Department of Chemistry, University of Illinois, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: John A. Katzenellenbogen, Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801. E-mail: jkatzene{at}uiuc.edu.
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ABSTRACT |
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INTRODUCTION |
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Dimer formation is thought to be essential for normal receptor function, because mutations that interfere with dimerization generally result in receptors that are insoluble or transcriptionally inactive (3). The principal dimerization interface of NRs is in the ligand-binding domain E and involves a large contact area that, in the case of the estrogen receptor- (ER
), encompasses about 15% (1703 Å2) of the surface area of each monomer (4). There is some contact between monomer units in the DNA binding domain C, but this interface is rather small, varies with different NRs, and is thought to contribute only to a minor degree to dimer stability (5).
ER has been reported to exist as a dimer even in the absence of ligand, and the dimer interaction of liganded ER ligand binding domain (ER
-LBD) is strong and resistant to high levels of denaturants (6). ER dimers have been estimated, by indirect methods, to have an equilibrium dissociation constant (Kd) of about 23 nm (7), although dimer affinity varies from NR to NR (7, 8, 9). There is evidence that the strength of the dimer interaction is regulated by ligand binding, although this issue has not been studied in a systematic fashion (7, 8, 9).
In this report, we describe convenient fluorescence resonance energy transfer (FRET)-based methods for measuring the thermodynamic and kinetic stability of ER-LBD dimers, using receptors that are chemically labeled with a single fluorophore in a site-specific manner. Using a kinetic FRET-based technique called "monomer exchange" (10), we can study the effect of ligand and coactivator binding on kinetics of ER
-LBD dimerization. We find that the rate of monomer exchange of unliganded receptor is remarkably slow and is slowed even further by ligand binding. Each class of ligand character (agonists, mixed agonist-antagonists, and pure antagonists) is found to have a characteristic effect on the rate of monomer exchange.
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RESULTS |
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Mutational studies suggest that either a cysteine-to-alanine or a cysteine-to-serine mutation at positions 381, 417, or 530 has minimal effect on ER activity (15, 16, 17, 18, 19). To maintain the environment of the binding pocket, we chose serine as a conservative replacement for solvent-exposed cysteines in our constructs. To enable site-specific fluorophore labeling of the ER-LBD, we have, in all cases, mutated cysteine 381 to serine and then separately mutated either cysteine 417 or cysteine 530 to serine, leaving a sole reactive cysteine, either at 530 or 417, respectively. For convenience, we have designated these ER constructs as C530 and C417 (with the bold italic type to indicate that they are mutant ERs with a single-reactive cysteine at that particular residue).
Our ER C530 and C417 constructs have an N-terminal His6-tag through which they can be purified over nickel-nitrilotriacetic acid (Ni-NTA) resin. We found it convenient to label these ER preparations while they were attached to the Ni-NTA resin. In this manner, excess cysteine-specific fluorophore could be removed from the receptor simply by washing the resin, before elution of the receptor. We used cysteine-specific fluorophores 5-iodoacetamidofluorescein (a FRET donor) and tetramethylrhodamine-5-maleimide (a FRET acceptor). Inclusion of the nonnucleophilic thiol reductant, tris(carboxyethyl)phosphine (TCEP), to maintain the ER-LBD in a fully reduced state, proved to be important for efficient labeling. The level of labeling, determined by matrix-assisted laser-desorption ionization mass spectroscopy (MALDI MS), was 9095% mono labeling, with no multiple labeling evident (data not shown).
We confirmed the site of ER-LBD labeling by analysis of the trypsin digestion pattern of receptor. Trypsin cleaves ER
-LBD at K467 to produce a 19-kDa N-terminal fragment (304467) and a 10-kDa C-terminal fragment (468554), the latter of which is then further cleaved to a 7-kDa fragment (468529/531) (20, 21). When fluorophore-labeled C530 ER
-LBD was treated with trypsin, only the 10-kDa and 7-kDa bands on SDS-PAGE were fluorescent, whereas when we trypsin digested C417 ER
-LBD, only the 19-kDa band was fluorescent (data not shown).
