Estrogen Receptor Residues Required for Stereospecific Ligand Recognition and Activation
Wayne P. Bocchinfuso and
Kenneth S. Korach
Receptor Biology Section Laboratory of Reproductive and
Developmental Toxicology National Institute of Environmental Health
Sciences National Institutes of Health Research Triangle Park,
North Carolina 27709
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ABSTRACT
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The mouse estrogen receptor (mER) has been shown
to exhibit stereospecific binding of certain stilbene estrogen
agonists. The region of the mER involved in the stereochemical
recognition of ligands was further defined using a stilbene isomer,
Indenestrol B (IB). The IB compound has a chiral carbon bearing an
ethyl substituent, and the wild type uterine mER has been shown to bind
the enantiomers, IB-S and IB-R, with similar
affinity. The wild type mER expressed in yeast exhibited a very minor
preference for IB-S in transactivation (1.5-fold lower
half-maximal dose than IB-R). The IB enantiomers could then
be used to determine whether stereochemically distinct compounds with
similar transcriptional activity utilize different amino acids in AF-2
for transactivation. Mutant mERs with glycine substitutions at Met521,
His528, Met532, and Val537 were expressed in yeast and measured for
IB-S- and IB-R-induced transactivation and
ligand binding. The M532G mER showed a 124-fold and 50-fold reduction
in transactivation induced by IB-S and IB-R,
respectively, without a corresponding change in their ligand-binding
affinities. Therefore, Met532 is required for transactivation induced
by both IB enantiomers but does not discriminate based on
stereospecificity. In contrast, the H528G mER displayed a gross change
in stereospecific ligand recognition as illustrated by a 110-fold
reduction in transactivation by IB-S and only a 8.8-fold
decrease in activity by IB-R. As a result, H528G mER
displayed a switch in ligand preference such that IB-R was
now 8-fold more active than IB-S in transactivation.
Therefore, His528 appears largely involved in transactivation
specifically induced by IB-S but has a minimal influence in
IB-S ligand binding. The remaining mutant mERs displayed
wild type ligand binding and transactivation properties for the IB
enantiomers illustrating no stereospecific recognition. These results
imply that individual IB enantiomers bind to the mER with similar
affinity but utilize at least one different amino acid within the AF-2
domain for signal transduction. The binding of stereochemically
distinct ligands may alter the tertiary structure of the mER and cause
repositioning of the AF-2 region that mediates transcription of
specific genes and/or affect the binding of receptor-associated
proteins, such as coactivators, which could influence transcription.
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INTRODUCTION
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The effect of different ligand structures in eliciting estrogenic
hormonal responses in mouse uterine tissue has been analyzed using
diethylstilbestrol (DES) and several DES metabolites that are
structural analogs (1, 2, 3, 4). These studies were important because DES,
which has high estrogenic biological activity, is associated with
toxicity and carcinogenesis of the reproductive tract in females, and
DES metabolites have the potential to function similarly (5, 6). The
DES analogs Indenestrol A (IA) and B (IB), which are stereoisomers,
possess estrogen agonist activity as shown by the induction of uterine
DNA synthesis, and estrogen-responsive genes such as the progesterone
receptor (PR), glucose-6-phosphate dehydrogenase (G6P-DH), ornithine
decarboxylase (ODC), and lactoferrin (LTF) (1, 2, 7).
These DES analogs share a partial structural similarity to the
anti-estrogenic triphenylethylene compounds, including hydroxytamoxifen
(8). Different biological activities are seen with the
triphenylethylene compounds compared with steroidal agonists such as
17ß-estradiol (E2) (9). Many studies have attempted to
explain the mechanistic differences between agonist and
antagonist/receptor complexes on transcriptional activity through
altered estrogen receptor (ER) conformations (10, 11, 12, 13, 14, 15). Therefore, the
study of ER transcriptional activation by IA and IB compounds and the
ability of the ER to bind these compounds might provide insight into
the partial agonist activity of tamoxifen compounds.
