Ligands Specify Coactivator Nuclear Receptor (NR) Box Affinity for Estrogen Receptor Subtypes

Kelli S. Bramlett, Yifei Wu and Thomas P. Burris

Gene Regulation, Bone, and Inflammation Research Lilly Research Laboratories Lilly Corporate Center Indianapolis, Indiana 46285


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors (NRs) require coactivators to efficiently activate transcription of their target genes. Many coactivators including the p160 proteins utilize a short NR box motif to recognize the ligand-binding domain of the NR when it is activated by ligand. To investigate the ability of various ligands to specify the affinity of NR boxes for a ligand-bound NR, we compared the capacity of p160 NR boxes to be recruited to estrogen receptor (ER{alpha}) and ERß in the presence of 17ß-estradiol, diethylstilbestrol, and genestein. A time-resolved fluorescence-based binding assay was used to determine the dissociation constants for the 10 NR boxes derived from the three p160 coactivators for both ER subtypes in the presence of the each of the agonists. While the affinity of some NR boxes for ER was independent of the agonist, we identified several NR boxes that had significantly different affinities for ER depending on which agonist was bound to the receptor. Therefore, an agonist may specify the affinity of an NR for various NR boxes and thus regulate the coactivator selectivity of the receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor (NR) superfamily includes the receptors for the thyroid hormones, steroid hormones, as well as for lipophilic vitamins such as vitamin A and D. NRs are generally characterized as ligand-activated transcription factors; however, orphan receptors, some of which may not require a ligand for function, comprise a significant portion of the superfamily membership. These receptors regulate diverse physiological functions such as differentiation, development, homeostasis, and behavior in organisms ranging from nematodes to humans. Primarily functioning as either homodimers, as in the case with the steroid receptors, or heterodimers with the retinoid X receptor (RXR), NRs bind to specific DNA sequences in the regulatory regions of their target genes and regulate their transcription. In the case of the steroid receptors (class I receptors), ligand regulates their ability to dimerize and recognize the DNA response element. Ligand binding is also responsible for inducing a conformational change that leads to transcriptional activation of the target gene. Class II receptors that require RXR for function are believed to bind constitutively to their response elements as an RXR heterodimer. Some class II receptors actively silence transcription of their target genes in the absence of ligand, but similar to the steroid receptors, ligand causes a conformational alteration that leads to transcriptional activation (1, 2, 3, 4).

The conformational change in the ligand binding domain (LBD) of the NR in response to binding of hormone is responsible for recruitment of coactivator proteins that are required for transcriptional activation by the receptor (5, 6, 7). Members of the p160-steroid receptor coactivator (SRC) family of coactivators were originally characterized by biochemical methods in which proteins were identified that bound the estrogen receptor (ER) in a hormone-dependent fashion (8). Steroid receptor coactivator-1 (SRC-1) was cloned using the progesterone receptor LBD as bait in a yeast two-hybrid screen and was subsequently shown to be a general coactivator for the NR superfamily (9). Three proteins have been identified in the p160 coactivator family and include: 1) SRC-1/NCoA-1, 2) SRC-2/TIF-2/NCoA-2, and 3) SRC-3/p/CIP/AIB-1/TRAM-1/RAC-3/ACTR (10, 11, 12, 13, 14, 15, 16, 17, 18). The p160 proteins contain a short conserved NR interaction motif (NR box), which has the core sequence LXXLL (L = leucine and X = any amino acid) (17, 19). All of the p160s have a core NR interaction domain that contains three of these NR boxes arranged in tandem. A unique fourth NR box has been characterized at the extreme carboxy terminus of an alternatively spliced variant of SRC-1, SRC-1a (20, 21). NR boxes have also been identified in other classes of coactivator molecules including p300/CBP, TRAP 220/DRIP 205/PBP, PGC-1, and TRBP/ASC-2/RAP250 (22, 23, 24, 25, 26, 27, 28). Crystal structures of agonist-bound NRs complexed to the NR boxes have yielded considerable information about the mechanism of recognition. The tertiary structure of the LBD of NRs is well conserved and has been described as a three-layered sandwich composed of 12 {alpha}-helices with the ligand-binding pocket buried inside the globular structure (29, 30, 31). Ligand binding results in a conformational change with the most significant alteration occurring in the carboxy-terminal helix (helix 12; H12) that contains the AF-2 transactivation domain. H12 rotates from an exposed position directed away from the LBD of the apo-receptor to a position in which it is folded back along the surface of the receptor where it serves to seal the ligand-binding cavity of the receptor (29, 30, 31). This conformational change creates a groove on the LBD that serves as the surface for the {alpha}-helical LXXLL core of the NR box to recognize the agonist-bound receptor (32, 33, 34, 35, 36). The hydrophobic face of the LXXLL helical domain of the NR box makes direct contact with the nonpolar groove on the LBD that is created after binding of the ligand. The side chains of two highly conserved residues within the LBD, a glutamic acid residue within H12 and a lysine residue near the carboxy-terminus of H3, make specific contacts with the backbone amides and carbonyls of amino acids within the LXXLL helix and serve to form a charge clamp that specifies the acceptable size of the LXXLL {alpha}-helix and orientation by which it binds within the hydrophobic groove of the LBD (33, 34, 35, 36). The hydrophobic side chains of the leucine residues of the LXXLL helix face the hydrophobic groove while the side chains of the X residues are solvent exposed; thus little if any specificity is directed by the small LXXLL region. However, residues flanking this sequence on both the amino and carboxy termini also make significant contacts with the surface of the LBD (33, 34, 35) and have been shown to be important for determining preferential binding of various NR boxes to particular NRs, which may lead to NR preference for certain coactivators (17, 18, 35, 36, 37, 38, 39, 40, 41, 42, 43). Different ligands for a particular NR may also alter specificity for coactivator binding (43, 44), due to conformational differences in the LBD. These differences in the tertiary structure of the LBD due to the binding of different ligands are exemplified by a number of structures that have been solved with estrogen receptor (ER) isoforms bound to agonists such as 17ß-estradiol (E2), diethylstilbestrol (DES), genestein (GEN), and selective ER modulators (SERMs) such as tamoxifen and raloxifene (34, 45, 46, 47). Both of the agonists, E2 and DES, produce a similar conformational change in the LBD of ER resulting in the formation of the hydrophobic groove that is available for recognition by the NR box (34). Binding of the SERMs results in unique positioning of H12 so that this helix actually obstructs the groove by mimicking the LXXLL domain with the sequence LXXML from H12 of the LBD, which is conserved in all NRs (34, 45). Thus, the groove is unavailable for docking of the coactivator consistent with the SERMs functioning as AF-2 antagonists. GEN, a partial agonist, induces novel positioning of H12 that also obstructs the hydrophobic groove (46) suggesting that there may be some interference with NR box binding.

