(Received for publication, October 25, 1994; and in revised form, July 27, 1995)
From the
Previous studies using in vitro procedures have not
clearly established whether the estrogen receptor (ER) acts as a
monomer or dimer in the cell. We have used the yeast two-hybrid system
as an in vivo approach to investigate the dimerization of the
estrogen receptor in the absence and presence of estrogen and
anti-estrogens. This system is independent of ER binding to the
estrogen response element. Two vectors, expressing GAL4 DNA binding
domain-human ER and GAL4 transactivation domain-human ER, were
constructed. Control experiments showed that each fusion protein had a
high affinity binding site for estradiol-17 and could
transactivate an ERE-LacZ reporter gene in yeast similar to the wild
type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were
expressed in the yeast strain, PCY2, which carries a GAL1
promoter-lacZ reporter. ER dimerization was measured via
reconstitution of GAL4 through interaction of the fusion proteins,
which transactivates LacZ through the GAL1 promoter. When both ER
fusion proteins were expressed,
-galactosidase activity was
estradiol-17
-inducible. Furthermore, we showed that both tamoxifen
and ICI 182,780 also induced
-galactosidase activity, albeit lower
than that induced by estradiol-17
. These results strongly argue
that ER dimerization is ligand-dependent and the dimer can be induced
by estradiol-17
, tamoxifen, or ICI 182,780. We also treated the
yeast containing the two fusion proteins with estradiol-17
and
tamoxifen or ICI 182,780 simultaneously to determine the effects on ER
dimerization.
-Galactosidase activity was lower when the yeast was
treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen
than estradiol-17
alone. Taken together, we conclude that ER
dimerization is ligand (estradiol-17
, tamoxifen, or ICI 182,
780)-dependent, and we suggest that estradiol-17
-induced dimers
are destabilized when estradiol-17
is used with tamoxifen or ICI
182,780 simultaneously.
The estrogen receptor (ER) ()is an intracellular
protein that mediates the actions of estrogens in target cells. The ER
is a member of a superfamily of related nuclear proteins which includes
receptors for steroid hormones, thyroid hormones, vitamin D, the
retinoids, and a number of proteins with high sequence homology but as
yet unidentified ligands. These receptors are ligand-inducible
transcription factors which bind to their specific DNA targets, termed
response elements, to regulate transcription. Based on sequence
homology and other approaches, the estrogen receptor protein can be
divided into six functionally and physically independent domains
(A-F)(1, 2) . These domains are required for DNA
binding (region C), nuclear localization (region D), and steroid
binding (region E). The ER has two well characterized transcriptional
activation functions, AF-1, which is located in the N-terminal A/B
region, and AF-2, which is located in region E and whose activity is
ligand-dependent. The DNA binding domain and the hormone binding domain
have both been reported to contribute to ER dimerization.
Estrogen action on target cells involves a distinct pathway where estradiol freely diffuses across the cell membrane and binds to its receptor. This ligand-receptor complex is thought to homodimerize and bind tightly to the estrogen response element (ERE). After binding DNA, ER activates transcription of its target genes by as yet unknown mechanisms. In this system, there has been some controversy as to whether the ER acts as a monomer or a dimer in the cell. It has been traditionally believed that estrogen induces dimerization of ER and hence the DNA binding(1) . The ER has been shown to form stable homodimers in solution(3) , and several studies have given evidence that the ER and other nuclear receptors bind to response elements as dimers(4, 5, 6, 7) . However, Gorski et al.(8) have proposed a model where the ER protein binds to an ERE as a monomer or perhaps forms heterodimers with other nuclear proteins, as is the case with thyroxine receptors(9) . Most of the data for and against dimerization of ER have been shown using gel mobility shift assays or complex assays in vitro, where the assay itself requires ER to bind DNA. These assays have given conflicting evidence in demonstrating whether estrogen is required (4) or not required (10, 11, 12) for high affinity binding of the ER to the ERE. It still remains unclear if the ER can form a dimer in vivo, whether estrogen has any effects on dimerization and whether the dimer is formed before or after binding the ERE.
