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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The combinatorial nature of ER genomic signaling described through the tripartite model (2) has traditionally been viewed in a unidirectional manner. It is thought that the binding of ligand to receptor stabilizes ER dimerization and initiates the binding of the ligand·receptor complex to specific estrogen response elements (EREs) and the precisely controlled recruitment of tissue-specific coregulators to this ligand·receptor·DNA complex upstream of ER-regulated genes. The effector components of the tripartite model, namely the EREs and coregulator proteins (coactivators or corepressors), are typically portrayed as simple docking partners for specific topographical features on the surface of the ligand·receptor binary complex, thereby functioning as physical or enzymatic bridging components between the nuclear receptor (NR) and the transcriptional machinery (2).
Gorski and co-workers (3) provided some of the earliest evidence for an ERE-induced allosteric modulation of ER conformation, suggesting that the ERE promoter context of ER-regulated genes might stabilize specific ligand·receptor conformations. Recent studies suggest that ERE sequences behave as endogenous allosteric ligands, regulating receptor structure and function upstream of ER-regulated genes (4, 5, 6, 7). Additionally, Moore and co-workers (8), using a novel receptor-reassembly assay, have described an allosteric corepressor-mediated stabilization of specific NR conformations. Crystallographic evidence with HNF-4, a member of the NR family of transcription factors, illustrates that coactivator rather than ligand binding locks the active conformation of this receptor (9). Also, our laboratory has previously reported allosteric action of coactivator peptides on the ER
ligand binding domain (LBD) by conformationally locking agonist ligands within the ligand binding pocket, and thus selectively decreasing their rate of dissociation from the receptor (10). This closely regulated reciprocal cross-interaction among the mediators of ER action (i.e. ligand, ERE, and coregulator proteins) adds an interesting, new level of complexity to ER pharmacology, the precise details of which remain to be elucidated.
In this report, we present direct evidence for coactivator-induced stabilization of ER conformation, using fluorophore-labeled ER-LBDs. The results of our fluorescence anisotropy-based protease challenge and regional dynamic assays suggest that coactivator proteins work together with agonist ligands to stabilize an activated form of the receptor. Using pyrene excimer (excited state dimer) fluorescence, we find direct evidence for a novel coactivator-stabilized (costabilized) receptor conformation or state, which might correspond to an especially stabilized conformation of the ER
-LBD that supports a high level of transcriptional activity. Throughout this report, we will refer to this unique receptor conformation as the costabilized conformation. In addition, we find that different classes of coactivator proteins, p160 vs. mediator, elicit varying degrees of receptor stabilization. Remarkably, higher concentrations of coactivator proteins shift the conformation of apo- and antagonist-bound ERs toward this costabilized form, suggesting a mechanism that might contribute to tamoxifen (TAM) resistance in clinical settings. Thus, our biophysical results suggest that the p160 effector proteins are not simply bridging factors that when recruited to ER act downstream of the receptor; rather, they are active contributors to receptor structure and function.
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RESULTS |
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Coactivator Peptides Increase the Population of Agonist-ER Complexes with Helix 12 Stabilized in the Activated Position
Fluorescence anisotropy is a population-based approach (14); thus, alterations in the number of receptor complexes with a particular conformation will lead to changes in the observed anisotropy values. We have used a 15-residue peptide corresponding to nuclear receptor box-2 (NR-box-2*) region of the steroid receptor coactivator-1 (SRC-1) protein (10) to study the effects of coactivator peptides on the dynamic nature of receptor structure in solution. The relatively small size of the coactivator peptide (1.8 kDa compared with 60-kDa ER
-LBD dimer), the very short fluorescence lifetime of the MTMR fluorophore (
2 nsec), and the attachment of the fluorophore to the receptor instead of the coactivator peptide, ensure that any changes in fluorescence anisotropy are due to the SRC-1-NR-box-2*-mediated allosteric alterations in regional dynamics of the ER. Comparison of the fluorescence anisotropy values with and without SRC-1-NR-box-2* (9 µM) in the presence of ligands (5 µM) of various pharmacological characters shows an increase of approximately 1214 mA units (anisotropy x 1000) for agonist-bound receptor complexes [estradiol (E2), diethylstilbestrol, and estriol (E3)], suggesting a coactivator-induced increase in the degree of conformational rigidity near cysteine 530 and/or an increase in the proportion of receptor complexes with an activated conformation. Only a less than 3-mA unit increase was observed for apo and mixed agonist-antagonist ligands [trans-4-hydroxytamoxifen (TOT), and raloxifene], suggesting a minimal coactivator effect on the ligand·receptor complexes at this concentration of coactivator peptide (Fig. 1A
).