To investigate the ability of various ligands to specify the affinity of NR boxes for ligand-bound receptor, we compared the capacity of p160 coactivator NR boxes to be recruited to ER{alpha} and ERß in the presence of E2, DES, and GEN. Using a time-resolved fluorescence-based binding assay, we determined the dissociation constants for several natural NR boxes for both ER isoforms in the presence of the three agonists. The affinities of several NR boxes were specified by the ligand, while others remained constant independent of the nature of the ligand. In addition, we identified several NR boxes that had significant ER isoform preference, and, interestingly, the box that demonstrated one of the highest degrees of isoform preference is the extreme carboxy-terminal box of SRC-1, which is localized to a region that is present in an alternatively spliced variant of this coactivator.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-Dependent NR Box Recruitment
SRC/p160 proteins interact directly with members of the NR superfamily and act to potentiate the transcriptional activity of these transcription factors. Interaction with the NRs is mediated by short motifs known as NR boxes that contain a conserved core LXXLL sequence. Three members of the SRC/p160 coactivator family have been identified, and they contain three copies of an NR box arranged in tandem within a central NR interaction domain. An alternatively spliced variant of SRC-1 contains an additional NR box in its extreme carboxy terminus (Fig. 1AGo). Although the core LXXLL sequence is conserved, the amino acid residues flanking this sequence are not conserved and are believed to play a role in NR box recognition of selective NRs (17, 18, 35, 36, 37, 38, 39, 40, 41, 42, 43). Thus, the sequences immediately flanking the core LXXLL sequence may specify a coactivator’s affinity for a particular NR. It has been demonstrated that various ligands for an NR such as ER induce novel conformations within the LBD (34, 45, 46, 47, 48); therefore, we sought to determine whether distinct ligands had the ability to specify the affinity of various NR boxes for a particular NR. Using both ER{alpha} and ERß, we examined both receptor subtype selectivity and ligand selectivity in terms of the receptors’ ability to bind to NR boxes from the p160 coactivator family. We used a time-resolved fluorescent technique to detect binding of NR box peptides (Fig. 1BGo) to recombinant human ER{alpha} or ERß.



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Figure 1. NR Boxes of p160/SRC Family Proteins

A, The localization and nomenclature of the NR boxes of SRC-1, -2, and -3. B, Sequence alignment of the NR boxes of the p160/SRC family of coactivators. The leucines within the conserved LXXLL domain are boxed.

 
NR box II of SRC-1 was used in our initial validation of the NR box binding assay. As shown in Fig. 2Go, E2 dose-dependently induced SRC-1 NR box II peptide binding to both ER{alpha} and ERß. The potency of E2 was similar for both receptors with an EC50 of 3.1 nM for ER{alpha} and 3.0 nM for ERß. As expected, the antiestrogen, ICI 182,780, blocked the binding of the NR box to both ER subtypes. Interestingly, ERß, but not ER{alpha}, exhibited significant ligand-independent binding to the SRC-1 NR II peptide. The ligand-independent portion of the binding could be blocked by the ER antagonist ICI 182,780 (Fig. 2BGo) and by unlabeled SRC-1 NR box II peptide (data not shown), indicating that in the unliganded state, ERß retains an active conformation. Examination of greater than 20 different NR boxes confirmed that ERß retains a basal active conformation while ER{alpha} does not (data not shown).



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Figure 2. Validation of the Time-Resolved Fluorescence NR Box Peptide Binding Assay

A, The europium-labeled NR box peptide, LTERHKILHRLLQE, based on the NR box II of SRC-1 binds in an E2 dose-dependent manner to human ER{alpha}. The antiestrogen, ICI 182,780 (100 nM), efficiently antagonizes the peptide interaction. B, The NR box peptide also binds to human ERß in an estrogen-dependent fashion, and the binding of the peptide is blocked by addition of 100 nM ICI 182,780. ICI 182,780 was also able to block basal binding of the NR box peptide to ERß. C, Unlabeled wild-type NR box peptide competes for binding of the labeled peptide to ER{alpha} in the presence of 1,000 nM E2 in a dose-dependent manner. An NR box peptide with two mutations within the LXXLL domain, AXXLA, does not compete for binding. D, Unlabeled NR box peptide also competes for binding of the labeled NR box peptide to ERß in a dose-dependent manner. Although the mutant AXXLA peptide retains some ability to displace the LXXLL peptide, its binding affinity is significantly decreased. Representative experiments are shown.

 
The specificity of binding of the SRC-1 NR box II peptide was examined by competing the binding of the labeled peptide to E2-bound ER with either unlabeled wild-type SRC-1 NR box II peptide or a mutant peptide in which two of the leucine residues were mutated to alanines. Crystal structures of several NRs bound to NR boxes indicate that the hydrophobic surface of the LXXLL {alpha}-helix created by the leucine side chains make van der Waals contacts with the hydrophobic coactivator-binding groove in the LBD surface. Mutation of the leucines within the LXXLL core sequence has been shown to have a detrimental effect on the binding of the NR box or the entire coactivator protein to an NR (17, 19, 35, 38, 39, 41, 43, 49). Unlabeled wild-type SRC-1 NR II peptide (LXXLL) dose-dependently competed with labeled SRC-1 NR box II peptide for binding to E2-bound ER{alpha} and ERß (Fig. 2Go, C and D). Unlabeled mutant SRC-1 NR II peptide (AXXLA) was unable to compete for labeled SRC-1 NR box II peptide binding to E2-bound ER{alpha}; however, weak dose-dependent competition was detected with E2-bound ERß, indicating that ERß is more tolerant of these mutations in the LXXLL core sequence than ER{alpha}. The IC50 values for the 14-amino acid SRC-1 NR II peptide (LTERHKILHRLLQE) for both ER{alpha} and ERß were in the 1–4 µM range; however, we subsequently determined that NR box peptides with additional carboxy-terminal residues displayed a significant increase (10-fold) in affinity and were subsequently used in additional experiments (Fig. 1BGo).