Using similar in vitro assays, the action of anti-estrogen on the ER pathway has been investigated at the levels of dimerization and/or DNA binding of ER. The ER, bound with an anti-estrogen such as tamoxifen or ICI 164,384, was shown to form a ligand-ER complex which can bind the ERE(13, 14) . Furthermore, ICI 164,384 and tamoxifen have been shown to induce DNA binding (15) and activate ER transactivation in yeast(16) . However, Parker and co-workers (6, 17, 18, 19) reported that ICI 164,384 and ICI 182,780 prevent the ER from binding ERE. Based on their previous study of mutant mouse ERs and in vitro DNA binding ability of ER, which had assumed that an ER dimer is required for binding to DNA(6, 20) , they concluded that ICI 164,384 and ICI 182,780 prevent ER dimerization.
Since the effect of
ligand on the ER-ER dimerization is still unclear and the actions of
estrogen and anti-estrogens can be hypothesized to involve differences
in ER dimerization, which may affect ER/DNA binding, we have used the
yeast two-hybrid system, which is independent of the interaction of ER
with ERE, to study ER protein dimerization in vivo. The yeast
two-hybrid system has been described by Fields and
co-workers(21, 22) . It involves the expression of a
LacZ reporter gene under the control of a GAL4-activated promoter (GAL1
promoter) that depends on the reconstitution of GAL4 activity via
protein-protein interactions. This is accomplished by apposition of the
GAL4 DNA binding (GAL4 DB) and transcription activation (GAL4 TA)
domains via interaction of polypeptides fused to each domain. Colonies
containing interacting polypeptides are detected with a chromogenic
substrate for -galactosidase. This system has also been used
successfully in screening a cDNA library (23, 24, 25) and has turned out to be a
useful approach to study protein-protein interactions in vivo.
In our experiments, the human ER cDNA was cloned into the GAL4 fusion vectors, pPC62 (GAL4 DB) and pPC86 (GAL4 TA)(24) , to study the dimerization of ER and the effects of ligands on dimerization. Using this yeast two-hybrid system, we show that the ER-ER interaction is estrogen-dependent in vivo. We also show that the anti-estrogens, tamoxifen and ICI 182,780, can induce ER dimerization. Dimerization, however, is perturbed when estrogen is used with anti-estrogens simultaneously. The implications of these data on the ER-ER dimer are discussed.
The yeast two-hybrid system control vectors, GAL4 TA-bz-c-jun, GAL4 DB-bz-c-jun, GAL4 TA-bz-c-fos, and GAL4 DB-bz-c-fos, were kindly provided by Dr. Chevray(24) . The yeast ERE-lacZ reporter (YRpE2) used in the leaky yeast experiments were kindly provided by Dr. T. Butt(16) .
Transformed yeast were selected and then
cultured in synthetic medium (in the case of -galactosidase
activity assays, glucose was replaced with galactose). The agonist
and/or antagonists were added to the yeast liquid culture after the
cells grew to late log phase (A
>0.7 for the
culture in the galactose medium). After stimulation with estrogen
agonist or antagonists for a certain time, the yeast cells were
collected by low speed centrifugation. The yeast cells were resuspended
in an equal volume of Z-buffer (60 mM Na
HPO
, 40 mM NaH
PO
, 10 mM KCl, 1 mM
MgSO
, 50 mM
-mercaptoethanol, pH 7.0) and
placed on ice. The 1 ml of reaction mixture was made up of 0.1 or 0.05
ml of cells in Z-buffer (the value of
-galactosidase activity is
an average value of the duplicated assay). The cells in the reaction
mixture were permeabilized with one drop of 0.1% SDS and two drops of
chloroform. Then, the reactions were started with the addition of 0.2
ml of 4 mg/ml o-nitrophenyl-
-D-galactoside
(ONPG) at 30 °C and stopped by adding 0.5 ml of 1 M
Na
CO
.
-Galactosidase activity was
determined with the values at A
and A
using the following equation: U = 1000
[(A
) -
(1.75
A
)]/[t
v
A
] (t =
time of the reaction (min); v = volume of yeast culture
used in the reaction mixture (ml)].
Figure 1:
The induction of -galactosidase by
estradiol-17
in the yeast (PCY2) carrying both GAL4 DB-hER and
GAL4 TA-hER fusion vectors. The yeast liquid culture was treated with
different concentration of estradiol-17
(
, 10
M;
, 10
M;
,
10
M;
, 10
M), and
-galactosidase was determined using ONPG
reaction. The yeast carrying only GAL4 DB-hER was also used as a
control (
, treated with 10
M of
estradiol-17
).
