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Endocrinology 145(6):2968 –2977
Copyright © 2004 by The Endocrine Society
doi: 10.1210/en.2003-0806
The Unusual Binding Properties of the Third Distinct
Teleost Estrogen Receptor Subtype ER␤a Are
Accompanied by Highly Conserved Amino Acid Changes
in the Ligand Binding Domain
M. B. HAWKINS
AND
P. THOMAS
Zoology Department, North Carolina State University (M.B.H.), Raleigh, North Carolina 27695; and University of Texas at
Austin, Marine Science Institute (P.T.), Port Aransas, Texas 78373
Three forms of estrogen receptor: ER␣, ER␤ (ER␤b), and a
second ER␤, ER␤a (formerly ER␥) are present in teleost fish.
All ER␤as share amino acid changes in the ligand binding
domain that may influence ligand specificity and receptor
function. We compared binding specificities of the three ERs
of the teleost fish, Atlantic croaker Micropogonias undulatus.
Bacterially expressed Atlantic croaker (ac) ER␣, -␤b, and -␤a
fusion proteins showed specific, high affinity binding to 17␤[3H]estradiol, with Kd values of 0.61 ⴞ 0.013, 0.40 ⴞ 0.006, and
0.38 ⴞ 0.059 nM, respectively. Rank orders of binding were:
diethylstilbestrol ⬎⬎ ICI182780 > 4-hydroxytamoxifen >
ICI164384 > estradiol > zearalenone > moxestrol > tamoxifen > estrone > 17␣-estradiol > estriol > 2-hydroxyestrone ⴝ
genistein ⬎⬎ RU486 for acER␣; ICI182780 > diethylstilbestrol > 4-hydroxytamoxifen > estradiol > ICI164384 >
E
STROGENS HAVE CRITICAL roles in the development
and function of many tissues, in particular those of the
reproductive, nervous, and cardiovascular systems. Estrogens exert their effects via estrogen receptors (ERs) present
in target tissues. Nuclear ERs are members of the steroid/
thyroid/retinoic acid superfamily of ligand-activated transcription factors (1, 2). All members of this family share a
modular structure that consists of a variable trans-activation
domain (A/B), a highly conserved DNA binding domain (C
or DBD), a variable hinge region (D), a well-conserved ligand
binding domain (E or LBD), and a variable C-terminal region
(F) (1, 2). When ligand binds to the receptor, a conformational
change occurs, and the receptor dimerizes. The receptor/
ligand complex then is transported to the chromatin where
it interacts with nuclear cofactors and specific regulatory
regions of target genes to modulate their transcription (transactivation) (2– 4).
An important characteristic of estrogen-regulated proAbbreviations: ac, Atlantic croaker; DES, diethylstilbestrol;
17,20␤DHP, 17,20␤-dihydroxyprogesterone; E1, estrone; E2, 17␤-estradiol; 17␣E2, 17␣-estradiol; E3, estriol; ER, estrogen receptor; h, human;
H, helix; IC50, competitor concentration that causes 50% displacement of
[3H]estradiol; ICI164, ICI164384; ICI182, ICI182780; 11KT, 11-ketotestosterone; LBD, ligand binding domain; MOXE, moxestrol; 2OHE, 2hydroxyestrone; RBA, relative binding affinity; TAM, tamoxifen; TOH,
4-hydroxytamoxifen; wt, wild-type.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
genistein > moxestrol > tamoxifen > zearalenone ⴝ estrone >
estriol ⴝ 17␣-estradiol > 2-hydroxyestrone ⬎⬎ RU486 for
acER␤b; and estradiol > diethylstilbestrol > 4-hydroxytamoxifen > ICI182780 > ICI 164384 > estriol > genistein > moxestrol > zearalenone > estrone > 17␣-estradiol > RU486 > tamoxifen > 2-hydroxyestrone for acER␤a. acER␤a showed
higher relative binding affinities for estradiol, estriol, and
RU486 and lower relative binding affinities for synthetic estrogens and antiestrogens than previously characterized ERs.
Mutation of the conserved teleost substitutions (acER␤aPhe396)
to the ER␣ or ER␤b counterpart shifted diethylstilbestrol and
tamoxifen affinities toward those of wild-type acER␣ and
acER␤b, supporting the hypothesis that the positions with
conserved residue changes in teleost ERs are important to ER
structure and function. (Endocrinology 145: 2968 –2977, 2004)
cesses is that they are mediated by multiple nuclear ER
subtypes. The discovery of a second ER in rats, ER␤, transformed the field of ER research by providing additional
mechanistic explanations for the pleiotropic effects of estrogens (5). ER␤ is widely distributed and has since been identified in other mammals, including humans, as well as in
birds and fish (6 – 8). ER␤ and the original ER (ER␣) have
distinct, yet partially overlapping, distributions in estrogen
target tissues and also have different ligand binding affinities
and trans-activation properties (9 –12). The differences in
binding affinities, trans-activation properties, and distribution are being exploited to find compounds that behave as
estrogen agonists or antagonists in a tissue- and/or ER subtype-specific fashion (13–15). These selective ER modulators
and subtype-specific ligands are promising tools in the treatment of estrogen-related disease and cancer without some of
the deleterious side-effects. A more comprehensive understanding of the significance of structural differences among
the ER subtypes to their binding affinities for estrogenic
compounds is required to develop effective and specific
drugs for osteoporosis, breast cancer, hormone replacement
therapy, and many other conditions (16).
We recently identified three distinct ER subtypes in a
teleost fish, the Atlantic croaker (Micropogonias undulatus)
(17). We designated two of these subtypes Atlantic croaker
(ac) ER␣ and acER␤ based on their sequence homology and
phylogenetic relationships to previously identified ERs. Because the third subtype was genetically distinct from acER␣
2968
Hawkins and Thomas • Unusual Binding Properties of ER␤a
and acER␤, and others had proposed that fish may possess
an ER␥ (18), we designated this new subtype acER␥ (17). The
ER␥ subtype is present in other teleosts and arose from a gene
duplication of ER␤ early in the teleost lineage after the divergence of ray- and lobe-finned fishes. These three subtypes
were subsequently identified in zebrafish (19). The acER␤
ortholog in zebrafish is now designated ER␤b or ESR2b (previously ER␤1) to comply with official zebrafish nomenclature rules (20), whereas the acER␥ ortholog is designated
ER␤a or ESR2a (previously ER␤2). The ER␤a designation
used for zebrafish will be adopted here for the acER␥ subtype
described in our previous paper (17) to standardize ER subtype nomenclature and in recognition of the fact that this
subtype has not been identified subsequently in any other
vertebrate classes (20). Similarly, the subtype formerly referred to as acER␤ is renamed acER␤b.