The estradiol (E2) binding affinities of unlabeled, fluorescein-, and tetramethylrhodamine-labeled ER double mutants are listed in Table 1. Previous studies have shown that ERs with single and double C-to-A, or C-to-S mutations retain near wild-type E2 binding affinities (15, 16, 17, 18, 19). We have also found that our unlabeled and fluorophore-labeled C-to-S double-mutant ER
-LBDs exhibit near wild-type affinities for E2. Interestingly, the attachment of a fluorophore to cysteine 417 or 530 appears to increase the affinity of our labeled receptors for E2 relative to that of the unlabeled receptors (Table 1
). Some of our dimer stability experiments were performed in urea (see below); therefore, we also measured E2 binding affinity in up to 2 m urea. This concentration of denaturant causes less than a 3-fold reduction of E2 binding affinity (Table 1
), consistent with our previous investigations (Gee, A. C., and J. A. Katzenellenbogen, unpublished).
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The Kd values of ER-LBD dimer affinity in 2 m urea (Fig. 2C
) are in the absence of ligand (apo, 1.0 nm), with an agonist (E2, 0.33 nm), a mixed agonist-antagonist [trans-hydroxytamoxifen (TOT), 0.27 nm], and a pure antagonist (ICI 182, 780, 0.34 nm). Under these conditions (2 m urea), there is a 3- to 4-fold increase in ER
-LBD dimer affinity with these three ligands of different character.
FRET Can Be Used to Study the Kinetic Stability of ER-LBD Dimers
In the absence of denaturant, the ER-LBD dimer affinity is quite high (Kd <0.1 nm), and, as noted above, this makes it technically challenging to measure ligand effects on thermodynamic dimer stability using FRET under native conditions. Hence, to measure dimer stability under native conditions, we used a standard kinetic FRET technique termed "monomer exchange" (Fig. 3
and Ref. 10).
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A representative dimer kinetic stability experiment is shown in Fig. 4A. To maximize the FRET signal (in our experiments typically 3550%) we used a 4:1 ratio of acceptor to donor receptor, so that every donor-labeled monomer is more likely to find an acceptor-labeled monomer. To confirm that the FRET signal results from the formation of donor-acceptor dimers, we determined that we could abrogate the FRET signal by adding an excess of unlabeled ER, which would then undergo monomer exchange and disrupt the donor-acceptor dimers (data not shown).
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Ligand Binding Affects the Kinetic Stability of ER-LBD Dimers
Titration experiments allowed us to estimate ligand concentrations that fully saturate the receptor (Fig. 4B). Here, the EC50 values reflect relative ligand affinity, whereas, the efficacy of dimer kinetic stability (i.e. maximum half-life under saturating ligand conditions) indicates the degree to which a particular ligand induces a conformation that stabilizes the dimer. Shown in Fig. 4B
are both a high-affinity ligand [E2, relative binding affinity (RBA) 100%] and a low-affinity ligand (genistein, RBA 0.013%). The rate of monomer exchange becomes progressively slower as receptor becomes occupied and reaches a maximum at ligand saturation (Fig. 4B
). All monomer exchange experiments were conducted under saturating ligand conditions (1 µm for most ligands, and 5 µm for very-low-affinity ligands).
Using our convenient FRET-based dimer kinetic stability assay, we have systematically assessed more than 30 natural and synthetic ligands for their effects on dimer dissociation of ER-LBDs. As agonist ligands, we have used estrogens, phytoestrogens, and synthetic agonists having various RBAs (Table 2
). As mixed agonist-antagonist ligands, we have used raloxifene (RAL), TOT, tamoxifen, cyclofenil, and three of our novel ER subtype-selective antagonist ligands (furan propyl antagonist, furan ethyl antagonist, and pyrazole ethyl antagonist) (Table 2
and Refs. 23, 24, 25). Additionally, as pure antagonists, we have used ICI compounds ICI 182,780 and ICI 164,384.