-Galactosidase activity was measured as a
function of time after the yeast was stimulated with estradiol-17
.
The error bar stands for the S.D. of population (n = 8).
To determine the extent of ER
dimerization that takes place upon addition of estradiol-17, we
compared the levels of ER dimerization with those of the dimers formed
by Jun and Fos using the same system. Both Jun/Jun and Jun/Fos form
functional dimers in vivo, giving strong Ap-1
activity(33) . However, formation of the Jun/Fos dimer is
favored over the Jun/Jun dimer as described (24) and also shown
here (Fig. 2). The ER fusion protein dimer reconstituted about
10% relative
-galactosidase activity of that by the Jun/Fos dimer
and was 25 times stronger than that by the Jun/Jun dimer (Fig. 2). We conclude that the ability of ER to dimerize is
significantly enhanced by estradiol-17
. In the absence of
estrogen, ER seems to exist as a monomer (or forms a
heterodimer/oligomer with other proteins), but there seems to be little
or no direct ER-ER interaction.
Figure 2:
-Galactosidase activity induced by
the ER dimerization compared with that induced by the bz-Jun/bz-Fos
(100%) and the bz-Jun/bz-Jun dimers in the same yeast strain (PCY2).
The yeast, PCY2, was cotransfected with GAL4
DB-bz-c-jun/bz-c-fos and GAL4
TA-bz-c-jun/bz-c-fos. The yeast carrying GAL4
DB-bz-c-fos and GAL4 TA-bz-c-fos showed no
significant value of
-galactosidase. The bz-Jun/bz-Fos heterodimer
induced activity was about 20% of wild type GAL4. These data were
similar to Chevray and Nathans(24) . Asterisk, the
yeast was treated with 1 µM of estradiol-17
(final
concentration) for 12 h. The error bar represents the S.D. (n = 4).
Figure 3:
Binding assays of GAL4-hER fusion proteins
extracted from yeast (PCY2). Extracts were prepared from yeast PCY2
carrying GAL4 DB-hER or GAL4 TA-hER, and the ER binding capability was
determined using [H]estradiol-17
as ligand. A, saturation tests were used to determine the specific
binding of the fusion proteins: GAL4 DB-hER (solid circle and solid line) and GAL4 TA-hER (open circle and dashed line). The specific binding was obtained by subtraction
of nonspecific binding from total binding. Nonspecific binding was
determined in presence of 100 times molar excess of unlabeled
estradiol-17
. B, the specific binding was further
determined by Scatchard analysis to calculate the K
of GAL4 DB-hER (solid circle and solid
line) and GAL4 TA-hER (cross and dashed
line).
To determine if GAL4
DB-hER and GAL4 TA-hER were functional as compared with wild type hER,
we measured the ability of these proteins to transcriptionally activate
an ERE-lacZ reporter plasmid in hyperpermeable (leaky) yeast.
Using leaky yeast allowed us to eliminate any questions regarding the
ability of the yeast to take up estradiol-17. Unfortunately, a
leaky yeast strain is not available currently for use in the two-hybrid
system. ERE-lacZ was cotransformed into RS188N yeast either
with GAL4 DB-hER or with GAL4 TA-hER. The ability of each fusion
protein to activate transcription of the reporter gene was highly
regulated by estradiol-17
, showing similar activity in response to
the various concentrations of estradiol-17
, from 1 nM to
1 µM (Fig. 4). These data are in good agreement
with the transcriptional activity reported with the wild type hER used
in the same yeast strain(16) . Therefore, we can conclude that
GAL4 DB-hER and GAL4 TA-hER fusion proteins expressed in yeast are
functional; they bind estradiol-17
with high affinity and show
similar estrogen-dependent gene regulation as compared with the wild
type hER.
Figure 4:
ERE-lacZ transcriptional
activation by GAL4-hER fusion proteins in response to estradiol-17
in leaky yeast (RS188N). The GAL4-hER expression vectors together with
ERE-lacZ reporter were introduced into the leaky yeast strain
RS188N. The yeast carrying the GAL4 DB-hER (solid circle and dashed line) or GAL4 TA-hER (open circle and solid line) was treated with various concentrations of
estradiol-17
(10
to 10
M) for 6 h as described(16) . The
transcriptional activity of GAL4-hER fusion proteins, via
-galactosidase activity under the control of ERE, was determined
using the ONPG reaction described under ``Material and
Methods.'' The error bar represents the S.D. (n = 4). The longer time treatments of ligand (9, 12, and 24
h) showed similar results (data not shown).