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Coactivator Binding to Agonist·ER Complexes Contributes to the Formation of a Novel Receptor Conformation, an Active Costabilized Conformation
We next asked whether coactivator recruitment to agonist·ER complexes contributes to a conformation of the ER structural scaffold that is unique to the tripartite agonist·ER·coactivator complex and whether it might be possible to detect such an activated form of the LBD directly by spectroscopic means. Based on x-ray structures of ER-LBD, the intermonomer distance between cysteine 530 residues within agonist ER
-LBD structures (E2 and diethylstilbestrol) is approximately 16 Å, whereas in the mixed agonist-antagonist (TOT) structure this distance is approximately 34 Å (Fig. 2
). The C530-C530 intermonomer distance appears to be directly linked to the activated (wrapped-back) vs. partially inactivated (extended) positions of helix 12.
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The emission spectra of pyrene-labeled C530 receptor in the apo or various ligand-bound forms contain only the vibronically structured monomer (M) emission bands at 386 nm and 406 nm; they lack the unstructured excimer (E) emission at 496 nm (Fig. 3A). The lack of an excimer signal in the apo or ligand-bound forms suggests C530-C530 intermonomer distances greater than 10 Å or the improper alignment of the two pyrene-labeled residues within these complexes.
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Coactivator Proteins Display Varying Degrees of Potency and Efficacy in Producing Novel Costabilized Receptor Conformations
A number of different classes of coactivator proteins have been identified in NR signaling pathways (19); however, cataloging functional differences within these classes has been a challenging task. By determining the ratio of pyrene excimer (E, 496 nm) to monomer (M, 386 nm) intensities (E/M) (16), we can quantify the coactivator-induced conformational changes in the ER-LBD (production of the costabilized ER-LBD conformation), in solution and under equilibrium conditions (Fig. 4). Monitoring the costabilized conformation of ER within a ternary complex may provide the means for discriminating between specific (productive) and nonspecific (nonproductive) coactivator recruitment, highlighting functional differences between families of coactivator proteins and their selective recruitment to various agonist·receptor complexes.
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We also assayed shorter fragments of the p160 coactivator proteins containing single NR-boxes (19 residue peptides corresponding to the NR-box-2 regions of SRC-1 and SRC-3) (Fig. 4B). As expected, the single NR-box coactivator peptides of SRC-1 and SRC-3 had lower potency compared with their longer NRD counterparts (micromolar vs. nanomolar affinities), but curiously, both have higher efficacy in contributing to the production of the costabilized ER conformation (compare E/M ratios of Fig. 4
, A vs. B).
In addition to the peptides, our group has developed a small molecule coactivator mimic termed, a coactivator binding inhibitor (CBI) with a pyrimidine core scaffold bearing three branched alkane side chains that mimic the three leucines of the SRC coactivator protein LXXLL NR-boxes (24). Interestingly, the CBI compound binding fails to produce the costabilized conformation of the receptor (Fig. 4B), although we have shown that this CBI prevents coactivator peptide binding to the E2·ER
-LBD with a Ki (inhibition equilibrium constant) of 29 µM (24).
Agonist Ligand Potency Is Regulated by the Degree of Coactivator-Induced Stabilization of the Ligand·Receptor·Coactivator Ternary Complex
The tissue-specific potency of small molecule ligands with estrogenic hormonal activity can be attributed to a number of factors, including ligand affinity for the receptor and the cellular concentration of receptor subtypes and constellation of coactivators (2). Recent evidence suggests that the potency (affinity) of coactivator peptides for agonist·receptor complexes can depend on the particular agonist ligand-stabilized ER conformation, with not all agonists being equivalent in the recruitment of coactivator peptides (20, 25). There are technical limitations, however, in directly studying the efficacy (extent) of coactivator-mediated stabilization of various agonist·receptor complexes. We have tried to avoid these limitations by using our conformation-based coactivator recruitment approach: we saturated the ER with excess ligand (5 µM) and then directly measured the potency and efficacy of SRC-3-NRD recruitment through the formation of the novel costabilized receptor conformation that is detected by excimer emission from pyrene-labeled ER-LBD (Fig. 5
).