The ER␤as share a high degree of sequence similarity with
other ERs in the conserved domains of the molecule. However, there are several significant amino acid changes in these
domains that are shared by all of the cloned ER␤as (17). These
amino acids may have functional significance that distinguish ER␤a from ER␣ and ER␤b. Several of these diagnostic
residues are located near or within regions involved in ligand
binding in mammalian ERs (21–23). Mammalian ER␣ and
ER␤ show differences in binding affinity for various estrogens and estrogenic compounds, and these differences have
been attributed to specific amino acid substitutions in the
LBD of ER␤ (24). In addition, it has been proposed that
species differences in the binding properties of bacterially
expressed ER␣ genes are due to amino acid changes in the
LBD (25). In support of this hypothesis, mutations of specific
amino acids within the LBD of human, rat, and fish ER fusion
proteins expressed in vitro can alter ligand binding characteristics (26 –28). The identification of three genetically distinct ERs in one species that possess naturally occurring
amino acid substitutions within the LBD provides an unprecedented opportunity to investigate the role of specific
amino acid substitutions in determining ER ligand binding
characteristics.
The binding properties of the E and F domains of acER␣,
acER␤a, and acER␤b expressed in a bacterial expression system were investigated in the present study. Saturation and
competition binding studies were conducted with the three
recombinant proteins containing the LBDs of each receptor
subtype. The relative binding affinities (RBAs) of the ER␣,
ER␤a, and ER␤b subtypes to various steroid hormones, steroid hormone receptor-targeting drugs, and phytoestrogens
were compared to test whether differences in their amino
acid sequences are reflected in their ligand binding profiles.
To further test this prediction, we examined the ligand binding specificities of acER fusion proteins that were mutated at
one of these diagnostic amino acid positions (acER␤aPhe396).
The results support the hypothesis that the amino acid substitutions have led to changes in ligand preferences and
affinities. These amino acid changes may reflect distinct
physiological functions for ER␤a and also indicate some important positions to investigate in mammalian estrogen
receptors.
Endocrinology, June 2004, 145(6):2968 –2977 2969
Materials and Methods
Biochemicals
17␤-[2,4,6,7-3H]Estradiol ([3H]E2; 84 –93 Ci/mmol) was purchased
from Amersham Pharmacia Biotech (Piscataway, NJ). The steroids
17,20␤-dihydroxyprogesterone (17,20␤DHP; 4-pregnen-17,20␤-diol-3one), 2-hydroxyestrone (2OHE; 1,3,5(10)-estratrien-2,3-diol-17-one),
moxestrol (MOXE; 1,3,5(10)-estratrien-17␣-ethynyl-2,11␤,17␤-triol-11methyl ether), 11-ketotestosterone (11KT), estriol (E3), 17␤-estradiol
(E2), estrone (E1), 17␣-estradiol (17␣E2), and cortisol were purchased
from Steraloids (Newport, RI). Testosterone was obtained from SigmaAldrich Corp. (St. Louis, MO). The synthetic estrogen diethylstilbestrol
(DES) was obtained from Steraloids. The antiestrogens, tamoxifen
(TAM; trans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine) and 4-hydroxytamoxifen (TOH); the antiprogestin mifepristone (RU 486; 11␤-(4-dimethylamino)phenyl-17␤-hydroxy-17-(1propynyl)estra-4,9-dien-3-one) and the fungal metabolite, zearalenone,
were purchased from Sigma-Aldrich Corp. The isoflavone genistein
(4⬘,5,7-trihydroxyisoflavone) was purchased from Steraloids. ICI164384
and ICI182780 (ICI164 and ICI182) were gifts from Dr. A. E. Wakeling
(Zeneca Pharmaceuticals, Cheshire, UK). All other chemicals were reagent grade and purchased from general laboratory suppliers.
Construction of acER fusion proteins
The E and F domains [acER␣ amino acids (AAs) 211–523, acER␤b AAs
317– 674, and acER␤a AAs 287–565] of each acER cDNA were subcloned
into the pET-27b⫹vector (Novagen, Madison, WI) to create fusion proteins incorporating tags for purification and detection. The fragments for
subcloning were obtained by PCR of full-length cDNA clones using
sequence-specific primers incorporating restriction sites. The acER constructs were transformed into NovaBlue-competent cells and sequenced
in both directions to confirm their nucleotide sequence (University of
Chicago Clinical Research Center DNA Sequencing Facility). The constructs were then retransformed into BL21(DE3)-competent cells (Novagen) for expression. The retention of the insert after retransformation
was confirmed by restriction digestion.
Expression of acER fusion proteins
Host cells containing acER constructs were grown in Luria-Bertoni
media (pH 7.6; 30 ␮g/ml kanamycin, 37 C) to an approximate OD at 600
nm of 1.0. Cell cultures were cooled, and protein translation was induced
with 1 mm isopropyl-b-d-thiogalactopyranoside (IPTG). After induction, cells were incubated at 25 C for 16 –20 h. Cells were harvested by
centrifugation (1700 ⫻ g at 4 C for 30 min), and the cell pellet was stored
at ⫺70 C. Cell pellets were weighed and then resuspended in ice-cold
assay buffer [20 mm HEPES, 150 mm NaCl, 10% (wt/vol) glycerol, 1.5
mm EDTA, 6 mm monothioglycerol, and 10 mm NaMoO4] at a concentration of 3.5 ml/g pellet (⬃3.1 ⫻ 1010 cells/g). Lysozyme (10 mg/ml)
was added to the resuspended cells for a final concentration of 1 mg/ml.
The cell/lysozyme mixture was incubated on ice for 5 min and then
sonicated (12 1-sec bursts at 30% power). Protease inhibitor cocktail set
III (Calbiochem, San Diego, CA) was added (25 ␮l/ml lysate). The crude
bacterial lysate was then centrifuged (12,000 ⫻ g at 4 C for 30 min), and
the supernatant was aliquoted and stored at ⫺70 C.
Saturation analysis
The lysate was diluted in ice-cold assay buffer, and 350 ␮l were
incubated with 50 ␮l of varying concentrations of [3H]E2, giving final
concentrations of 0.12–9.6 nm. The dilution factor (1:5 to 1:15), which
resulted in saturation of binding at a final concentration of 2–3 nm
[3H]E2, was determined for each lysate preparation. Nonspecific binding was determined for each concentration of [3H]E2 by adding DES to
duplicate tubes for a final concentration of 10 ␮m. Four microliters of
ethanol (volume used for adding DES and other compounds) were
added to total binding tubes; this amount of ethanol (total, 0.5%) did not
influence binding. After overnight incubation at 4 C, free [3H]E2 was
removed from bound by incubation with an equal volume of dextrancoated charcoal (0.1% dextran and 0.5% charcoal) for 10 min at 4 C,
followed by centrifugation (3400 ⫻ g at 4 C for 10 min). The supernatant
2970
Endocrinology, June 2004, 145(6):2968 –2977
Hawkins and Thomas • Unusual Binding Properties of ER␤a
was poured into scintillation vials, and 5 ml CytoScint (ICN, Costa Mesa,
CA) scintillation cocktail were added. Total bound [3H]E2 was measured
in a liquid scintillation counter (Beckman Coulter, Fullerton, CA). Specific binding was determined by subtracting nonspecific binding from
total binding. The equilibrium Kd and binding capacity were calculated
by nonlinear regression analysis using a one-site binding equation
(PRISM software, GraphPad, Inc., San Diego, CA). Scatchard plots were
used to linearize the specific binding data (29).