Differential Recruitment of p160 Coactivator NR Boxes by ER Subtypes
To characterize the NR box preferences for ER subtypes, we first examined E2-dependent recruitment of each of the 10 NR boxes from the p160 coactivator family (Fig. 1BGo). ER{alpha} and ERß displayed unambiguous NR box preferences, and, interestingly, the two ER subtypes exhibited significant differences in their NR box selectivity (Fig. 3Go). For the SRC-1 NR boxes, ER{alpha} had a clear preference for SRC-1 NR box II with some recruitment detected for SRC-1 NR box IV. Recruitment of SRC-1 NR box I and SRC-1 NR box III by ER{alpha} was not detected. ERß had a much different pattern of selectivity with a very strong preference for SRC-1 NR box IV followed by SRC-1 NR box II with SRC-1 NR boxes I and III showing minimal recruitment. The selectivity of the ER subtypes for SRC-1 NR box IV may be significant since the fourth box is only expressed in an alternatively spliced variant of SRC-1, SRC-1a. Differences between ER{alpha} and ERß were also detected with respect to their selectivity for SRC-2 NR boxes. ER{alpha} displayed a clear preference for SRC-2 NR box II followed by SRC-2 NR box I and no detectable binding to SRC-2 NR box III. In contrast, ERß recruited all three SRC-2 NR boxes with nearly equal efficacy. The rank order of recruitment of SRC-3 NR boxes was identical for both ER{alpha} and ERß (NR box I>NR box 2>NR box 3), with the only difference being that ERß displayed some binding to SRC-3 NR box III while ER{alpha} did not. Examination of the average E2 EC50 values for peptides that were significantly recruited revealed an approximate 6-fold selectivity for ER{alpha} vs. ERß (3 ± 0.2 nM vs. 18 ± 1 nM). This degree of selectivity has been previously reported for E2 in both radioligand binding assays and in cell lines either transiently or stably expressing ER subtypes (50, 51, 52). Since the NR box interaction assay is a functional assay as it detects the recruitment of an NR interaction domain of a coactivator in response to a ligand, we would expect that the behavior of ligands would be similar to a cell-based assay rather than a ligand-binding assay that typically only detects displacement of a radioligand. The potency of E2 appears to be lower in our NR box interaction assay than according to previously reported binding affinity data (Kd = 0.2–0.6 nM) (50, 53, 54, 55) and some cell-based reporter assays (51, 56); however, the potency of E2 in our assay is comparable to reported transient cotransfection assays (50, 52) and to values that we currently obtain in cell-based assays that assess endogenously expressed ER. Since we are detecting the binding of an NR box peptide to ligand-bound receptor, the potencies may not necessarily correlate with the ability of a particular ligand to displace a radioligand or activate transcription in a cell-based reporter assay. This is especially true for the cell-based reporter assays since they assess the transcriptional activity of the receptor, combining the activities of both AF-1 and AF-2 and all of the coactivators that they may recruit, whereas the current study examined only the ability of the receptors to bind to NR boxes from the p160 coactivators.



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Figure 3. ER{alpha} Selectively Recruits a Subset of SRC NR Boxes That Is Distinct from That Recruited by ERß

A, E2-bound ER{alpha} displays SRC NR box binding selectivity. ER{alpha} has a clear preference for NR box II of SRC-1 and SRC-2; whereas NR box I of SRC-3 is preferred over NR box II of SRC-3. B, In contrast to ER{alpha}, ERß prefers NR box IV of SRC-1 over NR box II and interesting no selectivity was displayed for the SRC-2 NR boxes while the selectivity profile for SRC-3 remains unchanged. A representative of three independent experiments is shown.

 
p160 NR Boxes Have Different Affinities for ER{alpha} and ERß
The Kd values of each of the p160 NR boxes were determined for both E2-bound ER{alpha} and ERß. Saturation binding assays were conducted in which receptor was treated with a dose of E2 that would allow maximal NR box binding followed by titration of labeled NR box until saturation. With few exceptions, the preferences of the ER subtypes for the NR boxes based on the Kd values matched well with the selectivity described above based on the E2-dependent recruitment. The exceptions are attributable to differences in maximal binding (efficacy) of various NR box peptides that we detected in our saturation binding assays and are reflected as the maximal efficacy in ligand-dependent NR box recruitment assays described above (Fig. 3Go). Alterations in the conformation of the NR box binding groove of the LBD may not only affect the affinity of the NR box, but also the relative binding capacity of the site. Thus, discrepancies between selectivity based on affinity vs. efficacy may be due to the creation of a high-affinity, low-capacity binding groove vs. a low-affinity, high-capacity binding groove for a particular NR box. Of the SRC-1 NR boxes, SRC-1 NR box II has the highest affinity for ER{alpha} (Kd = 155 ± 21 nM) followed by SRC-1 NR box IV (Kd = 934 ± 259 nM) (Table 1Go). SRC-1 NR box I and NR box III displayed no detectable binding to ER{alpha}. Both SRC-1 NR box II and NR box IV display similar affinities to ERß (Kd = 204 ± 27 nM and Kd = 261 ± 75 nM, respectively) in contrast to the large degree of selectivity shown for NR box IV in the ligand-dependent recruitment assay (Table 2Go). Unlike ER{alpha}, both SRC-1 NR box I and NR box III showed weak but detectable affinity for ERß (Kd = 1,025 ± 190 nM and Kd = 2,060 ± 214 nM, respectively). The significant ER subtype selectivity for SRC-1 NR box IV identified above is also apparent when examining the dissociation constants (ER{alpha}, Kd = 934 ± 259 nM vs. ERß, Kd = 261 ± 75 nM); however, differences in the maximal binding of this NR box for the receptors amplifies the differential selectivity. The dissociation constants for the SRC-2 NR boxes retain the identical rank order as described above for ER{alpha} (NR box II>NR box I, NR box III no binding), but ERß shows some preference for SRC-2 NR box I above SRC-2 NR boxes II and III. SRC-3 NR boxes I and II have similar high affinities for ER{alpha} (Kd = 216 ± 26 nM and 182 ± 23 nM, respectively) while SRC-3 NR box III does not bind. In contrast, SRC-3 NR box III does bind to ERß (Kd = 849 ± 147 nM); however, both SRC-3 NR box I and NR box II display greater affinities (Kd = 136 ± 26 nM and 443 ± 97 nM, respectively). Northrop et al. (42) have also examined the relative affinity of p160 NR boxes for ERß using a competition scintillation proximity assay (42). In terms of the rank order of potency, our results for ERß compare favorably with this study with a few exceptions. We detected weaker binding of several boxes, including NR boxes II and III from SRC-2 and NR box III of SRC-3, than reported by Northrop et al. (42). These discrepancies may be due to differences in the techniques used to quantitate NR box affinity. We measured the dissociation constant of each individually labeled NR box peptide to ERß, while Northrop et al. (42) assessed the ability of these NR box peptides fused to the maltose binding protein (MBP) to compete for binding of a radiolabeled MBP-SRC-1 NR interaction domain fusion consisting of the three central NR boxes to ERß.