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Inhibition of the Coactivator Protein Allosteric Effects on ER Structure
Undesired hormonal activities mediated through the estrogen receptor signaling pathway have traditionally been inhibited at the ligand level through the binding of selective ER modulators (SERMs), such as TAM, raloxifene (28, 29), and more recently, by a new class of ER pure antagonist ligands, Faslodex (ICI 182,780) (30, 31). Both in vitro and in vivo studies have demonstrated that antagonist compounds compete with agonist ligands, and elicit receptor conformations that prevent recruitment of key coactivator proteins (19, 32). This decrease in coactivator recruitment leads to the inhibition of the potent transcriptional activity of ER.
We find that the addition of increasing concentrations of antagonist ligand can successfully compete with an agonist ligand (50 nM E2) and disrupt the coactivator-mediated stabilization of ER-LBD (Fig. 6A
). We tested three ER antagonist ligands having varying relative binding affinities [(RBAs) with estradiol affinity set at 100%] for ER
; TAM (0.9%), TOT (140%), and ICI 182,780 (79%). As expected, all three antagonist ligands reverse the stabilizing effects of the coactivator protein on the receptor, as measured by the pyrene E/M ratio, and they do so with a range of IC50 values mirroring the relative affinity of these ligands for the receptor (Fig. 6A
). The hydroxylated and active metabolite of TAM, TOT shows an approximately 100-fold higher potency than TAM in reversing coactivator stabilization of receptor, in close agreement with the RBA value differences of these two ligands.
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Comparing Different in Vitro Coactivator Recruitment Techniques
It has been noted that there can be a discrepancy between cell-based ER transactivation assays and current in vitro coactivator binding studies, suggesting that coactivator binding affinities measured through current in vitro protein-protein interaction techniques might not be an appropriate model for receptor activity (7, 20). Specifically, significant levels of coactivator recruitment have been observed with apo-ER using ELISA or FRET-based in vitro recruitment studies, which do not correlate with the low transcriptional output of the receptor in the unliganded state. Also the ER transactivation studies demonstrate a higher transcriptional output from TAM-bound receptor compared with the apo receptor, which appears to contradict the coactivator potency trend typically observed with the current in vitro coactivator recruitment approaches (7, 33, 34). The cell-based ER transactivation results are in good agreement with the partial agonist-antagonist pharmacology observed with TAM in clinical settings (29). We compared a proximity-based (FRET) approach with our ER conformation-based (pyrene excimer) approach in terms of the coactivator recruitment profiles with apo, E2, SERM, and pure antagonist ligands.
We used a FRET-based coactivator recruitment assay with MTMR-labeled ER and an eight-residue LXXLL peptide labeled with a FRET quencher (QSY-7) (Fig. 7A). The signal output of this technique is not directly monitoring the ER conformational change and hence is incapable of directly quantifying the activation state of the receptor. The nonproductive assembly of the coactivator peptide onto the receptor, therefore, would also be detected with this technique. The results show that the order of coactivator recruitment affinity from highest to lowest is E2 > Apo > ICI 182,780 = TOT (Fig. 7A
). The E2·ER complex exhibit a 2.5-fold higher coactivator affinity compared with apo-ER, whereas the TOT·ER complex has 31-fold lower affinity compared with apo-ER. This trend in coactivator recruitment affinity is in good agreement with other classical in vitro studies, where the agonist ligand increased recruitment and antagonists decrease recruitment compared with apo receptor (32), but it is inconsistent with higher ER transcriptional activity observed with TAM compared with apo receptor conditions (33). A possible explanation for this discrepancy could be that this classical (proximity based) in vitro coactivator recruitment study detects both specific (productive) and nonspecific (nonproductive) recruitment of coactivator on the receptor complex, and thus is unable to accurately mimic the pattern of ER transactivation observed in cells.