Competition analysis
Assays were performed essentially as described for the saturation
binding experiments. All compounds were diluted in ethanol. Four
microliters of each compound dilution were added to each tube before
adding lysate and saturating amounts of [3H]E2 (2–3 nm). Total binding
and nonspecific binding were determined by adding either ethanol or
DES to additional duplicate tubes. All assays were repeated at least once
(except for 11KT and 17,20␤DHP, which did not bind), and duplicate or
triplicate tubes for each competitor concentration were run in all assays.
The IC50 values were calculated using nonlinear regression curves for
single site competitive binding analysis. IC50 is the competitor concentration that causes 50% displacement of [3H]E2. The data are expressed
as percent total specific binding of [3H]E2 vs. log of the competitor
concentration. The RBA for each competitor was calculated as the ratio
of the IC50 for E2 to that of the competitor. The RBAs for the mutant
constructs were calculated as the ratio of the IC50 for each compound for
the acER␤a fusion protein to that of the mutant ER fusion protein. All
analyses were performed using PRISM software (GraphPad, Inc.).
Sequence alignments
The LBDs of all ERs given in Table 2 were aligned to acER␤a using
the NCBI database, BLAST. The alignments were compared with the
CLUSTALX 1.5 alignment published previously (17) to identify the
amino acids corresponding to each diagnostic position. Teleost ER␤s
that possess 10 of 11 ER␤a diagnostic amino acids in the LBD are
considered to be ER␤a in our discussion (17).
Site-directed mutagenesis
The acER␤a expression construct was mutated at Phe396 (TTY) to Ile
(ATH) as in the ER␤s and to Met (ATG) as in the ER␣s. Mutations were
performed at Bio S&T, Inc. (Montreal, Canada), and each mutation was
confirmed by sequence analysis. The mutated constructs were transformed into BL21(DE3)-competent cells and then prepared and assayed
identically to the wild-type constructs.
Results
Saturation analysis
Saturation analysis of [3H]E2 binding to the acER␣,␤a,␤b
fusion proteins demonstrated there was high affinity and
saturable binding to all three ER subtypes (Fig. 1). Nonlinear
regression analysis gave Kd values of 0.61 ⫾ 0.013, 0.38 ⫾
0.059, and 0.40 ⫾ 0.006 nm for acER␣, acER␤a, and acER␤b,
respectively (n ⫽ 2 or 3). Transformation of the data using
Scatchard analysis was linear, indicating a single class of
binding sites (Fig. 1).
Ligand specificity
The IC50 values and RBAs for each compound are given in
Table 1. Curves used to calculate IC50 values are shown in
Figs. 2-6. In general, the three acER fusion proteins bound
FIG. 1. Saturation binding of [3H]E2 to acER fusion proteins: A,
acER␣; B, acER␤a; and C, acER␤b. Crude bacterial extracts were
incubated in the presence or absence of 10 ␮M DES for 18 h at 4 C.
Unbound radioligand was removed as described. Shown are specific
binding data from a representative experiment (mean ⫾ SE for each
concentration; n ⫽ 2 or 3). Specific bound radioligand was calculated
by subtracting nonspecific bound counts from total bound counts. Kd
values were determined from the curve shown, which represents the
fit of the data by nonlinear regression analysis to a one-site binding
equation. Inset, Scatchard plot transformation of the data.
Hawkins and Thomas • Unusual Binding Properties of ER␤a
Endocrinology, June 2004, 145(6):2968 –2977 2971
TABLE 1. IC50 and RBA of various competitors for acER␣, acER␤a, and acER␤b fusion proteins
Compound
E2
DES
E1
Estriol
17␣E2
2OHE
Moxestrol
TAM
TOH
ICI164
ICI182
Genistein
Zearalenone
RU 486
Testosterone
11KT
Cortisol
17,20␤DHP
acER␣
acER␤a
acER␤b
IC50 (nM)
RBA (%)
IC50 (nM)
RBA (%)
IC50 (nM)
RBA (%)
4.9
.1
48.9
126.5
51.2
197.2
10.2
19.3
1.9
3.5
.7
201.1
5.1
wb
nb
nb
nb
nb
100
4898
10
3.9
9.6
2.5
48
25.4
262
141
706
2.4
97
wb
nb
nb
nb
nb
2.7
2.8
93.1
27.1
127.1
1114
32.3
257.6
4.1
13.4
7.5
29.8
58.5
218.3
nb
nb
nb
nb
100
96
2.9
9.8
2.1
.2
8.3
1.0
65
20
36
9
4.6
1.2
nb
nb
nb
nb
2.7
.8
76.7
155.2
166.3
897.4
23.6
55.8
1.8
5.5
.8
14.8
72.8
wb
nb
nb
nb
nb
100
315
3.5
1.7
1.6
.3
11
4.8
144
49
324
18
3.6
wb
nb
nb
nb
nb
Each value is the mean of at least two competition assays. nb, No significant displacement of 2–3 nM [3H]E2. wb, Less than 50% displacement
of [3H]E2 at the highest concentration tested.
FIG. 2. Competitive binding of major natural and synthetic estrogens to acER fusion proteins: A, acER␣; B, acER␤a; and C, acER␤b. Crude
bacterial extracts were incubated with saturating amounts (2–3 nM) of [3H]E2 and increasing concentrations of competitors. Unbound
radioligand was removed as described. Shown are specific binding data from a representative experiment (mean ⫾ SE for each concentration;
n ⫽ 2 or 3). Specific bound radioligand was calculated by subtracting nonspecific bound counts from total bound counts. IC50 values were
determined by nonlinear regression analysis using an equation for competition for a single binding site. The results for the competitors E2,
E1, E3, and DES are shown.
steroids and other compounds as expected for ERs, but there
were some notable distinctions in their binding profiles.
Physiological and synthetic estrogens
All three acERs bound E2 (all compound abbreviations are
defined in Materials and Methods) similarly with IC50 values
of 4.9 nm for acER␣ and 2.7 nm for acER␤a and acER␤b (Fig.
2). DES has a 50-fold greater (relative) affinity than [3H]E2 for
acER␣ and a 3-fold greater affinity for acER␤b. However, the
DES IC50 for acER␤a is 2.8 nm, a value equal to or slightly
lower than that of E2 for acER␤a (Table 1).
The estrogen metabolite E3 had a 2.5- to 5.8-fold greater
affinity for acER␤a than for acER␣ and acER␤b (Fig. 2 and
Table 1). In addition, the rank of E3 in the order of ligand
preferences was much higher for acER␤a. E3 was the 6th best
competing ligand for acER␤a but the 11th compound for
acER␣ and ␤b.
E1 had a 10-fold lower affinity than E2 for acER␣ and a
30-fold lower affinity for acER␤a and acER␤b (Fig. 2 and
Table 1). acER␣ also had 2- to 5-fold higher affinities than
acER␤a and acER␤b for 17␣E2, 2OHE, and MOXE, with IC50
values of 51.2, 197.2, and 10.2 nm, respectively (Fig. 3 and
Table 1).