Figure 5:
Induction of -galactosidase
activity in the yeast carrying both GAL4 DB-hER and GAL4 TA-hER fusion
vectors by estrogen agonist and antagonists. The yeast liquid culture
was treated with different concentration (M) of
estradiol-17
, ICI 182,780, or tamoxifen as indicated. The activity
was determined at different times after the yeast was stimulated with
hormone. The error bar stands for the S.D. of population (n = 8).
Figure 6:
Induction of -galactosidase activity
in the yeast carrying both GAL4 DB-hER and GAL4 TA-hER fusion vectors
treated with a mixture of estrogen agonist and antagonists. Yeast
cotransformed with GAL4 DB-hER and GAL4 TA-hER was exposed
simultaneously to a combination of varying concentrations of estrogen
and anti-estrogens. After 6, 9, and 12 h, the activity of
-galactosidase was determined in each treatment group. Relative
-galactosidase activity is presented. Asterisk, the blank
control (without any treatment); Double asterisks, 0.3%
ethanol treatment was used as a vehicle control. The error bar stands for the S.D. of population (n =
8).
Figure 7:
Levels
of GAL4 fusion proteins in the yeast treated with estradiol-17,
ICI 182,780, or tamoxifen. The GAL4 DB-hER (756 amino acids) and GAL4
TA-hER (747 amino acids) fusion proteins were detected using anti-hER
antibody H222 using Western blot analysis. The protein samples were
extracted from the yeast carrying only GAL4 TA-hER (lane 1),
GAL4 DB-hER (lane 2), or both GAL4 TA-hER and GAL4 DB-hER (lanes 3-12). The yeast carrying both fusion proteins
had been treated with 10
M of
estradiol-17
(lanes 4-6), ICI 182,780 (lanes
7-9), or tamoxifen (lanes 10-12) for
different time as indicated. The yeast carrying the GAL4 DB fusion
vector (pPC 62) was used as a negative control (lane
0).
Previous studies using in vitro procedures have not
clearly established whether the ER acts as a monomer or dimer in the
cell. We have used the yeast two-hybrid system as an in vivo approach to examine the dimerization of the ER in response to
estrogen and anti-estrogens. Because the yeast two-hybrid system is
performed in vivo, the proteins involved are more likely to be
in their native conformations than in an in vitro assay. In
this system, the dimerization of the ER is ligand-dependent, as
measured by the reconstitution of GAL4 activity from GAL4-hER fusion
protein interaction. Estradiol-17 is quite effective in inducing
dimerization of the ER, whereas tamoxifen and ICI 182,780 also induce
ER dimerization, but less effectively.
Understanding the action of transcription factors such as the estrogen receptor in vivo will greatly enhance our ability to design therapeutic modalities for breast and uterine cancers. Analysis of steroid receptor interactions in a simple eukaryote, like Saccharomyces cervisiae offers the ease with which multiple analyses can be performed and the molecular genetics of the system exploited. The basic transcription machinery is remarkably conserved between mammals and yeast. The large subunit of RNA polymerase II from human cells and yeast show considerable homology(39, 40) . Besides the high degree of structural homology in the functional region, the TATA box-binding protein, TFIID, is functionally interchangeable between yeast and human TFIID(41) . As for the yeast transcriptional factors, GAL4 regulation functions in animal system(42) . Conversely, the human estrogen receptor has been shown to activate transcription in a hormone-dependent manner in yeast(30, 31, 43, 44) . Yeast have also been used as a system to overexpress functional ER (45) and to study the effects of estrogen agonist and antagonists on ER-dependent transactivation(16) .