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Table 1. Dissociation Constants (Kd) and Relative Binding Affinities (RBA) for p160 NR Boxes for ER{alpha}

 

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Table 2. Dissociation Constants (Kd) and Relative Binding Affinities (RBA) for p160 NR Boxes for ERß

 
Based on the dissociation constants, several NR boxes that display ER subtype selectivity when bound to E2 were identified (Table 3Go). The SRC-1 NR box IV box displays a high degree of selectivity for ERß over ER{alpha} (~3.6-fold), which may be an underestimate of the degree of functional selectivity since there is a large difference in the maximal binding of this NR box to the two receptors (Fig. 3Go). Several other NR boxes also appear to have a preference for ERß over ER{alpha} including SRC-2 NR box I, SRC-2 NR box III, and SRC-3 NR box III (Table 3Go). While SRC-2 NR box I has a 2.4-fold selectivity for ERß, both SRC-2 NR box III and SRC-3 NR box III bind reasonably well to ERß, but have no detectable affinity for ER{alpha}. Only two NR boxes were identified with ER{alpha} selectivity: SRC-2 NR II was 4.4-fold selective and SRC-3 NR II was 2.4-fold selective.


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Table 3. ER Subtype Specificity of p160 NR Boxes

 
Ligand-Mediated Selective Recruitment of NR Boxes
To determine whether different ER ligands could affect the ability of the receptor to recruit different NR box peptides, we compared the NR box binding pattern of both ER{alpha} and ERß when bound to E2, DES, or GEN. These three ligands were chosen for several reasons. They display structural diversity with E2 representing a steroid, while both DES and GEN are nonsteroidal compounds (Fig. 4Go). While E2 and DES display a slight ER{alpha} subtype preference, GEN has been characterized as having some degree of ERß specificity in both receptor binding and cell-based assays (50, 51, 52, 57). All three ligands display full agonist activity for ER{alpha}; however, GEN is only a partial agonist for ERß while both E2 and DES are full agonists (51). Thus, these ligands exhibit several distinct pharmacological properties when bound to ER that may be mediated by selectivity in NR box recruitment.



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Figure 4. Chemical Structure of Three ER Agonists Used in the Study

The structures of E2, DES, and GEN are illustrated.

 
Similar to E2 (Fig. 3Go), both DES and GEN recruited a subset of NR box peptides to both ER{alpha} and ERß in a dose-dependent manner (data not shown). A comparison of the effect of ligand on the maximal binding efficacies for each of the NR box peptides to ER{alpha} and ERß is shown in Fig. 5Go. Although the general pattern of NR box preference for E2 and DES was very similar to that for ER{alpha}, significant differences were noted with GEN. Both NR box I and II of SRC-3 are significantly recruited to E2- and DES-bound ER{alpha}; in fact, SRC-3 NR box I has the highest degree of efficacy for all of the NR boxes bound to ER{alpha} for both E2 and DES. In contrast, GEN-bound ER{alpha} recruits SRC-3 NR box I with less than half of the efficacy of either E2- or DES-bound ER{alpha}, and SRC-3 NR box II is not recruited at all (Fig. 5AGo). More subtle differences are noted for SRC-2 NR boxes, where NR box I has a low but significant level of recruitment to E2- and DES-bound ER{alpha}, but no binding to GEN-bound ER{alpha}. SRC-2 NR box II also displays reduced maximal binding to GEN-bound ER{alpha} relative to both E2- and DES-bound receptor (Fig. 5AGo). For ERß, the major differences between the ligands were again within the SRC-3 NR boxes. Binding of SRC-3 NR box I to ERß showed a great deal of dependence on the identity of the ligand (Fig. 5BGo). DES induced the greatest amount of binding of ERß to SRC-3 NR box I, which was approximately 1.8-fold greater than GEN-bound receptor and 3-fold greater than E2-bound receptor (Fig. 5BGo). These data clearly indicate that ligands can specify which NR boxes are recruited to the receptor as is the case with SRC-3 NR box II or the degree to which a NR box is recruited to the receptor.



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Figure 5. Ligands Specify the NR Box Binding Profile of the ERs

A, Maximal binding (1 µM of compound) of each of the NR box peptides from the SRC proteins for ER{alpha} is indicated. Differences in the ability of the ligands to induce recruitment of NR box peptides are readily apparent especially in the peptides derived from SRC-3. B, Maximal binding (1 µM of compound) of each of the NR box peptides from the SRC proteins for ERß is indicated. Like ER{alpha}, there are major differences in the ability of the ligands to induce recruitment of NR box I of SRC-3 to the receptor. A representative experiment is shown. The bars represent the mean of three individual determinations. Although the SEM is not indicated, they were typically less than 5% of the mean.

 
For each NR box that was significantly recruited to receptor, EC50 values were calculated and averaged to determine the relative potency of the agonists. As predicted from previous studies, GEN showed significant ERß selectivity with an EC50 of 471 ± 83 for ER{alpha} and 122 ± 53 nM for ERß (51, 57). DES also showed selectivity for ERß with an EC50 of 43.0 ± 18.5 nM for ER{alpha} and 7.1 ± 1.6 nM for ERß. This is in contrast to other studies that demonstrate that DES has a slight preference for ER{alpha} over ERß in radioligand binding and cell-based reporter assays (51, 57). The number of NR boxes recruited by agonist-bound receptor was also different when comparing either the receptor subtype or the agonist. For example, GEN-bound ERß was more efficient in recruiting a greater number of NR boxes than GEN-bound ER{alpha} (Fig. 5Go). In terms of agonist specificity, DES- and E2-bound ER{alpha} recruited more NR boxes than GEN-bound ER{alpha}.

Although we cannot make direct comparisons of maximal binding of NR boxes between ER{alpha} and ERß due to differences in specific activity of the receptors, some conclusions can be made based on the rank order of preference of the various NR boxes between the two receptors. The most interesting difference occurs in SRC-1 where NR box IV recruitment by ER{alpha} is not impressive for any of the ligands; however, all three ligands induce very strong binding of NR box IV to ERß (Fig. 5Go). With the SRC-2 NR boxes, ER{alpha} prefers NR box II when bound to all three of the ligands, while ERß binds to each of the SRC-2 NR boxes with no overt preference. The rank order preference for binding to the SRC-3 NR boxes (NR box I>NR box II>NR box III) is conserved for all three ligands bound to both ER subtypes with the exception of the inability of GEN-bound ER{alpha} to recruit SRC-3 NR box II.

Ligands Alter ER Affinity for p160 NR Boxes
It is possible that different ER agonists may cause distinct conformations in the region of the LBD that interacts with the coactivator NR box, thus altering affinity for the receptor. To examine this possibility, we determined the dissociation constants for each of the NR boxes bound to both ER{alpha} and ERß in the presence of E2, DES, and GEN. The affinities of the NR boxes to ER{alpha} are shown in Table 1Go. Similar to efficacy data, SRC-1 NR boxes I and III did not have detectable affinity for ER{alpha} when bound to any of the agonists. SRC-1 NR box II, however, bound well to ER{alpha} with the highest affinity in the presence of E2. DES- and GEN-bound ER{alpha} also had affinity for this box, but with reduced relative binding affinity (27, 28, 29, 30, 31, 32, 33, 34, 35). Interestingly, whereas E2-bound ER{alpha} had detectable affinity for SRC-2 NR box I, we could not detect significant binding to DES- or GEN-bound ER{alpha}. Selectivity for E2-bound ER{alpha} was also apparent for NR box II from both SRC-2 and SRC-3. On the other hand, SRC-3 NR I had similar affinities to E2- and DES-bound ER{alpha} with reduced affinity to GEN-bound receptor.

