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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
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{at}ncsu.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, ERß (ERßb), and a second ERß, ERßa (formerly ER
) are present in teleost fish. All ERßas 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, -ßb, and -ßa fusion proteins showed specific, high affinity binding to 17ß-[3H]estradiol, with Kd values of 0.61 ± 0.013, 0.40 ± 0.006, and 0.38 ± 0.059 nM, respectively. Rank orders of binding were: diethylstilbestrol >> ICI182780 > 4-hydroxytamoxifen > ICI164384 > estradiol 
zearalenone > moxestrol > tamoxifen > estrone 17-estradiol > estriol > 2-hydroxyestrone = genistein >> RU486 for acER; ICI182780 > diethylstilbestrol > 4-hydroxytamoxifen > estradiol > ICI164384 > genistein > moxestrol > tamoxifen > zearalenone = estrone > estriol = 17-estradiol > 2-hydroxyestrone >> RU486 for acERßb; and estradiol diethylstilbestrol > 4-hydroxytamoxifen > ICI182780 > ICI 164384 > estriol genistein > moxestrol > zearalenone > estrone > 17-estradiol > RU486 tamoxifen > 2-hydroxyestrone for acERßa. acERßa 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 (acERßaPhe396) to the ER or ERßb counterpart shifted diethylstilbestrol and tamoxifen affinities toward those of wild-type acER and acERßb, supporting the hypothesis that the positions with conserved residue changes in teleost ERs are important to ER structure and function. |
Introduction |
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An important characteristic of estrogen-regulated processes is that they are mediated by multiple nuclear ER subtypes. The discovery of a second ER in rats, ERß, transformed the field of ER research by providing additional mechanistic explanations for the pleiotropic effects of estrogens (5). ERß is widely distributed and has since been identified in other mammals, including humans, as well as in birds and fish (6, 7, 8). ERß 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, 10, 11, 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, 14, 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 acERß based on their sequence homology and phylogenetic relationships to previously identified ERs. Because the third subtype was genetically distinct from acER and acERß, 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 ERß early in the teleost lineage after the divergence of ray- and lobe-finned fishes. These three subtypes were subsequently identified in zebrafish (19). The acERß ortholog in zebrafish is now designated ERßb or ESR2b (previously ERß1) to comply with official zebrafish nomenclature rules (20), whereas the acER ortholog is designated ERßa or ESR2a (previously ERß2). The ERßa 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 acERß is renamed acERßb.
The ERßas 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 ERßas (17). These amino acids may have functional significance that distinguish ERßa from ER and ERßb. Several of these diagnostic residues are located near or within regions involved in ligand binding in mammalian ERs (21, 22, 23). Mammalian ER and ERß 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 ERß (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, 27, 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, acERßa, and acERßb 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, ERßa, and ERßb 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 (acERßaPhe396). 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 ERßa and also indicate some important positions to investigate in mammalian estrogen receptors.
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Materials and Methods |
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-ethynyl-2,11ß,17ß-triol-11-methyl ether), 11-ketotestosterone (11KT), estriol (E3), 17ß-estradiol (E2), estrone (E1), 17-estradiol (17E2), and cortisol were purchased from Steraloids (Newport, RI). Testosterone was obtained from Sigma-Aldrich 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; 11ß-(4-dimethylamino)phenyl-17ß-hydroxy-17-(1-propynyl)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, acERßb AAs 317–674, and acERßa 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 µg/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 x 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 x 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 µl/ml lysate). The crude bacterial lysate was then centrifuged (12,000 x 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 µl were incubated with 50 µl 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 µM. 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 dextran-coated charcoal (0.1% dextran and 0.5% charcoal) for 10 min at 4 C, followed by centrifugation (3400 x g at 4 C for 10 min). The supernatant 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 17,20ßDHP, 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 acERßa 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 acERßa 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 ERßs that possess 10 of 11 ERßa diagnostic amino acids in the LBD are considered to be ERßa in our discussion (17).
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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 |
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,ßa,ßb 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, acERßa, and acERßb, respectively (n = 2 or 3). Transformation of the data using Scatchard analysis was linear, indicating a single class of binding sites (Fig. 1).
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. Curves used to calculate IC50 values are shown in Figs. 2–6. In general, the three acER fusion proteins bound steroids and other compounds as expected for ERs, but there were some notable distinctions in their binding profiles.
