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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go) 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.

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go) 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 1Go, Fig. 2AGo).


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Table 1. Transcriptional Activity of Wild Type and Mutant mERs

 


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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{Delta}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.

 
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. 2BGo and Table 2Go). 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 1Go and 2Go).


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Table 2. Ligand-Binding Assay of Wild Type and Mutant mERs

 
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. 1Go). 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 1Go). 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 1Go). The V537G mER also bound the compounds with a relative affinity equivalent to wild type mER (Table 2Go).

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 1Go and 3Go and Fig. 3AGo). 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 2Go and Fig. 3BGo). 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 2Go and 3Go). 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 1Go and 3Go and Fig. 4AGo). 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 1Go). The switch in preference for IB-R by H528G mER was accompanied by a minor reduction in IB-S ligand binding affinity (Table 2Go and Fig. 4BGo). There was minimal effect on transactivation induced by DES and DES binding by H528G mER (Tables 2Go and 3Go).


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Table 3. Comparison of Wild Type and Mutant mER Transactivation

 


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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. 3Go.

 


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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. 3Go.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go), 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-{alpha} and holo-retinoic acid receptor-{gamma} 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 {alpha}-helical structure, then the amino acids chosen for mutation would reside on the same face of the {alpha}-helix as demonstrated previously using an {alpha}-helical face map of human ER (hER) residues 515–535, which are analogous to mER residues 519–539 (22). In the mER, His528 and Met532 correspond to residues Arg396 and Leu400 in human retinoic acid receptor-{gamma} 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. 1Go) 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. 5Go). 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. 5Go).



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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.

 
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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{Delta}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 1–407, prb 1–1122, pep 4–3, leu 2, trp 1, ura 3–52), 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).


    ACKNOWLEDGMENTS
 
The authors gratefully acknowledge Dr. Lars Pedersen for constructing the model in Fig. 5Go. 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.


    FOOTNOTES
 
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 B3–02, 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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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