Antiestrogens
TOH, the hydroxylated metabolite of TAM, bound acER␣
and acER␤b with a greater affinity than E2, with IC50 values
near 1.8 nm (Fig. 4 and Table 1). TOH had the third highest
affinity overall for acER␤a, with an IC50 of 4.1 nm and an RBA
65% that of E2.
The pure ER antagonist ICI164 had a 1.4-fold greater affinity for acER␣ than E2, with an IC50 of 3.5 nm (Fig. 4). The
relative affinities of ICI164 for acER␤a and acER␤b were both
lower than that for E2 with IC50 values of 13.45 and 5.5 nm,
respectively. acER␣ had a 7-fold and acER␤b had a 3-fold
greater relative affinity to the antiestrogen ICI182 than to E2.
In contrast, the affinity of ICI182 for acER␤a was one third
of that for E2, with an IC50 of 7.5 nm (Table 1).
2972
Endocrinology, June 2004, 145(6):2968 –2977
Hawkins and Thomas • Unusual Binding Properties of ER␤a
FIG. 3. Competitive binding of estrogens to acER fusion proteins: A, acER␣; B, acER␤a; and C, acER␤b. Assays were conducted as described
in Fig. 2. The results for the competitors E2, 2OHE, MOXE, and 17␣ E2 are shown.
FIG. 4. Competitive binding of antiestrogens to acER fusion proteins: A, acER␣; B, acER␤a; and C, acER␤b. Assays were conducted as described
in Fig. 2. The results for the competitors E2, TAM, TOH, ICI164, and ICI182 are shown.
FIG. 5. Competitive binding of phytoestrogens to acER fusion proteins: A, acER␣; B, acER␤a; and C, acER␤b. Assays were conducted as
described in Fig. 2. The results for the competitors E2, genistein (GEN), and zearalenone (ZEAR) are shown.
TAM competed for binding to acER␣ and acER␤b much
better than to acER␤a (Fig. 4). TAM was the second worst
binder to acER␤a (13th out of 14), whereas it was the 8th best
competitor for acER␣ and acER␤b. TAM bound to acER␣
best, with a 5-fold higher affinity than that to acER␤b and a
25-fold higher affinity than that to acER␤a, with an IC50 of
19.3 nm (Table 1).
Naturally occurring estrogenic compounds
Zearalenone had a more than 20-fold greater affinity for
acER␣ than acER␤b and acER␤a, with IC50 values of 5.1, 72.8,
and 58.5 nm, respectively (Fig. 5 and Table 1). In contrast, the
affinity of genistein for acER␣ was the lowest of all of the
compounds with measurable binding tested, whereas the
affinities for acER␤a and acER␤b were 3- to 6-fold greater,
with IC50 values of 29.8 and14.8 nm, respectively.
Nonestrogenic ligands
Interestingly, the antiprogestin RU 486 bound to acER␤a
with a higher affinity than that of TAM, with an IC50 of 218.2
nm (Fig. 6 and Table 1). RU 486 was unable to displace 50%
of the [3H]E2 with acER␣ or acER␤b, although there was some
displacement at a concentration of 100 ␮m competitor (Fig. 6).
The C19 steroids (testosterone and 11KT) and the C21 steroids
(cortisol and 17,20␤DHP) were unable to displace 50% of the
[3H]E2 binding at the highest concentration tested (10 ␮m).
Hawkins and Thomas • Unusual Binding Properties of ER␤a
Endocrinology, June 2004, 145(6):2968 –2977 2973
FIG. 6. Competitive binding of nonestrogenic ligands to acER fusion proteins: A, acER␣; B, acER␤a; and C, acER␤b. Assays were conducted
as described in Fig. 2. The results for the competitors E2, mifepristone (RU 486), cortisol (CORT), 17,20␤DHP (17,20␤), 11KT, and testosterone
(T) are shown.
Site-directed mutagenesis
Mutation of acER␤aPhe396 to the acER␤b residue Ile (acER␤aPhe-Ile) decreased the IC50 of DES from 3.8 to 1.2 nm
(Fig. 8A). The RBA of DES for acER␤aPhe-Ile was 316%
of that of acER␤a-wild-type (wt). The IC50 of TAM for
acER␤aPhe-Ile decreased from 310 to 260 nm, and the RBA
was 119% that of acER␤a-wt (Fig. 8B). Mutation of acER␤aPhe396 to the acER␣ residue Met (acER␤aPhe-Met) decreased the IC50 of DES from 3.8 to 2.5 nm (Fig. 8A). The RBA
of DES for acER␤aPhe-Met was 152% that of acER␤a-wt. The
IC50 of TAM for acER␤aPhe-Met decreased from 310 to 220
nm, and the RBA was 141% that of acER␤a-wt (Fig. 8B).
Discussion
As expected for ERs, there were general similarities in the
ligand binding profiles among the three croaker ER fusion
proteins and to other ERs. For example, E2, DES, TOH,
ICI164, and ICI182 were the top five competitors for all three
croaker receptors. These findings are consistent with those
previously reported for ERs from other species (9, 11, 25, 30).
However, the ligand preferences and RBAs of the acERs for
these compounds showed notable differences. Specifically,
acER␤a binding differed markedly from that of acER␣,
acER␤b, and previously analyzed ERs. For acER␣ and
acER␤b, the IC50 values for DES, ICI182, and TOH were 1.5to 50-fold lower (i.e. higher relative affinity) than the IC50
values for the natural ligand, E2. This is in general agreement
with previous findings for other ERs. In contrast, for acER␤a
the IC50 values for these compounds were 2.5- to 28-fold
higher than those for E2. A particularly striking difference
between acER␤a and other ERs is acER␤a’s lower affinity for
TOH (1.5-fold higher IC50 than E2). TOH consistently shows
a higher RBA than the endogenous ligand, E2, for other ERs
in mammals, birds, lizards, and fish (9, 11, 25, 30). This is the
first demonstration of an ER with a lower affinity to TOH
than to E2.
The RBA of the antiestrogen ICI164 for acER␤a is only 20%
that of E2. This is in contrast to previous findings for ERs of
other species and for croaker ER␣ and ER␤b, where ICI164
ranks either above or just below E2 in binding ability. In
addition, the antiestrogen ICI182 has a 10- to 20-fold lower
affinity for acER␤a than it does for acER␣ and acER␤b. Both
of these compounds have a side-chain at the 7 position on the
E2 skeleton, and perhaps this substitution is less tolerated by
acER␤a.
E3 was the sixth best competitor for acER␤a overall and
was considerably better at competing than E1, which is in
contrast to findings for other nuclear ERs, where E3 ranks
below E1 and is usually among the poorer competitors
tested. An exception is the channel catfish ER␤b, where E3
has a higher affinity than E1 (11). However, this receptor does
not appear to be an ER␤a, as it contains only one of the amino
acids (acER␤aV317) in the LBD diagnostic of ER␤as (Table 2)
(17). E3 is produced by the ovary of teleost fish (31). In
humans, E3 is present at high levels in the placenta (32) and
is produced by adipose tissue in men and postmenopausal
women (33). A distinct binding preference for an endogenous ligand such as E3 may reflect a novel function for the
ER␤a subtype.