The IA compound has a single chiral carbon with a methyl group and
exists as a mixture of enantiomers (16). The IA-S enantiomer
was demonstrated to be much more biologically active than
IA-R, and the mouse estrogen receptor (mER) was subsequently
shown to bind IA-S with high affinity similar to DES (2, 4, 16). Removal of the methyl group from IA-S resulted in a
15-fold lower binding affinity similar to the weakly active
IA-R. Thus, the greater biological activity of
IA-S was attributed to its higher mER binding affinity, due
to the presence and orientation of the methyl substituent. This earlier
report demonstrated that the mER ligand-binding site has the capacity
to distinguish between and bind ligands with a particular
stereochemistry (i.e IA-S vs.
IA-R). Subsequently, site-directed mutagenesis of the mER
ligand-binding domain revealed that Met532 specifically functioned in
IA-S-induced transactivation but not in IA-S
ligand binding (17).
The IB compound has a chiral carbon bearing an ethyl group (Fig. 1
) and also exists as a mixture of S and
R enantiomers. The uterine mER has been shown to bind the
IB-S and IB-R enantiomers with similar affinities
that are both slightly lower than DES (16). Although the mER binds the
IB enantiomers with similar affinity, direct gene transactivation
induced by the individual IB enantiomers has not been measured.
Therefore, it is important to determine whether the activities of
IB-S and IB-R mimic their ligand-binding
affinities, and whether each enantiomer utilizes different amino acids
in AF-2 for transducing the hormonal signal. A series of mER mutants
with individual amino acid substitutions in the ligand-binding domain
were used to further delineate the region of the mER responsible for
stereochemical recognition of the IB enantiomers and transactivation.
Transcriptional activity was assessed using a vitellogenin
A2 estrogen response element (ERE) linked to a
ß-galactosidase reporter gene construct in a yeast expression
system.

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Figure 1. Chemical Structures of E2, DES, and IB
Enantiomers
Note the different stereo-projections of the ethyl group at the chiral
carbon, designated by the asterisk, in
IB-S and IB-R.
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RESULTS
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The biological activity of the individual IB enantiomers has not
been assessed previously because it was assumed they would be
equivalent based on their similar binding affinities to the uterine
mER. However, biological potency is not only determined by
ligand-binding affinity but requires transcriptional activation of the
receptor and target gene expression (18). Thus, it was important to
determine the relative transcriptional activity induced by the IB
enantiomers in a yeast assay system expressing wild type mER. The
effects of IB enantiomer structure (Fig. 1
) on the ligand binding and
transactivation properties of the mutant mERs could then be compared
with wild type mER to identify amino acids involved in these
functions.
A yeast culture expressing wild type mER was exposed to increasing
amounts of DES, IB-S, and IB-R to generate
dose-response transactivation curves for each compound. Wild type mER
was most responsive to DES with a dose of 0.6 nM required
to induce half-maximal transactivation. IB-S and
IB-R were approximately 2- and 3-fold less active than DES,
respectively, as demonstrated by their greater concentrations required
for half-maximal activation of wild type mER. Furthermore,
IB-S was 1.5-fold more active than IB-R, and both
compounds induced maximal stimulation similar to DES (Table 1
, Fig. 2A
).

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Figure 2. Transactivation and Ligand-Binding Analyses Using
DES and the IB Enantiomers for Wild Type mER
A, Yeast cells were transformed with the wild type mER expression
vector (PG-MS) and the reporter plasmid p SERE containing a consensus
vitellogenin ERE linked to the lacZ gene. The cells were
incubated with increasing concentrations of each ligand at 30 C for
2 h. The cells were harvested, and ß-galactosidase activity was
detected using o-nitrophenyl
ß-D-galactopyranoside as substrate and measured by a
spectrophotometer at OD420. The activity induced by the
various compounds was expressed as fold stimulation over the vehicle
control value determined by ethanol induction. Each data point is the
mean ± SD of three determinations. B, A 100-µl
aliquot of yeast cytosol was incubated with 7.5 nM
[3H]E2 and increasing amounts of unlabeled
competitors at 4 C for 18 h. Receptor-bound
[3H]E2 was isolated by hydroxylapatite
adsorption of receptor and quantified by scintillation counting. The
data are expressed as percent of [3H]E2 bound
relative to the amount of total specific
[3H]E2 bound in the absence of competitor set
at 100%. Each data point is the mean ± SD of three
determinations.