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Our novel furan and pyrazole ligands, which are available in both agonist and antagonist forms within a structurally homologous series, enable us to investigate the issue of dimer stability and ligand character. Interestingly, the ER-LBD dimer stability is lower for the higher-affinity furan and pyrazole agonists than it is for their corresponding lower-affinity antagonists (Table 2
and Fig. 5
). This suggests that dimer kinetic stability appears to be more a function of ligand character (i.e. agonist vs. antagonist) than ligand affinity or ligand core structure. This trend is also seen with the generally greater dimer stability of ER
when it is bound with traditional antagonist vs. agonist ligands and is supported by an earlier report in which a chromatographic technique was used to monitor the rate of wild-type ER
-LBD monomer exchange (7).
Some interesting structure-activity relationships were observed from the ligands monitored for dimer kinetic stability effects on ER-LBD (Fig. 5
). The presence of a basic side chain (piperidine or dimethylamino-ethoxy) on antagonist ligands caused a substantial increase in dimer kinetic stability compared with structurally similar ligands (agonists) lacking this basic side chain [furan and pyrazole series; also tamoxifen and TOT vs. diethylstilbestrol (DES)]. Additionally, an 11ß substituent enhanced dimer kinetic stability compared with a similar ligand lacking a substituent at this position [CME2 vs. E2, or 11ß-ethyl estriol (Et-E3) vs. E3]. The addition of a 17
-ethynyl group decreases dimer stability in the estriol series, although it increases ligand binding affinity [11ß-ethyl-17
-ethynyl estriol (Et-EE3) vs. Et-E3].
Coactivator Peptides Selectively Stabilize Agonist-Bound ER-LBD Dimers and Provide a Functional Assay to Identify Ligand Character Based on Dimer Stability
Our FRET-based monomer exchange assay provides a convenient measure of ligand-induced dimer kinetic stability in a highly purified in vitro model system. Having obtained an initial structure-activity relationship profile of ligand-induced ER dimer stability, we wanted to see whether coactivator peptides, which should bind only to agonist-ER complexes, would enhance and sharpen the structure-activity relationship. Additionally, coactivators are present in in vivo systems; therefore, adding them to our in vitro model system makes it more similar to the in vivo situation.
The addition of a coactivator peptide containing residues 629831 of SRC-1 (encompassing LXXLL NR boxes 13) or of a 15-residue peptide containing only the NR box-2 of SRC-1 coactivator protein, caused up to a 2-fold kinetic stabilizing effect on E2-ER-LBD dimers (Fig. 6A
). In terms of this effect, the longer SRC-1 fragment containing three NR boxes has a potency (EC50) about 100-fold higher than the single NR box-2 peptide of SRC-1. Both coactivator peptides, as expected, failed to stabilize antagonist-bound ER
LBD (ICI 182, 780-ER
-LBD dimers, as shown in Fig. 6B
). Thus, the coactivator peptide-enhanced ER dimer stabilization is highly selective for agonist-bound receptor (Fig. 6C
). To our knowledge, this is the first direct evidence for coactivator-mediated nuclear receptor dimer stabilization. A mechanistic rationale for this process will be presented in Discussion.
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DISCUSSION |
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The site-specific labeled, fluorescent ER-LBD double C-to-S mutants that we have prepared have shown near wild-type characteristics in ligand binding and coactivator recruitment studies (Table 1
and Fig. 1
), consistent with reports of full-length ERs with multiple cysteine-to-alanine or cysteine-to-serine mutations also maintaining near wild-type binding affinity and transcriptional activity (17, 18). Curiously, the unlabeled cysteine-to-serine mutants have lower binding affinity than the labeled mutants (Table 1
). Others have reported lower affinity in these mutants and have ascribed this to a change in the overall lipophilic balance of ER (change in protein hydration due to C-to-S mutations) (18). Perhaps, addition of the lipophilic fluorophore to the remaining reactive cysteine restores the lipophilic balance of ER that has been perturbed by the cysteine-to-serine mutations.