Interestingly, mifepristone (RU 486) competed well with
[3H]E2 for binding to acER␤a, but caused only slight displacement of [3H]E2 from the other two acERs at the highest
concentration tested. This is the first evidence that RU 486 has
relatively high binding activity for an ER and raises the
possibility that this compound has estrogenic or antiestrogenic actions via binding to ERs. RU 486 is a 19-nortestosterone derivative and is a potent antiprogestin (34). RU 486
also has weak estrogenic activity in human breast cancer cells
(35) and rat uterine myocytes (36). It is not known whether
RU 486 exerts these effects in mammals via classical ER
pathways. Alternatively, these effects may be indirect via
aromatase inhibition (37). It seems important to investigate
the potential estrogenic actions of RU 486 further because of
its clinical uses in humans (34).
Both acER␤a and acER␤b arose from a duplication of an
ER␤ gene early in the teleost lineage and consequently share
a higher degree of amino acid identity with each other than
they do to acER␣ (17). It is therefore not surprising that for
some compounds acER␤a has similar binding affinities to
acER␤b and mammalian ER␤s. For instance, there is less than
a 2-fold difference in RBA between acER␤a and acER␤b for
E2, E1, 17␣E2, 2OHE, MOXE, genistein, and zearalenone.
acER␤a, acER␤b, and human ER␤ (hER␤) have RBAs for
genistein greater than those for zearalenone, a feature opposite that of ER␣s (30). In addition, rat ER␤ has a 7-fold
2974
Hawkins and Thomas • Unusual Binding Properties of ER␤a
Endocrinology, June 2004, 145(6):2968 –2977
TABLE 2. Amino acids in vertebrate ERs corresponding to positions diagnostic for teleost ER␣ and ␤a subtypes (13)
Human ER␣ amino acid
acER␤a[␣*]amino acid
L349
L324
[M166*]
M421
F396
Y526
H495
C530
M499
M342
V317
L409
S384
D411
S386
K416
S391
T460
L435
S470
E439
Q502
T471
ER␤as
M. undulatus (ER␤a)
S. aurata (ER␤)
P. olivaceous (ER␤)
O. latipes (ER␤)
O. niloticus (ER␤II)
C. carpio (ER␤)
D. rerio (ER␤a,2)
O. mykiss (ER␤)
C. auratus (ER␤1)
L
L
L
L
L
L
L
L
L
F
F
F
F
F
F
L
F
F
H
H
H
H
H
H
H
H
H
M
M
M
M
M
M
M
M
M
V
I
V
V
V
I
V
V
V
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
L
L
L
L
L
L
L
L
L
E
E
E
E
D
E
E
E
E
T
T
T
T
T
T
T
T
T
ER␤bs
M. undulatus (ER␤)
A. japonica (ER␤)
I. punctatus (ER␤)
D. rerio (ER␤b,1)
C. auratus (ER␤2)
L
L
L
L
L
I
L
I
I
I
S
S
S
S
S
R
R
R
R
R
M
M
V
M
M
K
K
K
K
K
N
N
N
N
N
Q
S
N
N
T
T
T
M
S
S
E
E
E
E
E
L
A
A
I
I
ER␤s
S. acanthias (ER␤)
Tetrapod ER␤s (n ⫽ 10)
L
L
I
I
Y
L
C
C
M
M
I
V
Ia
D
D
Q
K
P
P
E
E
Db,c
T
V
I
I
D
D
T3
ER␣s
M. undulatus ER␣
Fish ER␣s (n ⫽ 13)
M
M
M
M
Y
Y
C
C
M
M
I1,2,3
Tetrapod ER␣s (n ⫽ 9)
L
M
Fi
Lii
Y
C
M
Vi
L
Iii
D
D
D
E1,2
N4
K
Mi
Rii
S
S
T
Siii
G
G
S1,2,5,6
N3
S
Td,e,f,g
Mb,c
R
R
R
Qiv
Sequences were aligned to acER␣ and acER␤a using BLAST and compared to alignments published previously (13). The corresponding hER␣
amino acid, and acER␤a or ER␣* amino acid are given at the top of each column. Diagnostic positions previously investigated in mammalian
ERs and discussed in the text are underlined. Amino acid numbering for human ER positions is the same as in Ref. 18. The amino acid positions
for ERs that differ from the majority of their group have subscripts. Accessions for subscripts: a, C. jaccus, CAA70546.2 (ER␤); b, cow,
AF110402(ER␤); c, human, AF215937(ER␤); d, chicken BAA88667(ER␤); e, C. japonica AAC36463.2 (ER␤); f, S. vulgaris AF113513(ER␤); g,
O. aries AF177936 (ER␤); 1, O. niloticus AAD00245(ER␣); 2, C. gariepinus CAC37560(ER␣); 3, I. punctatus AF253505 (ER␣); 4, O. mykiss A37197
(ER) 5, C. auratus AAL12298(ER␣); 6, D. rerio AAK16740(ER␣); i, C. uniparens BAB79437 (ER); ii, X. laevis 625330 (ER); iii, C. japonica
AF442965(ER␣); iv, human AAA52399 (ER␣). Accessions for teleost ERs designated ER␤a: M. undulatus, AF298182 (ER␤a, ER␥); S. aurata,
AF136980(ER␤); P. olivaceous, BAB85623(ER␤); O. niloticus, AAD00246 (ERII); O. latipes, BAB79705(ER␤); C. carpio, BAB91218(ER␤); D.
rerio, NM_180966 (ER␤2,␤a); O. mykiss, CAC06714(ER␤); C. auratus, AF061269(ER␤1). Accessions for teleost ER␤bs: M. undulatus (ER␤b,
ER␤) AF298181; A. japonica, BAA19851(ER␤); I. punctatus, AF185568(ER␤); D. rerio, AJ414566, AF349413.3 (ER␤1, ER␤b); C. auratus,
AF177465(ER␤2). ER␤s: S. acanthias, AAK57823(ER␤); S. scrofa, AF267736; rat, CAA05631; mouse, 008537. Teleost ER␣s: M. undulatus,
AF298183; P. major, BAA22517; S. aurata, AF136979; P. olivaceous, BAB85622; O. aureus, CAA63774; O. latipes, BAA86925; H. trimaculatus,
AF326201; S. salar, CAA61999. Tetrapod ER␣s: C. crocodilus, BAB79436; chicken, CAA27433; T. guttata, AAB81108; rat, CAA43411; mouse,
AAA37580.
higher RBA for genistein than rat ER␣, but zearalenone was
not investigated (9).
The binding profile of acER␣ is most similar to that of
rainbow trout ER␣ (25). The compounds DES, TOH, and
ICI164 are all better competitors than E2 for rainbow trout
and croaker ER␣. In addition, zearalenone has an RBA approaching that of E2 (96% for croaker and 82% for trout) for
both fish ER␣s. TAM and E1 have RBAs between 25% and
10% for both ERs, whereas E3 and genistein have RBAs less
than 10%.