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Increasing Concentration of Coactivator Proteins as a Possible Mechanism of TAM Resistance
To our surprise, in the presence of a relatively high concentration (5.3 µM) of SRC-3-NRD, the TOT-bound receptor shows a clear and robust excimer signal with a 4.7-fold higher E/M ratio compared with the absence of SRC-3-NRD, suggesting that the binding of coactivator protein to the receptor can be specific and productive (Fig. 8A). A titration of high SRC-3-NRD concentrations shows that the E/M ratios increase more rapidly with the ternary TOT·ER·coactivator complex than with the binary apo-ER·coactivator complex (Fig. 8B
).
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A similar pattern was seen with SRC-1-NRD; however, Med220-NRD binding resulted in excimer formation to the same extent in both the TOT and E2·receptor·Med220-NRD tripartite complexes, suggesting a unique mechanism of interaction for the mediator family of coregulatory proteins compared with the p160 family (Fig. 7B) (21).
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DISCUSSION |
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Here, we have shown that the stabilizing effect of coactivator binding to an agonist·ER complex is manifest by an increase in fluorescence anisotropy of the MTMR-labeled receptor and an increase in resistance of receptor toward proteolysis (Fig. 1). The results from both of these assays suggest that the presence of a coactivator peptide causes a selective allosteric increase in the population of agonist-bound receptors with helix 12 conformationally locked in the agonist position leading to increasing helicity near residue 530 (13). More significantly, we have found that agonist·pyrene-labeled-C530-ER·coactivator complex develops a strong pyrene excimer emission, a phenomenon that we presume arises from a further conformational stabilizationeffected upon coactivator bindingthat enables the two pyrene fluorophores on the receptor dimer to form the excited state·ground state molecular contact complex that is required to produce the excimer emission. The presence of this excimer signal clearly highlights a novel ER conformation within the tripartite agonist·ER·coactivator complex that is absent in the binary agonist·ER conformation of the receptor (Fig. 3
).
We have termed the conformation of the LBD in this situation as active or costabilized, terms we feel are appropriately descriptive becausefrom the perspective of the ER-LBDthis is the conformation of the critical tripartite complex required for the transcriptional activation of estrogen-responsive genes. Our results clearly illustrate that coactivators are key contributors to the conformation of the ER; they do not merely aid in the locking of the ligand-stabilized conformation of the receptor (9). This finding is in contrast to the current hypothesis of NR action, which presumes that coactivator protein binding would not produce additional conformational changes in the receptor (9). It is of note that x-ray crystal structures also have not shown a novel coactivator-induced NR conformation; however, these structures are static pictures of crystallized protein components and are not well suited to reveal changes in the dynamics of protein structure, changes that are revealed here by protease sensitivity and specific fluorescence excimer emission methods. Thus, the molecular details and biological importance of coactivator protein contributing to novel NR conformations remain to be elucidated. Nevertheless, because in all of the crystal structures of ER agonist complexes the C530 positions in the dimer are separated by ca. 16 Å, whereas pyrene excimer emission requires that the two fluorphores be in parallel planes separated by approximately 10 Å, one can imagine that this costabilized conformation differs in important wayswith reduced C530-C530 distancefrom the conformations that have been observed by x-ray crystallography.
Generation of the excimer band has practical implications as well, because it enables one to study in a convenient and quantitative fashion, how changes in ligand and coactivator structure and concentration affect formation of this active receptor conformation and what types of inhibitors prevent its formation. This technique might provide a molecular sensor for elucidating how the structural features of coactivator components (number of NR-boxes and the flanking residues around the LXXLL motifs) control ER structural dynamics.
Ligand Structure and Coactivator Structure Each Play Essential yet Independent Roles in Formation of the Active ER-LBD Conformation
We find that the pyrene excimer emission, indicative of an active conformation of the ER-LBD, is most readily observed when an agonist ligand and p160 coactivators are both bound to the ER forming a ternary complex (Fig. 3). Ligand binding alone does not suffice, but interestingly high concentrations of coactivator proteins (SRC-1-NRD and SRC-3-NRD) are capable of inducing costabilized conformation in the binary apo-ER·coactivator complex, suggesting that the coactivator proteins behave as potent secondary endogenous ligands capable of controlling receptor structural dynamics in the absence of the primary estrogenic ligands (Fig. 8B
).