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The relative ligand-binding affinities of the wild type mER for the
different compounds were determined by competing
[3H]E2 with increasing concentrations of
unlabeled DES, IB-S, and IB-R and comparing the
ligand concentrations required to displace 50% of
[3H]E2 from the mER-binding sites
(IC50). The wild type mER displayed the greatest affinity
for DES and bound IB-S and IB-R with 3- and
5-fold less affinity, respectively (Fig. 2B
and Table 2
). Furthermore, IB-S was bound with 1.6-fold
greater affinity than IB-R. These observations are in
agreement with the ligand- binding data reported for mER isolated from
mouse uterus (16). Therefore, the relative transcriptional activities
of the compounds were reflected by their relative ligand-binding
affinities to the wild type mER (Tables 1
and 2
).
Mutant mERs containing individual amino acid substitutions in the
ligand-binding domain were used to delineate the region of the receptor
that is required for transactivation by the IB compounds and to
determine whether IB-S and IB-R utilize different
amino acids in AF-2 for mER transcriptional activation. The amino acids
targeted for mutation were Met521, His528, Met532, and Val537 because
it was thought that their side chains might provide hydrophobic
interactions with the methyl and ethyl groups on the IB compounds (Fig. 1
). The mutant mERs have been shown to be expressed at levels very
similar to wild type mER by Western analysis (17) and were subjected to
the same functional analyses as described with wild type mER. The
transactivation profiles for M521G and V537G mERs generated by DES,
IB-S, and IB-R were similar to wild-type mER, as
illustrated by their doses required for half-maximal transactivation
(Table 1
). These mutant mERs also exhibited a minor preference for
IB-S over IB-R in transactivation as demonstrated
by a 1.5- to 1.8-fold lower dose of IB-S required for
half-maximal transactivation (Table 1
). The V537G mER also bound the
compounds with a relative affinity equivalent to wild type mER (Table 2
).
In contrast, M532G mER displayed a 124-fold and 50-fold reduction in
transactivation induced by IB-S and IB-R,
respectively, as demonstrated by the greater doses required for
half-maximal activation (Tables 1
and 3
and Fig. 3A
). As a result, the M532G mER exhibited a small but
noticeable change in stereochemical preference (1.5-fold) for
IB-R over IB-S. However, there was no change in
the ligand-binding affinity of M532G mER for either IB enantiomer
(Table 2
and Fig. 3B
). Transactivation induced by DES was reduced to a
much lesser degree (8-fold) than the IB compounds with no change in
DES- binding affinity to M532G mER (Tables 2
and 3
). Interestingly,
H528G mER displayed a 110-fold reduction in transactivation induced by
IB-S but only an 8.8-fold loss in activity induced by
IB-R (Tables 1
and 3
and Fig. 4A
). As a result, there was an
8-fold preference for IB-R over IB-S in
transactivation, which also reverses the stereochemical preference for
IB-S exhibited by wild type mER (Table 1
). The switch in
preference for IB-R by H528G mER was accompanied by a minor
reduction in IB-S ligand binding affinity (Table 2
and Fig. 4B
). There was minimal effect on transactivation induced
by DES and DES binding by H528G mER (Tables 2
and 3
).

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Figure 3. Transactivation and Ligand-Binding Analyses Using
DES and the IB Enantiomers for M532G mER
The ß-galactosidase reporter assay (A) and the competition
ligand-binding analysis (B) were performed as described in Fig. 3 .
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Figure 4. Transactivation and Ligand-Binding Analyses Using
DES and the IB Enantiomers for H528G mER
The ß-galactosidase reporter assay (A) and the competition
ligand-binding analysis (B) were performed as described in Fig. 3 .
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DISCUSSION
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The IB enantiomers were measured for their ability to activate
yeast-expressed mER and were shown to induce ß-galactosidase
expression with half-maximal doses that are similar to DES. The
transcriptional activity of these compounds corresponded to their
relative binding affinities to yeast-expressed mER, which is in
agreement with uterine mER ligand-binding characteristics (16). The mER
only slightly favored the IB-S enantiomer over
IB-R in transactivation, which contrasts with the 15-fold
greater activity of IA-S over IA-R (16). This
allowed us to determine whether stereochemically distinct compounds
with similar activities (IB enantiomers) could utilize different amino
acids in AF-2 for transactivation.