Whereas many of the NR boxes were selective for E2-bound receptor when evaluating ER{alpha}, the opposite appears to be the case with ERß with most of the NR boxes having greater affinity for DES- and GEN-bound receptor (Table 2Go). NR boxes II and III from both SRC-2 and SRC-3 have greater affinities for DES- and GEN-bound ERß than E2-bound receptor. SRC-1 NR box IV and SRC-3 NR box I have similar affinities for ERß regardless of the identity of the agonist. SRC-1 NR box II appears to be somewhat preferential to GEN-bound ERß, while NR box I from both SRC-1 and SRC-2 prefer E2- and DES-bound receptor over GEN-bound receptor. These data illustrate that the nature of the agonist bound to ER can play an important role in specifying the affinity of the LBD for the NR box.

Determination of the dissociation constants for each of the NR boxes for both ER{alpha} and ERß allows for direct comparison of the affinities of the boxes between the two receptors. Table 3Go shows the degree of receptor specificity for each of the p160 NR boxes with each agonist. As described above, considerable receptor selectivity was noted when E2 was used as the agonist. Interestingly, the identity of the agonist also appears to play a significant role in ER subtype selectivity of particular NR boxes. For instance, SRC-2 NR box II has a preference for ER{alpha} (4.39-fold) when E2 is the agonist, but this selectivity reverses when DES is used (4.2-fold ERß selective). For this particular box, GEN shows only a slight preference for ERß (1.8-fold). SRC-1 NR box II and SRC-3 NR box I showed no subtype selectivity with E2 but were ERß selective when either DES or GEN were used. Several NR boxes had preference for ERß independent of the identity of the agonists such as SRC-1 NR box IV and SRC-3 NR box III. None of the p160 coactivator boxes were ER{alpha} selective with all three agonists. Thus, depending on the agonist there may be selectivity for ER{alpha} or ERß or relative nonselectivity even when examining a single NR box.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coactivators are responsible for mediating the transcriptional activity of NRs. Since the cloning of the first NR coactivator, SRC-1, in 1995, a large number of additional coactivator proteins have been identified and characterized (5, 6, 7). It is unclear why such a large number are required for NR action; however, the SRC-1 and SRC-2 knock-out mice demonstrate that distinct NR coactivators may have specific physiological functions based on the different phenotypes that these mice exhibit (58, 59, 60). This may be due to the preferences that specific NRs exhibit for particular coactivators (18, 38, 61, 62, 63). Many coactivators utilize a short motif known as an NR box to bind to NRs. Coactivators may contain a single NR box such as PGC or multiple NR boxes as is the case for the p160 coactivators. Several investigators have demonstrated that various NR boxes display receptor selectivity, which provides a mechanism that coactivator proteins may utilize to discriminate between receptors (17, 36, 37, 38, 39, 41, 43, 64). For example, ER{alpha} has been shown to require an intact NR box II of SRC-1 for efficient coactivation, while the retinoic acid receptor required both NR boxes II and III for normal function (17, 36, 37, 43). In contrast, the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) and progesterone receptor required NR boxes I and II of SRC-1 (43). In the current study, we examined the ability of the NR boxes of the p160 coactivator family to be recruited to both ER{alpha} and ERß in a ligand-dependent manner. We also directly measured the dissociation constants for each of these NR boxes to both receptors. Consistent with previous studies indicating that NR box II of SRC-1 was required for ER{alpha}, our data demonstrate that ER{alpha} has very high affinity for this box relative to the other NR boxes of SRC-1. NR box II of SRC-2 has also been shown to be necessary for efficient interaction with ER{alpha} (41), which is also in agreement with the high affinity of this NR box we detected. Our data predict that for SRC-3, NR box I and NR box II will play an important role for coactivation of ER{alpha}. Interestingly, the NR box selectivity changes significantly for ERß. For SRC-1, NR box IV is dominant, but NR box II still retains significant activity. All of the SRC-2 NR boxes have a similar profile with none of the NR boxes predominating while the NR boxes of SRC-3 retain a similar rank order as they did for ER{alpha}. These NR box preferences may underlie the ability of various NRs to exhibit some degree of coactivator selectivity.

In addition, the differences in NR box preferences that we noted between ER subtypes suggest that coactivator selectivity may play a role in the differential actions of these receptors. One of the more significant differences we noted was the selectivity of SRC-1 NR box IV for ERß. Since NR box IV is only contained in the longer splice variant of SRC-1 (SRC-1a), ERß may preferentially interact with SRC-1a over SRC-1e. Thus, in tissues that express both ER subtypes and greater amounts of SRC-1a than SRC-1e, ERß may display dominance. ERß has been shown to modulate the transcriptional activity of ER{alpha}, and it was proposed that the relative expression levels of these ER isoforms may regulate the level of transcriptional response to ER ligands (65). Based on this observation, our data suggest that the SRC-1a/SRC-1e expression ratio may also be important for determining the level of transcriptional response to ER ligands by modulation of the transactivation potential of ERß; however, additional investigation will be required in the context of the full-length coactivators. Differential expression of the SRC-1 splice variants has been reported in the brain (66), and interestingly, regions that predominately express the SRC-1a isoform, such as the paraventricular nucleus of the hypothalamus, also predominately express the ERß subtype (67, 68). Variation in the ability of the SRC-1 isoforms to potentiate ER{alpha} activity has been previously reported; however, the differences were due to suppression of an activation domain in SRC-1a (22).