One of the current approaches for functional sorting of the coactivator proteins is to determine their ligand-dependent recruitment profiles and affinities with a particular NR (20, 35, 36). However, as Korach and co-workers (7) have suggested, under certain in vitro conditions, ER might recruit coactivators in a manner that is not transcriptionally productive; thus, coactivator affinity measured simply through conventional protein·protein proximity-based interaction assays might not necessarily relate to their transcriptional efficacy. A coactivator recruitment profile obtained through our pyrene-labeled C530-ER excimer formation assay, on the other hand, documents an aspect of receptor structure that goes beyond simple receptor·coactivator interaction. We believe that the ER excimer formation assay provides an assessment of a conformation of the LBD that is functionally productive, one in which the binding of a coactivator protein to an agonist·ER complex places helix 12 in a fully activated position and leads to the stabilization of an ER-LBD conformation with a C530-C530 intermonomer distance of less than 10Å, eliciting the pyrene excimer signal (Fig. 2). Curiously, the ER transactivation data supports the coactivator recruitment profile obtained through our excimer assay more closely than other proximity-based recruitment assays (e.g. FRET) (Fig. 7
) (33).
Coactivator proteins of different classes exhibit varying levels of binding potency to the E2·ER complex. Although the receptor interaction domains of SRC-1-NRD and SRC-3-NRD show somewhat different profiles of E2·ER activation, they are essentially similar to each other but distinctly different from Med220-NRD, as evidenced by the excimer signal development, with the latter being ligand independent with much lower potency and efficacy (Figs. 4A and 7B
). These differences might be directly linked to the distinctive biological roles of these two classes of coregulators in vivo. Kraus and co-workers (21), using cell-based and molecular genetic approaches, have recently reported functional differences between ER recruitment of SRC-2 and the mediator coactivator proteins, and they describe a functionally distinct role for Med220, because it is recruited to DNA-bound NRs with low affinity in a ligand-independent fashion, at a subsequent step after the binding and release of p160 proteins. The results of our pyrene excimer studies quantifying the capacity that various coactivator proteins have in eliciting a novel receptor conformation suggest that in addition to potency (affinity), the efficacy of E2·ER stabilization varies dramatically between and within different classes of coactivator proteins and that CBIs are incapable of providing this stabilization. Furthermore, the binding potency of the coactivator protein component (three NR-boxes, one NR-box, or small molecule CBI) to presumably the same coactivator groove on the receptor, is not the critical component in determining the efficacy that coactivator proteins have on E2·ER stabilization.
Of the coactivator components that we have studied, the single NR-box has intermediate potency, while exhibiting the highest efficacy of E2·ER stabilization (Fig. 4). Previous studies in our laboratory (10) and others (37, 38) have shown a 2:1 stoichiometry of receptor monomer to SRC-1-NRD (with three NR-boxes), and that in both SRC-1 and SRC-3, the NR-box-2 region exhibit high affinity for ER
-LBD (20). Two of the high affinity single NR-box-2 peptides may bind to an ER-LBD dimer (with a 1:1 peptide:monomer stoichiometry) and this may result in the higher level of receptor stabilization observed with these peptides containing a single NR-box-2, compared with the longer SRC proteins that as a 2:1 receptor:monomer SRC-1 complex with the ER dimer would need to utilize two different NR-boxes with varying affinities and efficacies of stabilization for the receptor. Thus, the coactivator content of cells might determine the actions of estrogens through varying effects on ER structural dynamics.