Mutation of Met532 effectively dissociated the function of IB
ligand binding from IB-induced transactivation. This mutation had no
effect on the binding of either IB enantiomer; however, it dramatically
reduced the activation by IB-R and IB-S, 50- and
124-fold, respectively. Therefore, Met532 is important for activation
induced by both IB enantiomers but cannot discriminate well between
them. This is in contrast to the ability of Met532 to discriminate
between IA-S and IA-R for transactivation (17).
Although DES is structurally similar to the IB enantiomers, there are
still some structural and conformational differences that could account
for the limited loss of DES-induced transactivation by M532G mER
compared with the major loss of activation by the IB enantiomers.
Conversely, His528 appears more involved in transactivation induced by
IB-S rather than IB-R, as demonstrated by the
effect of mutating this residue. The 110-fold loss of activity induced
by IB-S compared with only an 8-fold loss of IB-R activity in H528G mER
demonstrates that His528 is able to discriminate between the IB
enantiomers for transactivation. The H528G mER displayed a gross change
in stereospecific ligand recognition with respect to transcriptional
activation, such that IB-R was now the more active
enantiomer.
Collectively, the results demonstrate that individual enantiomers,
which differ only with respect to the conformation of a substituent on
a chiral carbon (i.e. ethyl group, see Fig. 1
), are capable
of binding to the mER with similar affinity but utilize at least one
different amino acid within the AF-2 domain for signal transduction.
These observations are consistent with the recent elucidation of the
crystal structures for the apo-retinoid X receptor-
and
holo-retinoic acid receptor-
ligand-binding domains, which
demonstrated that a ligand-induced conformational change in the
ligand-binding domain repositions helices 11 and 12 of the AF-2 region
and forms a transcriptionally active receptor (19, 20). Primary
sequence alignments of the aforementioned ligand-binding domains with
other members of the nuclear receptor superfamily demonstrated that
His528 and Met532 are situated within putative helix 11 of the ER (21).
If this region of the mER does exist as an
-helical structure, then
the amino acids chosen for mutation would reside on the same face of
the
-helix as demonstrated previously using an
-helical face map
of human ER (hER) residues 515535, which are analogous to mER
residues 519539 (22). In the mER, His528 and Met532 correspond to
residues Arg396 and Leu400 in human retinoic acid receptor-
that
interact with all-trans-retinoic acid (20). However, our
results suggest that His528 and Met532 are required for transactivation
induced by IB compounds and contribute little to their binding.
Therefore, it will be difficult to assess how His528, Met532, and other
residues in this helix are positioned without crystal structure data on
the ER in the presence and absence of different ligands.
Our data suggest that the common three-ring structure of the IB
molecule (see Fig. 1
) may impart a conformational change in helix 11 of
the mER AF-2 region that utilizes Met532 to transduce a signal. However
upon IB-S binding, the ethyl substituent in the S
position may create an additional conformational change in helix 11,
which positions His528 for signal transduction (Fig. 5
).
In general, the AF-2 region may be repositioned in slightly different
ways depending on the ligand bound; therefore, a different set of amino
acids in AF-2 could be used for activating or repressing transcription
of target genes to varying degrees. This could arise by 1) altering the
interaction of the AF-2 domain directly with the transcriptional
components or, 2) generating a particular ER conformation, which may
attract or repel the binding of specific receptor-associated proteins,
modifying the interaction with the transcriptional machinery and
influencing gene transcription (23, 24). It should be noted that these
two events are probably not mutually exclusive (Fig. 5
).

