistical analysis was
performed by unpaired t-test. ** indicates p < 0.01.
Figure 6. Representative view of whole uterus of ovariec-
tomized mice after vehicle (Veh), ICI (2 mg/kg), TAM (2
mg/kg) or E2 (2 mg/kg) treatments for three consecutive
days. (Reproduced from [33]).
Copyright © 2013 SciRes. OJEMD
in the AF2ERKI female [33]. We also demonstrated the
AF2ER function in a receptive uterus model. Estrogen
induces stromal cell proliferation but not epithelial cell
proliferation in the receptive uterus. In the AF2ERKI
uterus, ICI increased proliferation of stromal cells but not
epithelial cells similar to E2 treated WT [33].
3.2. ERα Antagonist Restored Male Fertility in
Antagonist reversal was also observed in AF2ERKI male
mice [34]. 21-day-old AF2ERKI male mice were treated
with TAM for 3 weeks, then used in a 2 month continu-
ous breeding study. 75% of placebo-treated WT males
were fertile during this period. Under the experimental
conditions, 83% of TAM-treated WT males were fertile.
In contrast, none of placebo-treated AF2ERKI males
sired litters (0%); however, 38% of TAM-treated AF2ERKI
males sired offspring, suggesting that TAM activated the
AF2ER to restore fertility. TAM treatment induced ef-
ferent ductal gene expression (Slc9a3, Aqp1, Aqp9, and
Car2) in AF2ERKI mice to the same level seen in WT
mice, and at the same time the dysmorphology of
AF2ERKI testis was resolved. TAM treatment restored
the sperm motility but had no effect on the sperm count
in AF2ERKI male mice [34].
3.3. ERα Antagonists Did Not Regulate
Estrogenic Responses in Pituitary
Ovariectomy releases negative feedback in WT female
mice, resulting in increased serum LH. Additionally, E2
replacement down regulates the serum LH in the WT
female. In contrast, the increased serum LH level in ova-
riectomized AF2ERKI mice was not regulated by ICI or
TAM treatment [33]. The level of Lhb mRNA in the ova-
riectomized AF2ERKI pituitary was not changed by ICI
or TAM in parallel with the serum LH level [33]. Prolac-
tin (Prl) is a well-known estrogen respon sive gene in the
pituitary [46,47]. The basal level o f Prl expression in th e
AF2ERKI pituitary was lower than WT and similar to the
αERKO pituitary. The expression of the Prl gene was not
regulated by ICI or TAM in the AF2ERKI pituitary,
whereas E2 activated the Prl gene expression in the WT
pituitary [33]. These results ind icate that the fun ctiona lity
of ERα AF-1 or AF-2 in the pituitary is different than
their activities in the uterus.
3.4. Molecular Mechanism of AF2ER-Mediated
Antagonist Reversal
We have explored the molecular mechanism of the an-
tagonist reversal activity of AF2ER. The AF2ER muta-
tion blocked the recruitment of p160 co-activators to the
AF-2 with any ligand [48], thereby disrupting the E2
(agonist) dependent transcription activ ation. Even though
AF2ER does not recruit the tran scription co-activato rs to
AF-2, ICI and 4OHT (antagonists) activate AF2ER. We
demonstrated that the truncation of the AF2ER N-ter-
minal region diminished antagonist dependent transcrip-
tion, thus the N-terminal localized transcription activity
(AF-1) is essential for the antagonist reversal activity
[33]. Several of our in vitro studies indicate that the
mode of antagonist-mediated AF2ER activation is simi-
lar to the partial agonist activity of 4OHT on WT ERα.
Namely, the overexpression of transcription co-activator,
p300/CBP enhanced both antagonist mediated AF2ER
activation and 4OHT mediated WT ERα activation
through the AF-1 domain of ERα in a similar manner
[33]. This observation supports the previous findings,
which suggested that 4OHT mediated partial agonistic
activity for WT ERα can only be mediated by AF-1 [10].