We also find that the significant differences in the affinity of a particular p160 coactivator protein binding to various agonist·ER complexes are highly dependent on the structural nature of the agonist ligand, with not all estrogenic ligands being equally effective in coactivator recruitment. In particular, it takes considerably higher concentrations of SRC-3-NRD to effect a stabilized, active conformation of ER-LBDs that are complexed with the agonists genistein and E1 compared with E2, E3, and CME2 (Fig. 5
). This indicates that the potency and efficacy of p160 coactivator-induced allosteric stabilization of the ER-LBD is less effective with some agonist ligands (genistein and E1), an observation we believe is a direct indication that the quality of coactivator interaction with the agonist·ER complex is also a determinant of ligand potency in terms of ER transactivation, in addition to, and independent of ligand affinity for the receptor (20). The rank order of the SRC-3-NRD costabilization of the various ER-agonist complexes follows the known rank order of their transcriptional effectiveness, with genistein and E1 being less effective activators of ER
transcriptional activity than E3, E2, and CME2.
We also show, using our ER conformation-based coactivator recruitment assay, that the tripartite agonist·ER·coactivator complex can be disrupted by two distinct mechanisms: 1) with an antagonist ligand through competition with the agonist ligand, and 2) with a CBI compound through competition with the coactivator protein (Fig. 6). It is of note that TAM may inhibit ER signaling through both of these mechanisms, as evident from recent crystallographic studies where TAM ligands reside within the ligand binding pocket and the coactivators binding groove of the receptor (personal communication with Dr. Thomas Burris, Eli Lilly, Indianpolis, IN). These results imply that the expression pattern and relative concentrations of different coactivator proteins might regulate the complex tissue-specific pharmacology of ER ligands.
High Concentrations of SRC-3-NRD Can Induce the Active Conformation of the ER-LBD even in TOT-Liganded ER or Apo-ER, a Model for the Development of TAM Resistance
TAM is one of the most widely used cytostatic anticancer agents in the treatment of ER-positive breast cancers (29, 31). Despite its clinical success and relatively low side effects (for a chemotherapeutic agent), TAM resistance develops in a large percent of women taking this partial agonist-antagonist ER ligand (39, 40, 41, 42). The development of TAM resistance is a major limitation in the treatment of ER-positive breast malignancies (30, 31). A tissue-specific increase in expression levels of coactivator proteins such as the amplified in breast cancer 1 (AIB1) coactivator protein (also known as SRC-3), has been implicated in the TOT·ER·coactivator tripartite complex formation and enhanced ER-reporter gene transcription, although direct molecular evidence for such binding interactions has been rather limited (34, 43).
It is clear that coactivator stabilization of the ER-LBD can occur even with certain SERM complexes and with apo-ER, despite the fact that it requires higher coactivator concentrations to stabilize SERM·ER than agonist·ER binary complexes (Fig. 8) (34, 43). Our results show, in fact, that the interaction of ER with TOT ligand and coactivator proteins is a dynamic process: there exists a competition between the effect of the TOT antagonist ligand to stabilize the inactivated form of the receptor, and the effect of the coactivator protein to stabilize receptor in the activated form. In the presence of low coactivator levels, the TOT antagonist ligand successfully stabilizes helix 12 in the inactivated extended form, preventing the costabilized receptor structure and precluding the excimer signal (Fig. 3B
). In the presence of high coactivator levels (low micromolar concentrations), however, the coactivator protein binding elicits a competing stabilization effect and places helix 12 of the TOT·ER complex in a partially activated conformation (compared with excimer signal of E2·ER complex), leading to shorter C530-C530 distances and the production of a clear, albeit weak excimer signal that is distinctly different than seen with apo-ER (Fig. 8A
).
The results presented here provide an in vitro model for the hypothesis that alterations in the cellular levels of coactivator proteins might be a factor contributing to TAM resistance observed in clinical settings, as has been suggested by a number of investigations (43). In the context of a cell, however, other signaling pathways [such as those involving MAPK (40, 41, 42) and PKA (44)] are thought to result in modifications (e.g. phosphorylations) of specific residues on the interacting partners, changing protein activities in a manner that enforces a receptor·coactivator interaction that supports the increased estrogenic effect of TAM seen in TAM resistance. Although we are obviously not replicating these covalent protein changes in our model system, our studies do establish that TOT·ER and the SRC-3-NRD, even without these proposed modifications, do have the inherent capacity to interact.