In this report, we constructed GAL4-hER fusion proteins to investigate ER dimerization in the yeast two-hybrid system. Since dimerization of the ER has been highly correlated to the DNA binding domain(46) , it has been difficult to study the dimerization and the DNA binding independently. Previous studies have relied upon the gel retardation assay to investigate dimerization of the ER in vitro. Evidence for the existence of the ER dimer has depended on its DNA binding ability(4, 6, 20) . In addition, the hormone binding domain has also been thought to contribute to ER dimerization(47) . Recently, using in vitro experiments, it has been reported that the human ER hormone binding domain dimerizes independently of ligand activation(35) . In contrast, other DNA binding assays have shown that ER is capable of binding as a monomer to a thyroid hormone response element consisting of an inverted palindrome without spacing(48) , and that ERE binding requires neither the ER-ER homodimer nor estrogen (49) . Furthermore, the ER also binds as a monomer to half-palindromic EREs(50) , and a half-site of ERE in the c-jun gene turns out to be a strong regulatory element in response to estrogen induction (51) . Therefore, it seems that the dimerization of ER is not directly correlated to its DNA binding capability and transactivation, and a DNA band shift or ER transactivation cannot be used reliably as an indicator for the existence of an ER dimer in vivo. We have exploited the yeast two-hybrid system, which is independent of this ERE binding requirement, as an in vivo system to investigate the dimerization of the ER.
We have clearly shown that
-galactosidase activity was under estrogen control through ER
interaction. Other steroid hormones such as progesterone,
medroxyprogesterone acetate, dexamethasone, and testosterone do not
induce ER interaction as measured in these yeast. Furthermore, we
examined the effects of tamoxifen and ICI 182,780 on ER dimerization,
and the results strongly argued that ER dimerization is
ligand-dependent in each case. However, the effective concentrations
(10
, 10
, 10
M) used in our experiments were much higher than the
mammalian physiological concentration (10
M). It is possible that the estrogen agonist and
antagonists cannot efficiently penetrate the yeast, especially through
the cell wall, and thus the low concentration was ineffective. Indeed,
most work using estradiol-17
in yeast to investigate ER has used
concentrations at 10
M, due to the low
permeability of yeast to various
compounds(30, 31, 52) . Recent work has been
done using hyperpermeable (leaky) yeast, in which the physiological
concentration (10
M) of estradiol-17
is enough to induce significant values of ER-dependent
transcription(16) . Using the same leaky yeast strain (RS188N),
we showed that the GAL4-hER fusion proteins were functional, being able
to transactivate an ERE
-galactosidase reporter at the
physiological concentration of estradiol-17
(Fig. 4). It is
evident that these ER fusion proteins respond to estrogen normally as
compared with wild type human ER expressed in yeast(16) .
Ligand binding assays show that the GAL4 DB-hER (K
= 0.84
10
M) and GAL4
TA-hER (K
= 1.4
10
M) fusion proteins each have a high affinity binding
site for estradiol, slightly lower than that reported for the wild type
hER expressed in MCF-7 cells (K
= 0.6
10
M) or in yeast (K
= 0.5
10
M)(30, 31) . It is reasonable to
suggest that the higher K
may have necessitated
higher concentrations of ligand needed to contribute to dimerization in
our assay, which would not be available if the in vivo concentration of ligand was lower than that in the medium due to
low permeability to these compounds in the PCY2 yeast strain. In
addition, the requirement of the high concentration of ligand could
also have resulted from the limitation of the sensitivity of the assay,
since we can only detect about 50% of the potential dimers formed. GAL4
DB-hER/GAL4 DB-hER, GAL4 TA-hER/GAL4 TA-hER, GAL4 TA-hER/GAL4 DB-hER,
and GAL4 DB-hER/GAL4 TA-hER can each dimerize, but only the last two
can be detected using this system. As for the low anti-estrogen-induced
-galactosidase activity, it might be argued that intake of
anti-estrogens into yeast is lower than that of
estradiol-17
(52) . However, the decreased activities in
the mixture of estradiol-17
and anti-estrogen strongly support our
contention that there is no significant difference between
estradiol-17
and tamoxifen or ICI in entering yeast.
A major limitation of our work is that we cannot quantitate the ER dimer in the yeast. We used the homodimerization of c-Jun or c-Fos as positive or negative controls, respectively, and compared these levels with those of ER-ER dimerization. Using the yeast two-hybrid system, Chevray and Nathans (24) have shown that c-Jun, but not c-Fos, can form stable homodimers. We can show through the yeast system that ER-ER dimerization is 11 times less efficient than Jun:Fos, but 25 times more effective than Jun:Jun interaction, a transcriptional factor that has been shown to act as a homodimer (Fig. 2). Our results show that there is no significant dimerization between c-Fos/c-Fos or ER-ER untreated with steroid hormones. Since approximately 40% of the hER expressed in yeast has been reported to be able to bind ligand(16) , it is possible that the dimerization of ER in estrogen-responsive cells may be somewhat stronger than that measured in our assay.