Various ligands for a particular NR are believed to have the ability to induce novel conformations within the LBD of the receptor, which was exemplified by recent reports of the crystal structure of ER bound to different ligands. The role that the identity of the ligand plays in the conformation of the LBD of ER is particularly apparent when one examines the position of H12. Whereas agonist binding, such as E2 or DES, places H12 on the surface of the LBD capping the binding pocket with the coactivator binding groove exposed, SERMs such as tamoxifen and raloxifene do not allow H12 to cap the binding pocket due to steric hindrance, and H12 blocks the hydrophobic groove by mimicking the NR box LXXLL motif (45). Binding of GEN, a partial ERß agonist, to the receptor results in suboptimal positioning of H12 along the NR-box binding hydrophobic groove similar to that found in the SERM structures (46). In this study, we examined the ability of E2-, DES-, and GEN-bound ER{alpha} and ERß to interact with various NR boxes from the p160 proteins. Our data indicate that the identity of the agonist plays an essential role in determining the relative binding efficacy and potency of the p160 family NR boxes to ER{alpha} and ERß. Due to these ligand-dependent alterations in the NR box binding activity, an agonist is able to determine the relative selectivity of several of the p160 NR boxes for the ER isoforms. It is interesting to note that although the crystal structure of ERß with GEN indicates that H12 binds along the NR box binding groove on the surface of the LBD (46), our data indicate that GEN-bound ERß binds very well to the majority of the p160 NR boxes. A SERM such as raloxifene sterically restricts positioning of H12 in the antagonist position due to the protrusion of the piperidine ring of the drug from the ligand binding cavity (45); however, GEN does not sterically hinder positioning of H12 possibly allowing for more flexibility in its positioning and possible displacement by an NR box. Recent two-hybrid data confirm that GEN-bound ERß binds to both p160 and nonnatural phage display-identified NR boxes (69). Similar to our data with the p160 NR boxes, these investigators demonstrate that ERß displays differences in its ability to recruit several of the nonnatural NR boxes depending on whether it is bound to E2 or GEN (69).

Coactivation is more complex than simple NR box peptide binding to the NR. NR boxes often exist in tandem copies within the coactivator polypeptide sequence, and it has been demonstrated that a polypeptide containing multiple NR boxes exhibits higher affinity for an NR dimer than a single NR box presumably due to cooperative binding (33, 42). We sought to characterize the variations in the conformation of the coactivator binding groove specified by various ER ligands as detected by selective NR box binding, and since the cooperative binding of the multimerized NR boxes could potentially interfere with this examination, we chose to characterize the binding of single NR box peptides. Our studies also do not address coactivation that is independent of NR box binding such as AF-1-dependent coactivation. However, our methods allow for sensitive characterization of a very specific event in the activation of ER, which is the creation of a NR box binding surface on the LBD in response to a ligand.

Consistent with our data indicating that a ligand can specify the affinity of the NR for an NR box, McInerney et al. (43) demonstrated that the identity of the agonist subtly altered the requirement for NR box I within SRC-1 for efficient coactivation of PPAR{gamma} . These data suggest that NR box selectivity designated by particular ligands may allow NRs to preferentially bind specific coactivators. Indeed, a role for ligand in coactivator specificity has been shown for both PPAR{gamma} and the vitamin D receptor (VDR). While the natural PPAR{gamma} agonist, 15-deoxy-{Delta}12,14 prostaglandin J2 (15d-PGJ2) induced binding of several classes of NR box-containing coactivators including TRAP 220/DRIP 205, p300, and all three p160s, the synthetic agonist, troglitazone, was unable to induce recruitment of any of these proteins (70). Consistent with this finding, SRC-1 and SRC-2 coactivated PPAR{gamma} when 15d-PGJ2 was used as a ligand, but not when troglitazone was used (70). The ability of a ligand to specify selective recruitment of a particular p160 family member has been demonstrated for VDR. While the natural hormone, 1{alpha},25-dihydroxyvitamin D3, mediated recruitment of all three SRC coactivator family members to VDR, the synthetic analog OCT (22-oxa-1{alpha}, 25-dihydroxyvitamin D3) induced interaction of VDR with only SRC-2 (44). Interestingly, OCT displays a tissue-selective profile retaining the antiproliferative activity of 1{alpha},25-dihydroxyvitamin D3, but lacking the hypercalcemic effects (71). Thus, selective coactivator recruitment may be an underlying mechanism for tissue/cell/promoter specificity of NR ligands.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
E2, GEN, and DES were purchased from Sigma (St. Louis, MO). ICI 182,780 was purchased from Tocris (Ballwin, MO). Biotin-labeled peptides were synthesized by Sigma-Genosys (The Woodlands, TX). The peptides were biotin labeled at the N terminus and the amino acid sequences of the peptides are illustrated in Fig. 1BGo.

Time-Resolved Fluorescence
Full-length recombinant baculovirus-expressed human ER{alpha} or ERß was purchased from PanVera (Madison, WI). White FluoroNunc MaxiSorp 96-well plates (Fisher Scientific, Pittsburgh, PA) were coated overnight at 4 C with 100 µl/well of 40 pmol/ml ER protein that had been diluted in 0.1 M NaHCO3. Receptor-coated plates were then blocked with 100 µl/well 7.5% BSA in TBS (0.1 M Tris HCl, 0.15 M NaCl) containing 20 µM diethylenetriamine-pentaacetic acid (DTPA-Sigma, St. Louis, MO) as a stabilizer. Blocking was carried out at room temperature for at least 1 h.

A peptide-europium (Eu) conjugate was prepared by incubation of 0.8 µl 1 mM biotin-labeled NR box peptide with 120 µl 0.1 mg/ml Eu-labeled streptavidin (Wallac, Inc./Perkin-Elmer Corp., Norton, OH) on ice for 30 min. After blocking, coated 96-well plates were washed three times with TBST (0.1 M Tris HCl, 0.15 M NaCl, 0.1% Tween) again containing 20 µM DTPA for signal stabilization. The peptide-Eu conjugate prepared previously was made up to 10 ml with DELFIA Assay Buffer (Wallac, Inc./Perkin-Elmer Corp.) and added to the receptor-coated 96-well plate at 90 µl/well. The E2 was diluted to 10x concentration in DELFIA assay buffer and added to appropriate wells at 10 µl/well. Plates were incubated with ligand and peptide conjugate for 1.5 h at room temperature and then washed five times with TBST + 20 µM DTPA. DELFIA Enhancement Solution (Wallac, Inc./Perkin-Elmer Corp.) was then added to the plate (100 µl/well) and incubated at room temperature with gentle shaking for 5 min. Plates were then read using a Wallac, Inc. Victor II plate reader (Wallac, Inc./Perkin-Elmer Corp.).

Relative fluorescence was determined by subtracting the fluorescence value obtained in the absence of ligand from the value obtained with ligand. This method yields negative relative fluorescence values if hormone-independent binding occurs that is displaced by an antagonist as illustrated in Fig. 2Go. Dose-response experiments were performed a minimum of three times. Representative experiments are shown in the figures, and the relative fluorescence values reported are derived from the mean of three individual experiments ± SEM.

To determine the dissociation constant (Kd) for each NR box, the time-resolved fluorescence technique was used with a fixed amount of E2, DES, or GEN (1 µM), and the NR box concentration was varied. For this assay the peptide-Eu conjugate was prepared by incubation of 2.1 µl 1 mM peptide with 17 µl Eu-labeled streptavidin on ice for 30 min. The volume of the peptide conjugate was then made up to 700 µl with DELFIA assay buffer containing 1 µM E2. Eight-point (in triplicate) dose-response curves were generated, and the results from three independent experiments were normalized and pooled to determine the dissociation constant. Data were analyzed using Prism 3.0 (GraphPad Software, Inc., San Diego, CA) and the Kd values were determined with a one-site binding model. Nonspecific binding was typically less than 1% of total binding.