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Figure 5. Model for mER Transactivation Induced by
Enantiomers of IB
The binding of ligand causes a conformational change in nuclear
receptors particularly in helices 11 and 12 of AF-2, which may then
become accessible to the transcriptional components and/or
coactivators. When IB-S or IB-R binds to
the mER, the common three-ring nucleus of the IB molecules may impart a
conformational change in helix 11 that utilizes Met 532 for
transactivation. However, when IB-S is bound, the ethyl
group in the S position may create a further
conformation change in helix 11 that positions both His528 and Met532
for activation. The ethyl group in IB-R does not
initiate activation through His528; therefore, mutation of this residue
has a much greater impact on IB-S-induced
activation.
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The ability of His528 in the mER to distinguish stereochemically
between the IB enantiomers in transactivation is consistent with
previous studies, which have shown that this region of the
ligand-binding domain has the capacity to distinguish between agonists
and antagonists (25, 26, 27, 28, 29). In particular, mutation of the hER at Leu540
(corresponding to mER Leu544) results in agonist activity by tamoxifen
and ICI 164,384 and was further shown to require an intact F domain in
mammalian cell lines (28, 29). Altogether, these results can be
explained by the tripartite model of steroid hormone receptor action,
which states that the biological response to a hormone signal is not
just based on ligand binding and ligand potency (23). A biological
response requires additional mechanistic events such as receptor
conformational changes especially in AF-2 and the binding of
coactivators or corepressors in a promoter- and cell type-specific
manner (23, 24).
This model may help explain why the isomers, IA and IB, differentially
induce the expression of specific genes. The biological activities of
these DES-related compounds have been previously analyzed based on
their ability to bind to the mouse uterine ER, activate DNA synthesis,
and differentially induce a variety of estrogen-responsive gene
products such as G6P-DH, ODC, LTF, and the PR in vivo (1, 2, 7). Although IA-S binds to the mER with the same affinity as
DES, IA-S does not induce uterine DNA synthesis or
estrogen-responsive gene products such as ODC to the same level as DES.
Racemic IB binds to the mER with an affinity similar to racemic IA and
DES; however, it can induce uterine DNA synthesis and ODC gene
expression close to DES-induced levels. Furthermore, IA-S is
effective at inducing G6P-DH activity, while racemic IB is not (1, 2),
and LTF expression is induced by DES and racemic IA but not racemic IB
(7). These observations imply that the induction of transcription
requires more than ligand binding to the ER and that activation of the
receptor may require a unique ligand-induced conformation in AF-2
coupled with a promoter-specific hormone response element and accessory
factors. Previous studies have also shown that the mER can bind to a
vitellogenin ERE by gel-shift assay in the presence of either DES, IA,
or IB (2). Therefore, differential activity of these compounds is not
due to the inability of the mER to interact with DNA when bound by a
particular ligand.
Transactivation assay systems in yeast and mammalian cells have been
useful for evaluating receptor-mediated gene regulation and for
studying the effects of ligand structure on ER activity (14, 30, 31).
Furthermore, DES analogs such as the IA and IB enantiomers are useful
compounds for the study of subtle ligand stereochemical differences on
ligand binding and transactivation (17). The results of this study
provide a basis for other experiments analyzing the effect of different
ER mutations on transactivation induced by a different series of
agonists.
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MATERIALS AND METHODS
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Materials
[3H]E2 (85 Ci/mmol) was purchased from
New England Nuclear (Boston, MA). Unlabeled E2 and DES were
obtained from Steraloids, Inc. (Wilton, NH). The IB compound was
synthesized as a racemic mixture by ChemSyn Laboratories (Lenexa, KS)
as previously described (4). Separation of the IB enantiomers was
performed by Dr. Kun Chae (NIEHS) as previously described (32).
Oxalyticase was obtained from Enzogenetics (Corvallis, OR).
Expression and Reporter Constructs
An EcoRI fragment of mER cDNA clone MOR 100 (a gift
from Dr. M. G. Parker, Imperial Cancer Research Fund, London)
containing the open reading frame was blunt-ended and fitted with
SalI linkers for insertion into the SalI site of
the yeast expression vector PG-1 (33), yielding the mER
expression plasmid PG-MS, as described previously (17). The reporter
plasmid p
SERE (34) containing a single consensus vitellogenin ERE
linked to the LacZ gene was kindly provided by Dr. K. R.
Yamamoto (University of California-San Francisco).