Site-Specific Fluorophore-Labeled ER-LBDs Enable the Direct Measurement of ER Dimer Stability and Ligand-Induced Changes in This Stability
Using our donor- and acceptor-labeled ER-LBD constructs in a FRET-based dimer formation assay, we have measured directly the dimer affinity of apo and ligand-bound receptors, under equilibrium conditions. To our knowledge, this is the first report showing a direct measurement of ER dimer affinity, and we find that the Kd for dimerization is much lower (<0.1 nm) than the previously reported values measured using indirect techniques (6, 7). In fact, due to technical limitations, we could not determine the Kd in the absence of denaturants. This very high receptor dimer affinity is consistent with the highly hydrophobic nature of the dimer interface (helices 10 and 11 of ER
-LBD) (4, 6, 32).
Under mild denaturing conditions (2 m urea, which causes only about a 3-fold loss in E2 binding affinity, Table 1), we could investigate how ligands of various agonist vs. antagonist character affect dimer affinity (Fig. 2C
). We find that E2-, ICI 182,780-, and TOT-occupied receptors have a 3- to 4-fold higher dimer affinity compared with apo ER
-LBD dimers (Fig. 2C
).
Using a different FRET-based assay, a monomer exchange assay, we have made a careful analysis of kinetic stability of the ER-LBD dimer and how this is affected by ligands having a broad range of affinity, structure, and agonist vs. antagonist character (Fig. 5
and Table 2
). The results indicate that the rate of dimer dissociation, which presumably reflects the manner in which the conformation of the ER-ligand complex affects the dimer interface, is not a simple function of ligand binding affinity, or ligand dissociation kinetics, but more reflects ligand character. In general, and especially where specific comparisons can be made (e.g. with structurally related pyrazole and furan agonists and antagonists), it appears that antagonists stabilize the ER
dimer more than do agonists. The degree of dimer stabilization within each class of ligand character, however, covers a range of values.
This large range (0.65- to 6.2-fold) of ligand-induced kinetic stabilization of ER-LBD dimers, encountered with steroidal estrogens, phytoestrogens, and nonsteroidal estrogens (all of which are considered to be agonists), may play a significant role in determining the lifetime of the ER dimer when it is bound to an estrogen response element (ERE) in a transcriptionally active complex in association with other downstream factors. The lifetime of this complex could be important in determining the in vivo potency and efficacy of a particular ligand and the selectivity with which it is capable of inducing the various biological responses that are regulated by ER.
In our FRET-based monomer exchange assay, the SERMs (TOT, RAL) and our furan and pyrazole antagonist ligands showed consistently higher dimer kinetic stability compared with E2 and the majority of other agonist ligands surveyed (Fig. 5). Interestingly, two-hybrid ER dimerization studies performed in vivo suggest the opposite, namely, that the dimers of agonist-bound receptors are more stable than those of antagonist-bound ones (9). This discrepancy could be due to the presence of endogenous coactivator proteins containing LXXLL sequence motifs within the cellular context of the in vivo studies. We have, in fact, observed that SRC-1 coactivator peptides selectively stabilize only agonist-bound ER dimers (Fig. 6
). Hence, our in vitro studies, which are performed using highly purified receptor in the absence of other interacting proteins, may provide a clearer picture of ligand-induced effects on receptor dimer structural stability. They do suggest that coactivator content of different tissues could play a role in the kinetic stabilization of transcriptionally active ER dimers, which, in turn, may offer an explanation for the complex tissue-selective pharmacology observed with ER ligands.
Structural Considerations May Account for the Different Effects of Agonists, SERMs, and Pure Antagonists on ER-LBD Dimer Kinetic Stability
The greater kinetic stability of ER-LBD dimers that is engendered by mixed agonist-antagonists might be due to two factors: 1) the dimer interface or contact region is enlarged and hence more stable in the ER antagonist complexes; and/or 2) ER conformational changes outside of the dimer interface result in the active destabilization of the ER agonist structures. We have examined both of these possibilities through modeling.