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and 2.7 nM for acERßa and acERßb (Fig. 2
). DES has a 50-fold greater (relative) affinity than [3H]E2 for acER and a 3-fold greater affinity for acERßb. However, the DES IC50 for acERßa is 2.8 nM, a value equal to or slightly lower than that of E2 for acERßa (Table 1).
The estrogen metabolite E3 had a 2.5- to 5.8-fold greater affinity for acERßa than for acER and acERßb (Fig. 2 and Table 1). In addition, the rank of E3 in the order of ligand preferences was much higher for acERßa. E3 was the 6th best competing ligand for acERßa but the 11th compound for acER and ßb.
E1 had a 10-fold lower affinity than E2 for acER and a 30-fold lower affinity for acERßa and acERßb (Fig. 2 and Table 1). acER also had 2- to 5-fold higher affinities than acERßa and acERßb for 17E2, 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 acERßb 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 acERßa, 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 acERßa and acERßb were both lower than that for E2 with IC50 values of 13.45 and 5.5 nM, respectively. acER had a 7-fold and acERßb had a 3-fold greater relative affinity to the antiestrogen ICI182 than to E2. In contrast, the affinity of ICI182 for acERßa was one third of that for E2, with an IC50 of 7.5 nM (Table 1).
TAM competed for binding to acER and acERßb much better than to acERßa (Fig. 4). TAM was the second worst binder to acERßa (13th out of 14), whereas it was the 8th best competitor for acER and acERßb. TAM bound to acER best, with a 5-fold higher affinity than that to acERßb and a 25-fold higher affinity than that to acERßa, 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 acERßb and acERßa, 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 acERßa and acERßb 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 acERßa 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 acERßb, although there was some displacement at a concentration of 100 µM competitor (Fig. 6). The C19 steroids (testosterone and 11KT) and the C21 steroids (cortisol and 17,20ßDHP) were unable to displace 50% of the [3H]E2 binding at the highest concentration tested (10 µM).
Site-directed mutagenesis
Mutation of acERßaPhe396 to the acERßb residue Ile (acERßaPhe-Ile) decreased the IC50 of DES from 3.8 to 1.2 nM (Fig. 8A). The RBA of DES for acERßaPhe-Ile was 316% of that of acERßa-wild-type (wt). The IC50 of TAM for acERßaPhe-Ile decreased from 310 to 260 nM, and the RBA was 119% that of acERßa-wt (Fig. 8B). Mutation of acERßaPhe396 to the acER residue Met (acERßaPhe-Met) decreased the IC50 of DES from 3.8 to 2.5 nM (Fig. 8A). The RBA of DES for acERßaPhe-Met was 152% that of acERßa-wt. The IC50 of TAM for acERßaPhe-Met decreased from 310 to 220 nM, and the RBA was 141% that of acERßa-wt (Fig. 8B).
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Discussion |
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, acERßb, and previously analyzed ERs. For acER and acERßb, the IC50 values for DES, ICI182, and TOH were 1.5- to 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 acERßa the IC50 values for these compounds were 2.5- to 28-fold higher than those for E2. A particularly striking difference between acERßa and other ERs is acERßa’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 acERßa is only 20% that of E2. This is in contrast to previous findings for ERs of other species and for croaker ER and ERßb, 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 acERßa than it does for acER and acERßb. Both of these compounds have a side-chain at the 7 position on the E2 skeleton, and perhaps this substitution is less tolerated by acERßa.
E3 was the sixth best competitor for acERßa 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 ERßb, where E3 has a higher affinity than E1 (11). However, this receptor does not appear to be an ERßa, as it contains only one of the amino acids (acERßaV317) in the LBD diagnostic of ERßas (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 ERßa subtype.
Interestingly, mifepristone (RU 486) competed well with [3H]E2 for binding to acERßa, 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 acERßa and acERßb arose from a duplication of an ERß 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 acERßa has similar binding affinities to acERßb and mammalian ERßs. For instance, there is less than a 2-fold difference in RBA between acERßa and acERßb for E2, E1, 17E2, 2OHE, MOXE, genistein, and zearalenone. acERßa, acERßb, and human ERß (hERß) have RBAs for genistein greater than those for zearalenone, a feature opposite that of ERs (30). In addition, rat ERß has a 7-fold 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 ERs. 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, acERMet166 is equivalent to hERLeu349 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 ERs identified to date (Table 2).