The molecular mechanism of the different efficiency of
dimer formation by the different ligands, however, is still unclear.
Since the ability of ER to discriminate between estradiol-17,
tamoxifen, and ICI has been shown previously by hormone binding, DNA
binding, and transactivation assays, it is likely that estrogen and
anti-estrogens bind ER and induce different conformational changes,
resulting in different effects upon dimerization. Considering that the
data from GAL4-hER fusion proteins are unrelated to ER binding to ERE,
our results suggest that the dimerization and the DNA (ERE) binding are
separate events. However, whether this is true in an
estrogen-responsive mammalian cell is still unclear. It is also unclear
if these dimers can bind DNA or are required for DNA binding for the
same reason. The dimerization could be affected by DNA (ERE), because
the hormone binding domain can be allosterically modulated by DNA (ERE) (53) . In fact, the evidence that the DNA binding domain is
important for dimerization (46, 54) may suggest that
the ERE may influence the efficiency of ER dimer formation. The
relationship among estrogen- or anti-estrogen-induced dimerization of
ER with DNA binding and transactivation deserves further investigation.
In our experiments, we showed that the dimerization is less effective when estrogen and anti-estrogen are used simultaneously than when estrogen is used alone. It is difficult to explain the ability of anti-estrogen alone to induce ER dimerization. However, the assay used in our experiments argues persuasively that anti-estrogens do induce ER dimerization, albeit to a lower extent. Among several possible explanations to our observation, we offer a model in which the yeast two-hybrid system is detecting protein-protein interaction(s) of one or more ER-associated proteins, which would not require direct ER dimerization, but where ER is involved. Only upon addition of ligand would the proper conformational change of ER take place to allow ER to then bind the protein in question. The ER-protein binding would then cause subsequent conformational changes in the binding protein, which would then allow, and be required for, the binding protein to dimerize, thus forming an oligomeric complex of GAL4 DB-hER, ER-binding protein:ER-binding protein, GAL4 TA-hER. Dimerization of the binding protein, but not direct dimerization of ER, would thus be detected. Recently, three estrogen receptor-associated proteins of 160, 140, and 80 kDa have been identified(55, 56) , each satisfying the requirements of this model. These estrogen receptor-associated proteins only bind to ER in the presence of estrogen, whereas tamoxifen and the pure anti-estrogens block ER-estrogen receptor-associated protein complex formation. This model suggests that ER-ER interaction takes place in a large complex with the aid of adapter proteins, such as the estrogen receptor-associated proteins.
The antagonistic activity of tamoxifen and the ICI compound may occur at several levels of the estrogen transactivation pathway(57) . It is obvious that the effects of tamoxifen and ICI compounds on ER dimerization and DNA binding alone are not enough to explain the antagonistic function of anti-estrogens. Instead, anti-estrogens may actually mimic estrogenic action somewhat at the two steps of dimerization and DNA binding. At other levels of the estrogen pathway, the differential structure of ER-anti-estrogen complexes bound to the ERE(14) , their different DNA bending abilities(13, 14, 58) , as well as the ligand dependence of ER induced changes in chromatin structure (59, 60) have all been proposed to be responsible for the antagonistic activity of anti-estrogens. In addition, the ICI-induced fast turnover of ER (61, 62) and the disruption of ER nucleocytoplasmic shuttling (63) have also been suggested to contribute to ICI antagonism. Therefore, it appears that anti-estrogenic action on the estrogen pathway may be more complex than previously believed.
In conclusion, our observations emphasize that ER dimerization is an estrogen-inducible event in vivo. Tamoxifen or ICI 182,780 also induces ER dimerization at a lower level, and tamoxifen or ICI 182,780 makes the dimerization ineffective when it is used with estrogen simultaneously. The establishment of this inducible ER-ER interaction within the yeast two-hybrid system sets up a good system to further investigate ER and its associated proteins, as well as a possible system to test effectiveness of drugs in disrupting ER dimerization. Further investigation using this system for ligand induced ER-ER dimerization will offer more detail in the understanding of the function of the ER dimer.