Transformation of Yeast Cells and Site-Directed Mutagenesis
The reporter plasmid was transformed into the protease-deficient
yeast strain BJ2168 (MAT a, prc 1407, prb 11122, pep 43, leu 2,
trp 1, ura 352), a kind gift from Dr. K. R. Yamamoto, by the lithium
acetate method (35) and selected by uracil auxotrophy. The mER mutants
with single amino substitutions in the ligand-binding domain were
created as described previously (17). The mER expression plasmid
containing either a wild type or mutant mER cDNA was then transformed
into the yeast cells, and double transformants containing both reporter
and expression plasmids were selected by tryptophan and uracil
auxotrophy.
Transactivation Assay
A transformed yeast culture was grown overnight at 30 C in
complete minimal dropout medium lacking uracil and tryptophan. The
yeast culture was diluted to OD600 = 0.3 and grown for
2.5 h before the addition of the appropriate compounds. The IB
enantiomers and DES were dissolved in ethanol, and 10 µl of an
individual compound solution were added to 1 ml of yeast culture such
that the concentration of ethanol did not exceed 1% by volume. The
working concentrations used for each compound are shown in the figures.
The yeast cultures were grown for an additional 2.5 h before
harvesting for measurement of ß-galactosidase activity. The yeast
cells were pelleted by centrifugation and resuspended in 1 ml Z buffer
(60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1
mM MgSO4, 40 mM
ß-mercaptoethanol, pH 7.0). An aliquot of the suspension was measured
for cell density at OD600. A 100-µl aliquot of yeast
suspension was then diluted to 1 ml with Z buffer and then treated with
12 µl 0.1% SDS and 15 µl chloroform. The samples were mixed by
vortexing and placed at 30 C for 15 min to permeabilize the cells (36).
The yeast cell suspension was then treated with 200 µl
o-nitrophenyl ß-D-galactopyranoside (4 mg/ml
in 0.1 M KH2PO4, pH 7.0) and
further incubated until chromogenic development. The reactions were
stopped by adding 500 µl of 1 M
Na2CO3 to the samples. Yeast suspensions were
then centrifuged to pellet the cellular debris, and the supernatant was
analyzed by spectrophotometry at OD420 to measure
ß-galactosidase activity (37). The activity induced by the various
compounds was expressed as fold stimulation over the vehicle control
value determined by ethanol induction.
ER-Binding Assay
The transformed yeast cells were grown to an OD600 =
1.0 and harvested. The cells were pelleted by centrifugation and washed
and incubated at 30 C for 30 min in sorbitol buffer (1.2 M
sorbitol, 40 mM potassium phosphate, 20 mM
ß-mercaptoethanol, pH 7.4). The cells were pelleted and then
resuspended in sorbitol buffer containing 15 µg/ml oxalyticase before
incubation at 30 C for 90 min. The spheroplasts were washed with
sorbitol buffer and lysed by hypotonic shock in TE buffer (10
mM Tris-Cl, 1 mM EDTA, pH 7.5). The cytosol was
isolated by centrifugation of the cellular lysate at 5000 x
g for 10 min. The competition binding assay was performed as
described previously using 100 µl cytosol, 7.5 nM
[3H]E2, and increasing concentrations of
unlabeled competitors at 4 C for 18 h (38). A 200-fold excess of
unlabeled DES was used to measure nonspecific binding of the
radiolabeled ligand. Receptor-bound [3H]E2
was isolated by hydroxylapatite adsorption of receptor and quantified
by scintillation counting (38).
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ACKNOWLEDGMENTS
|
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The authors gratefully acknowledge Dr. Lars Pedersen for
constructing the model in Fig. 5
. The helpful discussions with Dr. Beth
Sadler and Dr. Kun Chae and the assistance of Sylvia Curtis are greatly
appreciated. The authors thank Dr. Jeffrey Webster and Dr. Lars
Pedersen for critical reading of the manuscript.
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FOOTNOTES
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Address requests for reprints to: Kenneth S. Korach, Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, NIH, MD B302, P.O. Box 12233, Research Triangle Park, North Carolina 27709.
Received for publication January 23, 1997.
Revision received February 20, 1997.
Accepted for publication February 21, 1997.
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