Further investigation rev ealed that the antagonists induce
homodimerization of the AF2ER LBD, and the trunca-
tion of 41 amino acids of the C-terminal end (known as
F-domain) diminished this homodimerization [49]. In-
terestingly, 4OHT also induced homodimerization of
WT-LBD and F-domain truncation from the WT-LBD
strongly reduced this dimerization similar to AF2ER.
The prevention of dimer formation diminished the an-
tagonist dependent AF2ER activation and this dimeriza-
tion correlated with the ligand-dependent estrogen re-
sponsive DNA element binding activ ity [49].
One of the major antagonistic effects of ICI has been
shown bot h in vivo and in vitro to result from loss of ERα
protein through proteasome-mediated proteolysis [27,28].
As ICI is an agonist for AF2ER, we analyzed the effect
of ICI on AF2ER protein stability in vitro. As expected,
ICI induced the degradation of WT protein. On the con-
trary, ICI did no t indu ce AF2ER pro tein degrad ation [49 ].
In the ICI-treated AF2ERKI female, the level of uterine
AF2ER protein was not reduced, whereas the level of
WT ERα was markedly reduced by ICI treatment [33].
These results suggest that the AF2ER mutation prevents
the ICI-mediated proteolysis of ERα protein and blocks
loss of AF2ER protein in vivo and in vitro. Addition ally,
we confirmed that dimerization does not correlate with
the ICI-dependent ERα protein degradation [49].
4. Summary and Future Directions
We describe here that the AF-2 of ERα is a critical do-
main to maintain the estrogen dependent physiological
functions in female and male mice. Surprisingly, AF-2
core mutation disrupts the growth factor inducible cell
proliferation in the AF2ERKI uterus, a phenomenon that
has been recognized as a “ligand-independent” action of
ERα. This result indicates that the proper positioning of
helix 12 (AF-2) is neccesary for appropriate AF-1 func-
tionality. Interestingly, the AF-2 mutation reversed an-
tagonists to agonists. This antagonist dependent trans-
Copyright © 2013 SciRes. OJEMD
criptional activity is derived from AF-1 similar to TAM-
mediated WT ERα activation. These results suggest that
the AF2ER mutation disrupted helix 12 (AF-2) position-
ing with agonist or when unliganded, but do not affect
the configulation of the LBD with SERMs (e.g. TAM).
TAM regulates uterine gene expression in the AF2ERKI
female, however not in the pituitary. This suggests the
existence of differential tissue factors for AF-1 activation
between the uterus and pituitary. The antagonist reversal
activity in the AF2 ERKI mouse model will be a usefu l to
analyze the tissue selective agonist and antagonist ac-
tivity of SERMs.
We find that the AF2ER mutation prevents ICI- de-
pendent ERα proteolysis and that is an explanation for
AF2ER activation. ICI is now categorized as a SERD
which means SERDs may possess agonistic activity that
is covered by their strong proteolysis inducible activity.
SERD is a new concept of estrogen receptor modulation
and it is still ambiguous with SERMs. The AF2ERKI
model may be useful in classifying estrogen modulators
into SERDs and SERMs.
Our preliminary results suggest that long-term TAM
treatment to the AF2ERKI female prevents body weight
gain compared to the vehicle treated group. This suggests
that ERα AF-1 related activity is involved in preventing
female weight gain. It may be beneficial to screen estro-
genic chemicals that prefer AF-1 activity to control obe-
In conclusion, in vitro and in vivo experiments using
the AF2ER mutant will help determine the AF-1 versus
AF-2 dependent tissue selective action of estrogenic
chemicals as it relates to their physiological activities of
estrogen hormon es and estrogen receptors.
5. Acknowledgements
We thank Dr. Y. Mishina, Dr. M. K. Rey and Mr. G.
Scott for support to generate AF2ERKI mouse, Dr. E. M.
Eddy, Dr. K. S. Janardhan and Ms. E. H. Goulding for
analyses of AF2ERKI male mice, and Ms. S. C. Hewitt
and Dr. W. Winuthayanon for critical reading of this ma-
nuscript. This research was granted to K. S. K. (Z01ES
70065) by the Intramural Research Program of the NIH,
National Institute of Environmental Health Sciences.
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