    FOOTNOTES
 
Address requests for reprints to: Dr. Thomas P. Burris, Gene Regulation Research, DC0434, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285. E-mail: burris_thomas-p{at}lilly.com

Received for publication January 22, 2001. Accepted for publication March 2, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Burris TP 2000 The nuclear receptor superfamily. In: Burris TP, McCabe ERB (eds) Nuclear Receptors and Genetic Disease. Academic Press, New York, pp 1–57
  2. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  3. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839[Medline]
  4. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[Medline]
  5. Lonard DM, Nawaz Z 2000 Coactivators and corepressors. In: Burris TP, McCabe ERB (eds) Nuclear Receptors and Genetic Disease. Academic Press, New York, pp 389–408
  6. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coactivators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  7. Chen JD, Li H 1998 Coactivation and corepression in transcriptional regulation by steroid/nuclear hormone receptors. Crit Rev Eukaryot Gene Expr 8:169–190[Medline]
  8. Halachmi S, Marden E, Marin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated preteins: possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  9. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid receptor superfamily. Science 270:1354–1357[Abstract]
  10. Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domain of steroid receptors. Proc Natl Acad Sci USA 93:4948–4952[Abstract/Free Full Text]
  11. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transriptional coactivator for the AF2 transactivation domain of steroid, thyroid, retinoid and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
  12. Voegel JJ, Heine MJ, Zechel C, Chambon P, Gronemeyer H 1996 TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function of AF-2 of nuclear receptors. EMBO J 15:3667–3675[Abstract]
  13. Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan XY, Sauter G, Kallioniemi OP, Trent JM, Meltzer PS 1997 AIB1, a steroid receptor coactivator amplified in breast and ovarian cancer. Science 277:965–968[Abstract/Free Full Text]
  14. Chen H, Lin RJ, Schiltz RL, Chakravarti D, Nash A, Nagy L, Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300. Cell 90:569–580[Medline]
  15. Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear receptor-associated coactivator that is related to SRC-1 and TIF-2. Proc Natl Acad Sci USA 94:8479–8484[Abstract/Free Full Text]
  16. Takeshita Z, Cardona GR, Koibuchi N, Suen CS, Chin WW 1997 TRAM-1, a novel 160 kDa thyroid hormone receptor activator molecule exhibits distinct properties from steroid receptor coactivator-1. J Biol Chem 272:27629–27634[Abstract/Free Full Text]
  17. Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK, Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and mediates nuclear-receptor function. Nature 387:677–684[CrossRef][Medline]
  18. Suen CS, Berrodin TS, Mastroeni R, Cheskis BJ, Lyttle CR, Frail DE 1998 A transcriptional coactivator, steroid receptor coactivator-3, selectively augments steroid receptor transcriptional activity. J Biol Chem 273:27645–27653[Abstract/Free Full Text]
  19. Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature motif in transcriptional coactivators mediates binding to nuclear receptors. Nature 387:733–736[CrossRef][Medline]
  20. Takeshita A, Yen PM, Misiti S, Cardona GR, Liu Y, Chin WW 1996 Molecular cloning and properties of a full-length putative thyroid hormone receptor coactivator. Endocrinology 137:3594–3597[Abstract]
  21. Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1998 A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell 85:403–414
  22. Ren Y, Behre E, Ren Z, Zhang J, Wang Q, Fondell JD 2000 Specific structural motifs determine TRAP220 interactions with nuclear hormone receptors. Mol Cell Biol 20:5433–5446[Abstract/Free Full Text]
  23. Ko L, Cardona GR, Chin WW 2000 Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator. Proc Natl Acad Sci USA 97:6212–6217[Abstract/Free Full Text]
  24. Tcherepanova I, Puigserver P, Norris JD, Spiegelman BM, McDonnell DP 2000 Modulation of estrogen receptor-{alpha} transcriptional activity by the coactivator PGC-1. J Biol Chem 275:16302–16308[Abstract/Free Full Text]
  25. Rachez C, Gamble M, Chang CP, Atkins GB, Lazar MA, Freedman LP 2000 The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. Mol Cell Biol 20:2718–2726[Abstract/Free Full Text]
  26. Caira F, Antonson P, Pelto-Huikko M, Treuter E, Gustafsson JA 2000 Cloning and characterization of RAP250, a novel nuclear receptor coactivator. J Biol Chem 275:5308–5317[Abstract/Free Full Text]
  27. Zhu Y, Qi C, Jain S, Rao MS, Reddy JK 1997 Isolation and characterization of PBP, a protein that interacts with peroxisome proliferator-activated receptor. J Biol Chem 272:25500–25506[Abstract/Free Full Text]
  28. Lee SK, Anzick SL, Choi JE, Bubendorf L, Guan XY, Jung YK, Kallioniemi OP, Kononen J, Trent JM, Azorsa D, Jhun BH, Cheong JH, Lee YC, Meltzer PS, Lee JW, A nuclear factor, ASC-2, as a cancer-amplified transcriptional coactivator essential for ligand-dependent transactivation by nuclear receptors in vivo. J Biol Chem 274:34283–34293
  29. Wurtz J-M, Bourguet W, Renaud J-P, Vivat V, Chambon P, Moras D, Gronemeyer H 1996 A canonical structure for the ligand-binding domain of nuclear receptors. Nature Struct Biol 3:87–94[Medline]
  30. Moras D, Gronemeyer H 1998 The nuclear receptor ligand-binding domain: structure and function. Curr Opin Cell Biol 10:384–391[CrossRef][Medline]
  31. Weatherman RV, Fletterick RJ, Scanlan TS 1999 Nuclear-receptor ligands and ligand-binding domains. Annu Rev Biochem 68:559–581[CrossRef][Medline]
  32. Feng W, Ribeiro RCJ, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 280:1747–1749[Abstract/Free Full Text]
  33. Nolte RT, Wisely GB, Westin S, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor {gamma}. Nature 395:137–143[CrossRef][Medline]
  34. 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[Medline]
  35. Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev 12:3343–3356[Abstract/Free Full Text]
  36. Mak HY, Hoare S, Henttu PMA, Parker MG 1999 Molecular determinants of the estrogen receptor-coactivator interface. Mol Cell Biol 19:3895–3903[Abstract/Free Full Text]
  37. Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO J 17:232–243[Abstract/Free Full Text]
  38. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ, Stallcup MR 1998 Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 12:302–313[Abstract/Free Full Text]