Curiously, in breast cancer, the onset of resistance to hormone therapy occurs more rapidly (35 yr) with TAM than with arimidex (an aromatase inhibitor) (41, 42, 45, 46). It is possible that the apo-ER conditions, as would be expected with arimidex therapy, are less prone to therapeutic resistance through an increase in coactivator protein levels than are seen with TAM therapy. These studies, along with recent cell-based ER transactivation findings, suggest a higher transcriptional output from the TOT·ER compared with the apo-ER complex in the presence of increased coactivator concentration in cells (34). Our pyrene E/M ratios, in agreement with these findings, show that the coactivator stabilization effects are stronger in the TOT·ER complex compared with the apo-ER complex (Fig. 8B). The apo-ER shows 27% lower E/M ratios at 5.3 µM SRC-3-NRD compared with TOT·ER (Fig. 7B
). The decreased levels of the costabilized conformation of the receptor observed in the absence of ligand is most likely due to the less structured features of the apo receptor, leading to longer C530-C530 distances in the dimer (13, 47). Interestingly, our findings also show that, in the presence of SRC-3-NRD, the pure antagonist, ICI 182,780·receptor complex has 30% lower E/M ratios compared with TOT·ER, suggesting a lower coactivator effect on ER when it is complexed with this pure antagonist than with the SERM TOT (Fig. 7B
). This is consistent with the therapeutic success of ICI 182,780 (Faslodex) in the treatment of TAM-resistant breast cancers (30, 31).
Spectroscopic Probes on NR-LBDs Can Function as Sensors of Conformation at this Critical Nexus of Ligand, Protein, and DNA Interactions
Using pyrene fluorescence, we have developed an exquisitely sensitive in vitro model system that clearly highlights the contributing effects of coactivator proteins on the stabilization of ER conformational dynamics. Our findings illustrate that coactivator protein recruitment is not simply a consequence of ligand-stabilized ER conformations; instead, these accessory proteins behave as secondary, cell-specific, endogenous ligands for the receptor and contribute to the formation of novel receptor topographical features that might be recognized by the cellular machinery leading to the transcription of ER-regulated genes. X-ray crystallographic studies with NRs show high structural similarities among this family of ligand-regulated transcription factors, suggesting that coactivator proteins might have similar contributing effects on the structure and function of other NRs.
Although this is a biophysical model system, it can be used to differentiate between the functionally distinct p160 and mediator families of coactivator proteins. An extension of this work would be to use our system as an in vitro model of TAM resistance by carefully quantifying the effects of various cofactors on the TOT·ER structural dynamics in the presence of different receptor mutations and/or posttranslational modifications (acetylation, phosphorylation, methylation).
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MATERIALS AND METHODS |
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Expression, Purification, and Site-Specific Fluorophore-Labeling of ER-LBD Constructs
The expression, purification, and site-specific MTMR, or N-(1-pyrene)-maleimide labeling of single reactive-cysteine ER-LBD (residues 304554) construct (C530, having C381S/C417S mutations) was conducted as described previously (11, 12, 13). The ligand binding characteristics of the unlabeled, and fluorophore-labeled ER constructs were reported previously (11, 12, 13). These constructs are labeled stoichiometrically, and they retain ligand binding and coactivator recruitment activities.
Coactivator Preparations
The SRC-1-NRD construct in pET-15b containing three NR-box regions and encoding for amino acids 629831 was described previously (10). A similar size region of SRC-3-NRD (residues 627829) was PCR-amplified using primers designed for the specific sites and subcloned into pET-15b plasmid using BamHI and NdeI restriction sites. A pET-15b construct of Med220-NRD (residues 527714) containing two NR-box regions was prepared using similar conditions. The DNA sequences were confirmed by dideoxy sequence analysis. The coactivator-NRDs were expressed and purified in the same fashion as was ER-LBD.
The preparation of the 15-amino acid peptide corresponding to SRC-1-NR-box-2* corresponding to amino acids (683696) was described previously (10). The 19-amino acid peptides corresponding to SRC-1 (amino acids 683701) and SRC-3 (amino acids 689706) NR-box-2 regions were synthesized by the University of Illinois Biotechnology Center. The eight-amino acid QSY-7-NR-box peptide (ILRKLLQE) was synthesized and labeled at the N terminus with QSY-7 carboxylic acid, succinimidylester (Molecular Probes, Eugene, OR) by the University of Illinois Biotechnology center. All peptides were purified using C18 reverse-phase HPLC and peptide quality was determined with analytical HPLC and mass spectroscopy (University of Illinois Biotechnology Center).