An overlay of the E2-(an agonist, green) and TOT-bound (a mixed agonist-antagonist, red) ER-LBD x-ray structures show that the two structures of ER
-LBD overlay well, especially in the dimer interface region of helices 10 and 11 (Fig. 7A
). Thus, from these structures there is no evidence of an enlarged/stabilized dimer interface in the antagonist ER structures, although it is possible in solution, where ER conformations are more dynamic, that this is the case.
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Previous studies have suggested a possible dimer disruption and high cellular turnover rate effected by the ICI antiestrogens on full-length ER (35). Our dimer kinetic stability assays show that the ICI compounds engender the most rapid monomer exchange rates of all antagonists and SERMs surveyed (Fig. 5B). They cause about 35% less kinetic stabilization of ER
-LBD dimers compared with the SERMs (TOT and RAL). Pike et al. (36) recently reported the x-ray crystal structure of ICI 164,384-bound ERß-LBD, in which helix 12 is not resolved and is therefore highly mobile. Thus, in the ICI structure, the residues C terminal of helix 12 might have a dimer-destabilizing effect that is intermediate between that of the agonist ligands (active destabilization enforced by the agonist position of helix 12 pointed toward the dimer interface) and the SERMs (active destabilization prevented by helix 12 being in the extended antagonist position pointing away from the dimer interface). This could account for the more modest effect of the ICI pure antagonists on stabilizing the ER
-LBD dimers.
This structural model provides a satisfying rationale for the different degrees to which agonists, mixed agonist-antagonists, and pure antagonists stabilize ER dimers, in general. The fact that the dimer stabilities observed within each of these three classes overlap one another, however, suggests that a factor that is specific to the ligand, irrespective of its pharmacological class, is involved. We can only speculate that the different ligand structures of the members within each of these biocharacter classes have subtle effects on the conformation of the dimer interface that contribute to dimer stability.
Changes in the receptor conformation could differentially influence the dimer stability of activated receptors (agonist-bound) and could be a possible innate mechanism to turn off these receptors, so as to maintain homeostasis in the cell. To evaluate this hypothesis, we are currently working toward generating fluorescent ER constructs with both E and F domains.
Using Coactivator-Induced Dimer Stability to Define Ligand Agonist vs. Antagonist Character
In addition to gaining insight into ligand-induced perturbation in ER-LBD dimer interface structure and function, our convenient FRET-based monomer exchange assay can also be used to define the agonist vs. antagonist character of previously uncharacterized ligands. We have found that coactivator peptides selectively increase the dimer kinetic stability of agonist-bound ER
-LBD receptors (Fig. 6
) in a manner that clearly distinguishes them from antagonist-bound ERs. This selective increase in dimer kinetic stability is reminiscent of the effect that coactivator peptides have of reducing the agonist ligand dissociation rate, as demonstrated previously in our laboratory (30).
In recent studies, it has been reported that of the first three NR box regions of SRC-1, box-2 has the highest affinity for E2-, diethylstilbestrol-, or genistein-bound ER (37). When compared in our coactivator-enhanced dimer stability studies, the multivalent SRC-1 peptide (NR boxes 13) shows about a 100-fold higher potency (EC50) compared with the single NR box-2 peptide (Fig. 6A
). We and others have shown that there is a 1:1 stoichiometry between a nuclear receptor LBD dimer and a multivalent SRC-1 peptide, using solution and x-ray techniques (30, 38, 39). Thus, the higher potency of the longer SRC-1 peptide, in terms of increasing the kinetic stability of agonist-bound receptor dimers, might be caused by a dimer-tethering effect, induced by this multivalent coactivator peptide. The presence of multiple NR boxes within many coactivator proteins could be part of an integral mechanism whereby these multivalent proteins tether activated NR dimers and increase their kinetic stability. Regardless of the exact mechanism of this stabilization, either coactivator peptide (SRC-1-NR boxes 13, or SRC-1-NR box-2) will separate agonist from antagonist ligands in our dimer kinetic stability assay, thus providing a definitive identity to the character of any novel ER ligand.