Twenty-six amino acids within parts of helix 3 (H3), H6,
H8, H11, and H12 line the mammalian ER ligand binding
pocket and/or interact with bound E2 (22) (Fig. 7). These
residues are highly conserved across vertebrate ERs, including those of Atlantic croaker. However, some of the amino
acids lining the binding pocket and adjacent amino acids are
changed in croaker ERs (Table 2 and Fig. 7). Four residue
changes in the croaker ER subtypes are at positions surrounding the hER␣ pocket. These changes are conserved in
other fish ER subtypes, suggesting an important role for
these positions in determining species- and subtype-specific
binding characteristics (Table 2). For example, acER␣Met166
is equivalent to hER␣Leu349 that interacts with the A ring of
E2 in the hER␣ pocket (Fig. 7). This Leu to Met change is
found in all 14 fish ER␣s identified to date (Table 2).
A second residue in the euteleost ER␤s at the equivalent
position to hER␣Met421 is changed to acER␤aPhe396 and
acER␤bIle426. All but one of the nine identified euteleost
ER␤as possess a Phe(TTY) at this position (Table 2). The Met
to Ile change (ATG-ATH) in croaker ER␤b is shared by all but
one euteleost ER␤bs as well as all tetrapod ER␤s, including
hER␤. These mutations have therefore been highly conserved for at least 200 million yr, strongly suggesting a functional significance. The two exceptions in each of the ␤ clades
Hawkins and Thomas • Unusual Binding Properties of ER␤a
FIG. 7. Schematic of the crystal structure of the human ER␣ ligand
binding pocket (18) showing amino acid changes in croaker ERs.
Amino acids that make direct hydrogen bonds are blue. Residues that
line the hER␣ pocket or interact with E2 have arcs. Amino acids that
are known to alter ER-ligand interactions and are changed in teleost
ERs (Table 2) are red (18 –20, 24).
(Anguilla japonica and Danio rerio) have a Leu (CTN or TTR)
in the equivalent position (Table 2).
Two additional changes, ER␣Tyr526-acER␤aHis495 and
hER␣Cys530-acER␤aMet499, are conserved in all nine fish ERs
that we designate as ␤as in Table 2. The hER␣Cys530 position
is unchanged in tetrapod ␤s, but is changed to Arg in euteleost ␤bs. This teleost-specific change differentiates ER␤b
from the tetrapod ER␤ as well as from the third teleost
subtype, ER␤a. The finding that amino acid changes in this
position alone can distinguish all of the vertebrate ER subtypes identified to date suggests that it is a key residue in the
evolution of ER subtypes and thus may have an important
role in the development of the pleiotropic actions of
estrogens.
Crystallographic and mutagenesis studies of mammalian ERs and mutagenesis studies of fish ERs indicate that
these four residue changes may account for the differences
in ligand binding profiles we observed for the croaker ERs.
For example, the 17␣-hydroxyl of E2 hydrogen-bonds with
hER␣His524 (equivalent to acER␤aHis493) (22). The additional His at hER␣Tyr526 (acER␤aHis495) may help stabilize
the extra hydroxyl group of E3, resulting in the increased
relative affinity of this endogenous steroid for acER␤a.
Previous studies have demonstrated that the replacement
of hER␣Tyr526 (acER␤aHis495) with Ala raises the IC50 of
E2-induced transcriptional activity 4-fold (24), suggesting
that this position is important to E2 binding.
In this study genistein had a higher affinity for acER␤a and
acER␤b than for acER␣. This is analogous to findings for
hERs, where hER␣Met421 switches to hER␤Ile373. This residue is on the ␣ face of the cavity where the O4 of genistein’s
flavone ring lies (24). The same change occurs from acER␣ to
acER␤b (acER␣Met238 to acER␤bIle426), whereas acER␤a
changes to Phe396. It has been proposed (20) that the change
in the slightly polar Met for the less polar Ile allows for more
polar substituents at the distal end of the cavity, resulting in
Endocrinology, June 2004, 145(6):2968 –2977 2975
the higher affinity of hER␤ for genistein. As the Phe in
acER␤a is also nonpolar, it may account for the increased
affinity of genistein for acER␤a as well.
hER␣ Met421 (acER␤aPhe396) contacts DES and TOH on
their A⬘ and B rings, respectively, but does not contact E2
(23). In addition, the region of the pocket that includes this
residue changes conformation depending on the ligand.
acER␤a has at least a 10-fold lower affinity for DES, TAM,
and TOH compared with acER␣ and acER␤b, suggesting that
the mutation in the ER␤as to the larger and less polar Phe
may be responsible for its unusual binding affinities for these
compounds. The studies we present here provide further
evidence of the functional importance of this position to
ligand recognition. We show that mutation of acER␤aPhe396
to the acER␤b residue Ile increases the RBA of DES to 316%
of that of acER␤a (Fig. 8A). This increase accounts for 90% of
the difference seen in binding affinity between acER␤a and
acER␤b (Table 1). These data strongly suggest that this position is critical to the interaction of ERs with DES. Alternatively, the change of acER␤aPhe396 to the acER␣ amino acid
Met increased the RBA of DES to just 150% of acER␤a, only
5% of the 13-fold difference seen between the wt receptors.
The change from Phe to Met also seems to slightly increase
the affinity of the ER mutant construct for TAM, but this
increase is just 10% of the difference seen between acER␤a
and acER␣ (Fig. 8B). It is likely that a Met in this position
interacts with additional ␣-specific amino acids to produce the higher affinities that vertebrate ER␣s have for DES
and TAM.
The conserved ER␤a residue acER␤aMet499 might also
alter ligand binding characteristics for DES, TAM, and
TOH. Affinity labeling studies suggested that hER␣Cys530
(acER␤aMet499) might be involved in binding of the antiestrogen, TAM aziridine (38). However, site-directed mutagenesis and carboxymethylation studies contradict this finding
(28, 39). hER␣Cys530 is located in H11 adjacent to residues
known to be critical to ligand binding of the D ring of E2 (21,
28). Even if this residue is not directly on the face of the
cavity, Met (acER␤a) is larger and slightly more polar than
Cys (hER␣) and does not form disulfide bonds. Therefore,
this substitution may indirectly alter the size and shape of the
cavity such that compounds with diphenolic structures such
as DES, TAM, and TOH do not fit as well into the ER␤a
pocket.
acER␣ binds E2 with about a 2-fold weaker affinity than
acER␤a or acER␤b. Zebrafish ER␣ also has nearly a 2-fold
weaker affinity for E2 than ER␤a, but unlike croaker, ER␤b
also has a 2-fold weaker affinity for E2 (19). In the rainbow
trout ER␣ (and in all other euteleost ER␣s; Table 2),
there is a conservative substitution of hER␣Leu349 to a
Met(acER␣Met166). Reciprocal mutagenesis at this position
causes a temperature-dependent 2-fold lowering of Kd for
the rainbow trout ER␣ (26). This substitution may therefore
be responsible for the lower affinity of E2 for acER␣. In
contrast, acER␣ binds 17␣E2, E1, and 2OHE better than
acER␤a and acER␤b, albeit binding is low for all three compounds compared with that for E2. It is possible that
acER␣Met166 may play a role in these differences as well, but
relative affinities to other estrogens in the reciprocal mutagenesis system were not evaluated (26).