Fluorescence Anisotropy-Based Regional Dynamics and Trypsin Challenge Studies
The regional dynamics studies using MTMR-labeled C530-ER-LBD were conducted as described previously (13). Experiments were conducted with 5 µM ligand where indicated and 9 µM SRC-1-NR-box-2* or vehicle. The unit change in mA (anisotropy x 1000) with and without coactivator peptide were measured using the Spex II (model IIIc) fluorometer.
The trypsin challenge studies using MTMR-labeled C530-ER-LBD were conducted as described previously (13). Experiments were conducted with 10 µM ligand and 10 µM SRC-1-NR-box-2* or vehicle, whereas the anisotropy was followed with time using the Victor (2) V multilabel HTS counter.
FRET-Based Coactivator Peptide Recruitment Studies
MTMR-labeled C530-ER-LBD and QSY-7-NR-box peptide (ILRKLLQE) were used in the FRET assays. The QSY-7 is a nonfluorescent FRET acceptor for the MTMR-labeled ER. A 300-µl solution of ER (25 nM), with ligand (10 µM) or vehicle, and 0.3 mg/ml chicken ovalbumin in Tris-glycerol (pH 8.0) buffer was placed in separate wells of a black round bottomed 96-well Nunc polypropylene plate and incubated in the dark for 1 h at room temperature, after which the donor emission was measured with a 544/15 excitation and 590/10 emission filter pair using the Victor (2) V multilabel HTS counter. From a serial dilution of QSY-7 coactivator peptide in Tris-glycerol (pH 8.0) buffer, a 3-µl sample was removed and mixed with the 300 µl donor ER (<1.0% dilution). The samples were incubated for an additional hour at room temperature before measuring the donor emission again. Percent FRET data were calculated based on the donor intensity (50) with the following equation:
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Pyrene Excimer-Based Coactivator Recruitment Studies
All experiments were conducted with 30 nM pyrene-labeled C530-ER-LBD, 0.3 mg/ml chicken ovalbumin in Tris-glycerol (pH 8.0) buffer. The concentrations of ligand, coactivator-NRDs, coactivator-NR-boxes, or CBI for different experiments are shown in figure legends. The receptor was allowed to equilibrate with ligand and/or coactivator for 1 h at room temperature, in the dark. A sample from each experiment was loaded into a 5.0 x 5.0 mm quartz fluorescence cuvette and placed into the sample chamber of the Spex Fluorolog II (model IIIc) fluorometer. The sample chamber was held at a constant 25 C, whereas the sample was excited at 339 nm (5.0-mm slits), and emission monitored from 370570 nm (2.5-nm slits) under magic angle conditions (51). The excimer (E) to monomer (M) ratios were calculated based on a previously described procedure (16) using the (E) intensity at 498 nm and the (M) intensity at 386 nm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online January 20, 2005
Abbreviations: apo-ER, Unliganded ER; C530-ER, C417S/C381S double ER-LBD mutant; CBI, coactivator binding inhibitor; CME2, 11ß-chloromethyl estradiol; E1, estrone; E2, 17ß-estradiol; E3, estriol; ER, estrogen receptor; E/M, pyrene excimer/monomer intensity ratio; ERE, estrogen response element; FRET, fluorescence resonance energy transfer; K6, inhibition equilibrium constant; LBD, ligand binding domain; MTMR, tetramethylrhodamine-5-maleimide; NR-box-2*, SRC-1-NR-box-2 (amino acids 683696); NR, nuclear receptor; NRD, nuclear receptor domain region of coactivators that includes three NR-boxes for SRC-1-NRD and SRC-3-NRD and two NR-boxes for Med220-NRD; RBA, relative binding affinity; SERM, selective ER modulator; SRC, steroid receptor coactivator; TAM, tamoxifen; TOT, trans-4-hydroxytamoxifen.
Received for publication November 15, 2004. Accepted for publication January 13, 2005.
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REFERENCES |
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