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Met421 is changed to acERßaPhe396 and acERßbIle426. All but one of the nine identified euteleost ERßas possess a Phe(TTY) at this position (Table 2). The Met to Ile change (ATG-ATH) in croaker ERßb is shared by all but one euteleost ERßbs as well as all tetrapod ERßs, including hERß. 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 ß clades (Anguilla japonica and Danio rerio) have a Leu (CTN or TTR) in the equivalent position (Table 2).
Two additional changes, ERTyr526-acERßaHis495 and hERCys530-acERßaMet499, are conserved in all nine fish ERs that we designate as ßas in Table 2. The hERCys530 position is unchanged in tetrapod ßs, but is changed to Arg in euteleost ßbs. This teleost-specific change differentiates ERßb from the tetrapod ERß as well as from the third teleost subtype, ERßa. 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 hERHis524 (equivalent to acERßaHis493) (22). The additional His at hERTyr526 (acERßaHis495) may help stabilize the extra hydroxyl group of E3, resulting in the increased relative affinity of this endogenous steroid for acERßa. Previous studies have demonstrated that the replacement of hERTyr526 (acERßaHis495) 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 acERßa and acERßb than for acER. This is analogous to findings for hERs, where hERMet421 switches to hERßIle373. 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 acERßb (acERMet238 to acERßbIle426), whereas acERßa 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 the higher affinity of hERß for genistein. As the Phe in acERßa is also nonpolar, it may account for the increased affinity of genistein for acERßa as well.
hER Met421 (acERßaPhe396) 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. acERßa has at least a 10-fold lower affinity for DES, TAM, and TOH compared with acER and acERßb, suggesting that the mutation in the ERßas 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 acERßaPhe396 to the acERßb residue Ile increases the RBA of DES to 316% of that of acERßa (Fig. 8A). This increase accounts for 90% of the difference seen in binding affinity between acERßa and acERßb (Table 1). These data strongly suggest that this position is critical to the interaction of ERs with DES. Alternatively, the change of acERßaPhe396 to the acER amino acid Met increased the RBA of DES to just 150% of acERßa, 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 acERßa 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 ERs have for DES and TAM.
The conserved ERßa residue acERßaMet499 might also alter ligand binding characteristics for DES, TAM, and TOH. Affinity labeling studies suggested that hERCys530 (acERßaMet499) might be involved in binding of the antiestrogen, TAM aziridine (38). However, site-directed mutagenesis and carboxymethylation studies contradict this finding (28, 39). hERCys530 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 (acERßa) 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 ERßa pocket.
acER binds E2 with about a 2-fold weaker affinity than acERßa or acERßb. Zebrafish ER also has nearly a 2-fold weaker affinity for E2 than ERßa, but unlike croaker, ERßb also has a 2-fold weaker affinity for E2 (19). In the rainbow trout ER (and in all other euteleost ERs; Table 2), there is a conservative substitution of hERLeu349 to a Met(acERMet166). 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 17E2, E1, and 2OHE better than acERßa and acERßb, albeit binding is low for all three compounds compared with that for E2. It is possible that acERMet166 may play a role in these differences as well, but relative affinities to other estrogens in the reciprocal mutagenesis system were not evaluated (26).
hERGlu353 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 acERßa 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 ß 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 acERßa is universal to all ERßas, and consequently which, if any of the conserved amino acids are responsible for this difference.
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 ERß, 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 ERßas, 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, acERßaPhe396 to the corresponding ER (Met) or ERßb (Ile) amino acid, shifted the binding affinities for DES and TAM toward those for the corresponding wt acER and acERßb. 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 ERßa 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 ERß 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.
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Acknowledgments |
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Footnotes |
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Abbreviations: ac, Atlantic croaker; DES, diethylstilbestrol; 17,20ßDHP, 17,20ß-dihydroxyprogesterone; E1, estrone; E2, 17ß-estradiol; 17E2, 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, 2-hydroxyestrone; RBA, relative binding affinity; TAM, tamoxifen; TOH, 4-hydroxytamoxifen; wt, wild-type.
Received June 27, 2003.
Accepted for publication February 17, 2004.
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