  39. Leers J, Treuter E, Gustafsson JA 1998 Mechanistic principles in NR box-dependent interaction between nuclear hormone receptors and the coactivator TIF2. Mol Cell Biol 18:6001–6013[Abstract/Free Full Text]
  40. Chang C-Y, Norris JD, Gron H, Paige LA, Hamilton PT, Kenan DJ, Fowlkes D, McDonnell DP 1999 Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors {alpha} and ß. Mol Cell Biol 19:8226–8239[Abstract/Free Full Text]
  41. Voegel JJ, Heine MJS, Tini M, Vivat V, Chambon P, Gronemeyer H 1998 The coactivator TIF2 contains three nuclear receptor-binding motifs and mediates transactivation through CBP binding-dependent and -independent pathways. EMBO J 17:507–519[Abstract/Free Full Text]
  42. Northrop JP, Nguyen D, Piplani S, Olivan SE, Kwan STS, Go NF, Hart CP, Schatz P 2000 Selection of estrogen receptor ß and thyroid hormone receptor ß specific coactivator mimetic peptides using recombinant peptide libraries. Mol Endocrinol 14:605–622[Abstract/Free Full Text]
  43. McInerney EM, Rose DW, Rlynn SE, Westin S, Mullen TM, Drones A, Inostroza J, Torchia J, Nolte RT, Assamunt N, Milburn MV, Glass CK, Rosenfeld MG 1998 Determinates of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation. Genes Dev 12:3357–3368[Abstract/Free Full Text]
  44. Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, Kitanaka S, Suzawa M, Yanagisawa J, Kato S 1999 Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog. Mol Cell Biol 19:1049–1055[Abstract/Free Full Text]
  45. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
  46. Pike ACW, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljunggren J, Gustafsson J-A, Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full agonist. EMBO J 18:4608–4618[Abstract/Free Full Text]
  47. Tanenbaum DM, Wang Y, Williams SP, Sigler PB 1998 Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci USA 95:5998–6003[Abstract/Free Full Text]
  48. McDonnell DP, Clemm DL, Hermann T, Goldmann ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol Endocrinol 9:659–669[Abstract]
  49. Gee AC, Carlson KE, Martini PGV, Katzenellenbogen BS, Katzenellenbogen JA 1999 Coactivator peptides have a differential stabilizing effect on the binding of estrogens and antiestrogens with the estrogen receptor. Mol Endocrinol 13:1912–1923[Abstract/Free Full Text]
  50. 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[Abstract/Free Full Text]
  51. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson J-A, Nilsson S 1998 Differential response of estrogen receptor {alpha} and estrogen receptor ß to partial estrogen agonists/antagonists. Mol Pharmacol 54:105–112[Abstract/Free Full Text]
  52. Gaido KW, Leonard LS, Maness SC, Hall JM, McDonnell DP, Saville B, Safe S 1999 Differential interaction of the methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane with estrogen receptors {alpha} and ß. Endocrinology 140:5746–5753[Abstract/Free Full Text]
  53. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  54. Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I, Chambon P 1989 The cloned human oestrogen receptor contains a mutation which alters its hormone binding properties. EMBO J 8:1981–1986[Abstract]
  55. Allan GF, Hutchins A, Clancy J 1999 An ultrahigh-throughput screening assay for estrogen receptor ligands. Anal Biochem 275:243–247[CrossRef][Medline]
  56. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  57. Kuiper GGJM, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson J-A 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  58. Xu J, DeMayo FJ, Tsai SY, Tsai MJ, O’Malley BW 1998 Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279:1922–1925[Abstract/Free Full Text]
  59. Weiss RE, Xu J, Ning G, Pohlenz J, O’Malley BW, Refetoff S 1999 Mice deficient in the steroid receptor coactivator 1 (SRC-1) are resistant to thyroid hormone. EMBO J 18:1900–1904[Abstract/Free Full Text]
  60. Xu J, Liao L, Ning G, Yoshida-Komiya H, Deng C, O’Malley BW 2000 The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development. Proc Natl Acad Sci USA 97:6379–6384[Abstract/Free Full Text]
  61. Schulman IG, Shao G, Heyman RA 1998 Transactivation by retinoid X receptor-peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) heterodimers: intermolecular synergy requires only the PPAR{gamma} hormone-dependent activation function. Mol Cell Biol 18:3483–3494[Abstract/Free Full Text]
  62. Zhou G, Cummings R, Li Y, Mitra S, Wilkinson HA, Elbrecht A, Hermes JD, Schaeffer JM, Smith RG, Moller DE 1998 Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer. Mol Endocrinol 12:1594–1604[Abstract/Free Full Text]
  63. Dowell P, Ishmael JE, Avram D, Peterson VJ, Nevrivy DJ, Leid M 1997 p300 functions as a coactivator for the peroxisome proliferator-activated receptor a. J Biol Chem 272:33435–33443[Abstract/Free Full Text]
  64. Needham M, Raines S, McPheat J, Stacey C, Ellston J, Hoare S, Parker M 2000 Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1 depends on residues flanking the motif. J Steroid Biochem Mol Biol 72:35–46[CrossRef][Medline]
  65. Hall JM, McDonnell DP 1999 The estrogen receptor ß-isoform (ERß) of the human estrogen receptor modulates ER{alpha} transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578[Abstract/Free Full Text]
  66. Meijer OC, Steenbergen PJ, DeKloet ER 2000 Differential expression and regional distribution of steroid receptor coactivators SRC-1 and SRC-2 in brain and pituitary. Endocrinology 141:2192–2199[Abstract/Free Full Text]
  67. Hrabovszky E, Kallo I, Hajszan T, Shughrue PJ, Merchenthaler I, Liposits Z 1998 Expression of estrogen receptor-ß messenger ribonucleic acid in oxytocin and vasopressin neurons of the rat supraoptic and paraventricular nuclei. Endocrinology 139:2600–2604[Abstract/Free Full Text]
  68. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor-{alpha} and -ß mRNA in the rat central nervous system. J Comp Neurol 388:507–525[CrossRef][Medline]
  69. Hall JM, Chang C, McDonnell DP 2000 Development of peptide antagonists that target estrogen receptor-ß interactions. Mol Endocrinol 14:2010–2033[Abstract/Free Full Text]
  70. Kodera Y, Takeyama K, Murayama A, Suzawa M, Masuhiro Y, Kato S 2000 Ligand type-specific interactions of peroxisome proliferator-activated receptor {gamma} with transcriptional coactivators. J Biol Chem 275:33201–33204[Abstract/Free Full Text]
  71. Abe J, Nakano T, Nishii Y, Matsumoto T, Ogata E, Ikeda K 1991 A novel vitamin D3 analog, 22-oxa-1,25-dihydroxyvitamin D3, inhibits the growth of human breast cancer in vitro and in vivo without causing hypercalcemia. Endocrinology 129:832–837[Abstract]