2976
Endocrinology, June 2004, 145(6):2968 –2977
FIG. 8. IC50 values for DES (A) and TAM (B) in competitive binding
assays with acER␤a-wt and acER␤a-mutant fusion proteins. The
amino acid Phe396 of the acER␤a-wt fusion protein was mutated to
Met (F to M) or Ile (F to I) and used in competitive binding assays as
described in Figs. 2– 6. Data are averaged from two independent
experiments (each performed in triplicate), with error bars representing the SEM. The RBA for each competitor was calculated as the
ratio of the IC50 for each compound with the wt construct to that of
the mutant construct.
hER␣Glu353 hydrogen-bonds with the 3-hydroxyl group
on the A ring of estrogens and is critical to the discrimination
of 3-hydroxyl estrogens from the C19 and C21 3-ketosteroids
(40). This Glu is 100% conserved among ERs, including the
acERs. It is therefore surprising that acER␤a binds RU 486,
given that this compound possesses a keto group at position
3 as in C19 and C21 steroids. The 17-hydroxyl orientation in
the ␤ position and the lack of the C19 methyl group in RU 486
must allow for some binding of this compound to ERs. It
would be interesting to know whether this affinity of RU 486
for acER␤a is universal to all ER␤as, and consequently which,
if any of the conserved amino acids are responsible for this
difference.
Hawkins and Thomas • Unusual Binding Properties of ER␤a
This study sheds light on the molecular nature of ligand
interactions with ERs, but does not address how these compounds, once bound to the croaker ERs, modulate transcription of estrogen-responsive genes. Compounds can be either
agonists or antagonists of ER actions depending on the conformational changes they induce in the receptor upon binding. These conformational changes alter the ability of ERs to
interact with cell-specific regulatory proteins and subsequently activate gene transcription (16). For instance, TAM
binds to both mammalian ER␣ and ER␤, but only activates
ER␣ (41). In addition, TAM and TOH do not activate any of
the zebrafish ERs, which is in contrast to findings for mammalian ERs (19, 42, 43). It is not known whether these differences seen in zebrafish are due to species differences in ER
trans-activation or ligand binding or due to the population of
coactivators present (or absent) in the transfected cells. However, it does seem likely that, like zebrafish, croaker ERs will
exhibit some trans-activation properties different from those
of mammalian ERs, because zebrafish and croaker share
many of the teleost-specific amino acid substitutions in regulatory regions of the molecule (17).
The three acERs provide a natural starting point for uncovering key amino acid positions involved in receptor function. The acERs represent three clades of ERs that diverged
more than 150 million yr ago. These groups, in particular the
ER␤as, possess distinct amino acid substitutions that arose
after their divergence and were then nearly all retained in the
members of the clade (Table 2). This strong degree of conservation suggests that these positions are critical to receptor
function. Evidence from receptor ligand studies of mammalian ERs allowed us to identify four conserved amino acid
substitutions in the acERs that might be involved in the
different binding profiles we observed in this study. Mutation of one of these substitutions, acER␤aPhe396 to the corresponding ER␣ (Met) or ER␤b (Ile) amino acid, shifted the
binding affinities for DES and TAM toward those for the
corresponding wt acER␣ and acER␤b. This is the first demonstration of a direct role for this position in ligand discrimination and supports the hypothesis that the amino acid
changes at this position have been highly conserved because
of its functional significance. Other conserved substitutions
within the fish ER␤a clade point to at least seven additional
amino acids that could be important (Table 2). These positions were largely overlooked in earlier mammalian studies
because of the sequence conservation between mammalian
ER␣ and ER␤ subtypes and a lack of direct ligand interactions. The availability of a native receptor model in which
residue changes have evolved together to create a functional
protein with novel binding properties could provide useful
information on the roles of these positions in mammalian
systems. More site-directed mutagenesis studies are needed
to determine the role that each of these positions plays in
ligand binding. This approach may ultimately lead to the
development of more ER subtype-specific agonists and antagonists and could also focus studies on trans-activation,
receptor dimerization, and cofactor recruitment.
Acknowledgments
We thank Dr. Yong Zhu for assistance with establishing the bacterial
expression system and Dr. John Godwin for laboratory support.
Hawkins and Thomas • Unusual Binding Properties of ER␤a
Received June 27, 2003. Accepted February 17, 2004.
Address all correspondence and requests for reprints to: Dr. Mary
Beth Hawkins, Department of Zoology, North Carolina State University,
Box 7617, Raleigh, North Carolina 27695. E-mail: beth_hawkins@
ncsu.edu.
This work was supported by University of Texas Marine Science
Institute, the Harry Page Marine Science Fellowship, the Houston Livestock Show and Rodeo Natural Sciences Fellowship and by Texas Sea
Grant R/ES-92 and EPA STAR Grant R827399 (to P.T.).
References
1. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/
thyroid receptor superfamily members. Annu Rev Biochem 63:451– 486
2. Hewitt SC, Korach KS 2002 Estrogen receptors: structure, mechanisms and
function. Rev Endocr Met Dis 3:193–200
3. Beato M, Sanchez-Pacheco A 1996 Interaction of steroid hormone receptors
with the transcription initiation complex. Endocr Rev 17:587– 609
4. Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and co-repressors in the
integration of transcriptional responses. Curr Opin Cell Biol 10:373–383
5. Kuiper G, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl
Acad Sci USA 93:5925–5930
6. Mosselman S, Polman J, Dijkema R 1996 ER␤: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49 –53
7. Foidart A, Lakaye B, Grisar T, Ball GF, Balthazart J 1999 Estrogen receptor-␤
in quail: cloning, tissue expression and neuroanatomical distribution. J Neurobiol 40:327–342
8. Todo T, Adachi S, Yamauchi K 1996 Molecular cloning and characterization
of Japanese eel estrogen receptor cDNA. Mol Cell Endocrinol 119:37– 45
9. Kuiper G, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S,
Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors ␣ and ␤. Endocrinology 138:
863– 870
10. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson JA, Nilsson S 1998
Differential response of estrogen receptor ␣ and estrogen receptor ␤ to partial
estrogen agonists/antagonists. Mol Pharmacol 54:105–112
11. Xia ZF, Gale WL, Chang XT, Langenau D, Patino R, Maule AG, Densmore
LD 2000 Phylogenetic sequence analysis, recombinant expression, and tissue
distribution of a channel catfish estrogen receptor ␤. Gen Comp Endocrinol
118:139 –149
12. Xia ZF, Patino R, Gale WL, Maule AG, Densmore LD 1999 Cloning, in vitro
expression, and novel phylogenetic classification of a channel catfish estrogen
receptor. Gen Comp Endocrinol 113:360 –368
13. Lonard DM, Smith CL 2002 Molecular perspectives on selective estrogen
receptor modulators (SERMs): progress in understanding their tissue-specific
agonist and antagonist actions. Steroids 67:15–24
14. Harris HA, Katzenbellenbogen JA, Katzenbellenbogen BS 2002 Characterization of the biological roles of the estrogen receptors, ER␣ and ER␤, in
estrogen target tissues in vivo through the use of an ER␣-selective ligand.