Fluorescent Nuclear Receptors: Probes for Monitoring Structure and Biological Functions
It is not clear whether ligands affect dimer stability of other NRs, such as the androgen and progesterone receptors (8, 33). Many steroid, nonsteroid, and orphan NRs have a low number of conserved cysteine residues in their LBDs (generally three to five cysteines per LBD) (8). Thus, the methodology that we have developed for ER should be applicable, as well, to the study of ligand-induced effects on dimer affinity and monomer exchange dynamics in these other NR systems.
We are in the process of using fluorescent ERß-LBDs to study homo- and heterodimers of both ER subtypes and the manner in which SERMs and ER subtype-selective ligands (both agonists and antagonists) modulate differential homo- and heterodimer stability. Fluorophore-labeled ERs can also be used for the development of other types of assays to characterize receptor conformation, conformational dynamics, and ligand or coregulator interactions.
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MATERIALS AND METHODS |
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Mutagenesis and Fluorescent ER Preparations
Cysteine-to-serine mutations at positions 381, 417, and 530 were introduced in a pET-15b human ER-LBD construct (304554) using Stratagene QuikChange Site-Directed Mutagenesis Kit with PfuTurbo DNA polymerase and the appropriate oligonucleotides. The corresponding C-to-S ER
-LBD constructs were subcloned in NdeI/BamHI sites of a newly double-digested pET-15b construct and sequenced. The His6-tagged ER
-LBDs were expressed from pET-15b vectors in BL21(DE3)pLysS E. coli and purified as described previously (29). Site-specific labeling of receptor was accomplished using 30:1 equivalents of cysteine-specific fluorophore to supernatant [3H]E2 binding activity while the purified His6-tagged ER
-LBD was still immobilized on the Ni-NTA resin. The labeling reaction was incubated overnight at 4 C in Tris-glycerol (50 mm Tris, 10% glycerol) pH 7.0 buffer, in the presence of 0.10.5 mm TCEP. Excess fluorophore was removed by washing the ER
-LBD-bound resin complex before eluting the fluorescent receptor. The level of derivatization was determined by comparing matrix-assisted laser-desorption ionization-mass spectroscopy spectra of unlabeled and labeled receptor.
Scatchard and Fluorescent GST Pull-Down Assays
The binding affinities of the derivatized ERs were determined directly with [3H]E2 and calculated by Scatchard analysis as described previously (42). The fluorescent GST pull-down studies were conducted using previously described protocols with the following modifications (27). Briefly, GST-His6-SRC-1 (629831) construct in pET-15b, generously provided by Dr. David Shapiro, was expressed in BL21(DE3)pLysS E. coli and purified using Ni-NTA resin following the same protocol as with the ER-LBD (29). The purified GST-His6-SRC-1 was bound to GSH resin and incubated with active fluorescent ER
-LBD in the presence of excess ligand. After 1 h incubation at 4 C, the GSH beads were washed three times, and bound proteins were eluted with 20 µl solution of elution buffer as described previously (27). The 20 µl elution was combined with 580 µl of Tris-glycerol (pH 8.0) buffer, placed in a 5.0 x 5.0-mm quartz fluorescence cuvette, and measured using a Fluorolog II (model IIIc) fluorometer (Jobin Yvon, Inc.). The fluorescein-labeled C530 receptor was excited at 488 nm (2.5-mm slits), and the emission intensity was monitored at 521 nm (2.5-nm slits). The tetramethylrhodamine-labeled C417 receptor was excited at 541 nm, and the emission intensity was monitored at 580 nm under similar conditions.
Kinetic Dimer Dissociation Assay
For the FRET-based monomer exchange assay, the purified, fluorescein (donor)- and tetramethylrhodamine (acceptor)-labeled ER-LBD preparations were diluted to 55 nm concentrations. A 5-µl solution of 10 µm ligand stock in Tris-glycerol (pH 8.0) buffer was added to a 45 µl solution of 55 nm donor ER placed in separate wells of a black round-bottom 96-well Nunc polypropylene plate, yielding a final donor ER concentration of 50 nm and 1 µm ligand. In a second plate, 25 µl of 10 µm ligand stock were added to a 225-µl solution of 55 nm acceptor ER placed in separate wells, yielding a final acceptor ER concentration of 50 nm and 1 µm ligand. Both plates were incubated at room temperature for 1 h before 200 µl were removed from the acceptor ER wells and the mixture was added to the 50 µl of donor ER-containing wells incubated with the same ligand, yielding a final concentration of 10 nm donor, 40 nm acceptor ER, and 1 µm ligand. After mixing, the 96-well plate was covered with a clear polyolefin sealing tape (Nalge Nunc International, Rochester, NY), and monomer exchange was monitored immediately by following FRET through the decrease, with time in the fluorescein (donor) emission intensity at 530 nm using a Molecular Devices Gemini XS fluorometer. The samples were excited at 485 nm, with a 515-nm cutoff filter in the excitation pathway, and the chamber was temperature controlled at 28 C. The experiments containing coactivator peptides were conducted under similar conditions with final concentration of 6 nm donor, 20 nm acceptor ER, 1 µm ligand, and proper coactivator peptide. The expression and purification of His6-tagged SRC-1-NR boxes 13 (residues 629831) and synthesis of SRC-1-NR box 2 (residues 683696) were conducted as described previously (30). Similar monomer exchange experiments were conducted with a 5 nm total ER concentration in cuvettes using a Fluorolog II (model IIIc) fluorometer under magic-angle (43) conditions. Monomer exchange kinetics observed with both fluorometers were very similar. To reduce any loss of intensity due to nonspecific sticking of protein to cuvettes or plates, a carrier protein (0.3 mg/ml of chicken ovalbumin) was added to all reaction mixtures. The collected data were fitted to a nonlinear regression, one-phase exponential decay function (y = plateau + span -t), using Prism 3.00. For all experiments, only data sets that could be fitted with an r2 value greater than 0.95 were used.
Thermodynamic ER Dimer Affinity Assay
For the FRET-based thermodynamic dimer formation assay, a 700-µl solution of 0.1 nm donor-labeled ER (in Tris-glycerol, pH 8.0, buffer and 0.3 mg/ml chicken ovalbumin, with or without 1 µm ligand) was placed in separate tubes. From a serial dilution of acceptor-labeled ER solution, a sample of less than10 µl was removed and mixed with the 700 µl donor ER (<1.5% dilution). Due to the rather slow dissociation rates of receptor dimers, the samples were incubated for 58 h at room temperature (in the dark), for reactions to reach equilibrium. A 600-µl solution of each sample was placed into a 5.0 x 5.0-mm quartz fluorescence cuvette and placed into the sample chamber of the Fluorolog II fluorometer. The sample chamber was held at a constant 25 C while the sample was excited at 488 nm (5.0-mm slits), and donor emission was monitored at 521 nm (2.5-nm slits). Each sample was measured until the standard error was below 1%, three emission data points were averaged for each sample, and each sample was prepared in duplicate. The following signal corrections were conducted for each sample: dark counts correction (to correct for any fluctuations in the photomultiplier tube), signal/reference correction (signal divided by a reference signal to correct for fluctuations in light source intensity), and blank subtraction. Percent FRET was calculated based on the donor intensity (22) with the following equation:
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: CME2, 11-ß-Chloromethyl estradiol; DES, diethylstilbestrol; E2, 17ß-estradiol; ER-LBD, estrogen receptor-
-ligand binding domain; ERE, estrogen response element; Et-E3, 11ß-ethyl estriol; Et-EE3, 11ß-ethyl-17
-ethynyl estriol; FRET, fluorescence resonance energy transfer; GSH, glutathione Sepharose; GST, glutathione-S-transferase; Kd, dissociation constant; Ni-NTA, nickel nitrilotriacetic acid; NR, nuclear receptor; RAL, raloxifene; RBA, relative binding affinity; SERM, selective ER modulator; SRC, steroid receptor coactivator; TCEP, tris(carboxyethyl)phosphine; THC, tetrahydrochrysene; TOT, trans-4-hydroxytamoxifen.
Received for publication July 16, 2002. Accepted for publication September 9, 2002.
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REFERENCES |
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