Endocrinology 143:4172– 4177
15. Harris HA, Albert LM, Leathbury Y, Malamas S, Mewshaw RE, Miller CP,
Kharode YP, Marzolf J, Komm BS, Winneker C, Frail DE, Henderson RA,
Zhu Y, Keith Jr JC 2003 Evaluation of an estrogen receptor-␤ agonist in animal
models of human disease. Endocrinology 144:4241– 4249
16. McDonnell DP 2003 Mining the complexities of the estrogen signaling pathways for novel therapies. Endocrinology 144:4237– 4240
17. Hawkins MB, Thornton JW, Crews D, Skipper JK, Dotte A, Thomas P 2000
Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts. Proc Natl Acad Sci USA 97:10751–10756
18. Enmark E, Pelto-Huikko M, Grandien K, Lagercrantz S, Lagercrantz J, Fried
G, Nordenskjöld M, Gustafsson JA 1997 Human estrogen receptor ␤-gene
structure, chromosomal localization, and expression pattern. J Clin Endocrinol
Metab 82:4258 – 4265
19. Menuet A, Pellegrini E, Anglade I, Blaise O, Laudet V, Kah O, Pakdel F 2002
Molecular characterization of three estrogen receptor forms in zebrafish: binding characteristics, transactivation properties, and tissue distributions. Biol
Reprod 66:1881–1892
20. Sprague J, Doerry E, Douglas S, Westerfield M 2001 The Zebrafish Information Network (ZFIN): a resource for genetic, genomic and developmental
research. Nucleic Acids Res 29:87–90
21. Ekena K, Weis KE, Katzenellenbogen JA, Katzenellenbogen BS 1997 Dif-
Endocrinology, June 2004, 145(6):2968 –2977 2977
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
ferent residues of the human estrogen receptor are involved in the recognition
of structurally diverse estrogens and antiestrogens. J Biol Chem 272:5069 –5075
Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O,
Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of
agonism and antagonism in the oestrogen receptor. Nature 389:753–758
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene
GL 1998 The structural basis of estrogen receptor/coactivator recognition and
the antagonism of this interaction by tamoxifen. Cell 95:927–937
Pike ACW, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom
O, Ljunggren J, Gustafsson JA, Carlquist M 1999 Structure of the ligandbinding domain of oestrogen receptor ␤ in the presence of a partial agonist and
a full antagonist. EMBO J 18:4608 – 4618
Matthews J, Celius T, Halgren R, Zacharewski T 2000 Differential estrogen
receptor binding of estrogenic substances: a species comparison. J Steroid
Biochem Mol Biol 74:223–234
Matthews JB, Clemons JH, Zacharewski TR 2001 Reciprocal mutagenesis
between human ␣(L349, M528) and rainbow trout (M317, I496) estrogen receptor residues demonstrates their importance in ligand binding and gene
expression at different temperatures. Mol Cell Endocrinol 183:127–139
Danielian PS, White R, Hoare SA, Fawell SE, Parker MG 1993 Identification
of residues in the estrogen-receptor that confer differential sensitivity to estrogen and hydroxytamoxifen. Mol Endocrinol 7:232–240
Ekena K, Weis KE, Katzenellenbogen JA, Katzenellenbogen BS 1996 Identification of amino acids in the hormone binding domain of the human estrogen
receptor important in estrogen binding. J Biol Chem 271:20053–20059
Scatchard G 1949 The attractions of proteins for small molecules and ions. Ann
NY Acad Sci 51:660 – 672
Kuiper G, Lemmen JG, Carlsson B, Corton JC, Safe SH, van der Saag PT, van
der Burg P, Gustafsson JA 1998 Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor ␤. Endocrinology 139:4252– 4263
Ponthier JL, Shackleton CHL, Trant JM 1998 Seasonal changes in the production of two novel and abundant ovarian steroids in the channel catfish
(Ictalurus punctatus). Gen Comp Endocrinol 111:141–155
Peter M, Dorr HG, Sippell WG 1994 Changes in the concentrations of dehydroepiandrosterone-sulfate and estriol in maternal plasma during pregnancy:
a longitudinal-study in healthy women throughout gestation and at term.
Horm Res 42:278 –281
Grodin JM, Siiteri PK, Macdonal. Pc 1973 Source of estrogen production in
postmenopausal women. J Clin Endocrinol Metab 36:207–214.
Baulieu EE 1989 Contragestion and other clinical-applications of RU-486, an
antiprogesterone at the receptor. Science 245:1351–1357
Jeng MH, Langanfahey SM, Jordan VC 1993 Estrogenic actions of RU486 in
hormone-responsive MCF-7 human breast-cancer cells. Endocrinology 132:
2622–2630
Dibbs KI, Sadovsky Y, Li XJ, Koide SS, Adler S, Fuchs AR 1995 Estrogenic
activity of RU-486 (mifepristone) in rat uterus and cultured uterine myocytes.
Am J Obstet Gynecol 173:134 –140
Schmidt M, Loffler G 1997 RU486 is a potent inhibitor of aromatase induction
in human breast adipose tissue stromal cells. J Steroid Biochem Mol Biol
60:197–204
Harlow KW, Smith DN, Katzenellenbogen JA, Greene GL, Katzenellenbogen BS 1989 Identification of cysteine-530 as the covalent attachment site of an
affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen-receptor. J Biol Chem 264:17476 –17485
Hegy GB, Shackleton CHL, Carlquist M, Bonn T, Engstrom O, Sjoholm P,
Witkowska HE 1996 Carboxymethylation of the human estrogen receptor
ligand-binding domain-estradiol complex: HPLC/ESMS peptide mapping
shows that cysteine 447 does not react with iodoacetic acid. Steroids 61:367–373
Ekena K, Katzenellenbogen JA, Katzenellenbogen BS 1998 Determinants of
ligand specificity of estrogen receptor-␣: estrogen versus androgen discrimination. J Biol Chem 273:693– 699
Watanabe T, Inoue S, Ogawa S, Ishii Y, Hiroi H, Ikeda K, Orimo A, Muramatsu M 1997 Agonistic effect of tamoxifen is dependent on cell type,
ERE-promoter context, and estrogen receptor subtype: functional difference
between estrogen receptors ␣ and ␤. Biochem Biophys Res Commun 236:140 –
145
Bardet PL, Horard B, Robinson-Rechavi M, Laudet V, Vanacker JM 2002
Characterization of oestrogen receptors in zebrafish (Danio rerio). J Mol Endocrinol 28:153–163
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie
F, Giguere V 1997 Cloning, chromosomal localization, and functional analysis
of the murine estrogen receptor ␤. Mol Endocrinol 11:353–365
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