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Molecular Endocrinology 17 (12): 2593-2602
Copyright © 2003 by The Endocrine Society

Molecular Sensors of Estrogen Receptor Conformations and Dynamics

Anobel Tamrazi, Kathryn E. Carlson and John A. Katzenellenbogen

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.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The ligand-induced conformation of a nuclear receptor ligand-binding domain (LBD) is a principal factor leading to transcriptional activity and determining the pharmacological response. Using the estrogen receptor (ER) LBD-labeled site specifically with a fluorophore, we demonstrate that effects of ligand binding on the conformation and dynamics of this domain can be studied directly, in a quantitative and convenient fashion, by various fluorescence methods. Estrogen ligands of different pharmacological character—agonists, selective ER modulators (SERMs), and pure antagonists—each produce distinctive spectroscopic signatures, characteristic of the conformational or dynamic features of their ER-LBD complexes. We can directly follow the equilibrium of helix 12 positions through the degree of local fluorophore rotational freedom and receptor helicity near the C terminus of helix 11. We observe differences even between ligands within a specific pharmacological class, such as the SERMs raloxifene and trans-4-hydroxytamoxifen, highlighting the ability of these fluorescent receptor sensors to detect unique ER conformations induced even by closely related ligands, yet ones that produce distinctive biological activities in estrogen target cells. Fluorophore-labeled LBDs can serve as versatile molecular sensors predictive of ligand pharmacological character and should be broadly applicable to other members of the nuclear receptor superfamily.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE NUCLEAR RECEPTOR (NR) superfamily comprises a large number of structurally related transcription factors whose activity is regulated by the interaction of hormonal ligands with their cognate ligand-binding domains (LBDs), and orphan receptors, whose ligands are currently not known. The activity of the estrogen receptor (ER) is regulated by ligands that vary from full agonists, such as the natural hormone estradiol (E2), to mixed agonist-antagonists (also termed selective ER modulators or SERMs), such as tamoxifen and raloxifene, used for breast cancer prevention and treatment (1, 2), to pure antagonists, such as ICI 182,780 or Faslodex, used in second line hormone therapy for SERM-resistant breast carcinomas (3, 4). The particular pharmacological character of each of these ligand classes derives from distinct conformations that they induce upon binding to the LBD of the receptor. These conformations, through their interactions with cellular cofactors and coregulator proteins, are responsible for controlling chromatin architecture and mediating the transcriptional activity of estrogen-responsive promoters (5, 6, 7, 8, 9).

The most detailed information on the conformation of ER and other NR-LBDs has come from x-ray crystallography. These investigations have shown that ligand-induced changes in the position of helix 12 control the shape and accessibility of a hydrophobic groove motif on the LBD surface that is a major site of coregulator interaction (5, 6, 7, 9, 10). In fact, the equilibrium between various helix 12 positions in the LBD is regarded as the molecular switch between the transcriptionally activated or inactivated forms of the receptor (11). NR-LBD crystal structures, however, are challenging to obtain; they do not reveal dynamic features of the LBD structure in solution, nor are they always predictive of the full pharmacological character of the bound ligand (12). Coregulator recruitment methods and designer binding proteins (monobodies) used to characterize particular ligand-induced conformational changes in NR-LBDs are indirect and provide only secondary information regarding structural features of the receptor (9, 13, 14). Dynamic stabilization studies can directly measure the extent of receptor stabilization by ligands and coregulators; however, they lack structural detail regarding the position of helix 12 adopted by the LBD (15, 16).

Fluorescence is a powerful method for characterizing protein conformation and dynamics. In this report, we present a biophysical approach by which one can directly monitor the conformations and dynamics of the estrogen receptor using site-specific fluorescent-labeled ER{alpha}-LBDs. These fluorophore-labeled receptors function as versatile molecular sensors through which distinctive spectroscopic signatures for ligand-induced changes in ER conformation can be obtained rapidly, quantitatively, and in solution. We show how these spectroscopic signatures are modulated in a characteristic manner by the binding of ER ligands from distinct pharmacological classes and how they might serve as versatile predictors of the pharmacological nature of novel ligands bound to the receptor.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Site-Specific Fluorescent-Labeled ERs as Molecular Sensors of Receptor Conformations and Dynamics
To study the conformation and dynamics of the LBD, we labeled our bacterially expressed ER{alpha}-LBD constructs (residues 304–554) with cysteine-specific fluorophores site-specifically at either cysteine 417 or 530; we refer to these constructs as C417-ER and C530-ER, respectively (17). These fluorophore-labeled constructs retain their native functionality in terms of ligand binding affinity and coactivator recruitment profiles (17). The two sites chosen for fluorophore labeling are natural cysteines, located either between helices 7 and 8 (C417) or near the C terminus of helix 11 (C530) (6, 18) (Fig. 1Go). The former site (C417) is in an ER-LBD region that has a similar helical conformation in all published ER structures and thus is considered to be a conformational control site. By contrast, the latter site (C530) undergoes distinct changes in orientation and secondary structure in different ER{alpha}-ligand structures (being helical in agonist complexes and in a loop in antagonist complexes), and thus is considered to be a conformationally sensitive site (6, 10) (agonist and antagonist compound structures are shown in Fig. 2Go).



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  		Fig. 1. Structural Transitions of ER{alpha}-LBD Dimers between Estradiol (E2) and TOT Bound Complexes

Residues 417 and 530 are illustrated in the agonist (E2, panel A) and mixed agonist-antagonist (TOT, panel B) conformations of ER. The positions of helix 12 are highlighted with rectangles in both structures. Figures were generated with Sybyl 6.7 (Tripos, St. Louis, MO) from the corresponding research collaboratory for structural bioinformatics protein data bank (RCSB-PDB, files names: 1ERE for E2 and 3ERT for TOT).

 


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  		Fig. 2. Structures and RBAs of Ligands used in this Study

A, Agonist ligands. B, Mixed agonist-antagonist or selective estrogen receptor modulators (SERMs). C, Pure antagonist ligands. RBAs for binding to the human ER{alpha}-LBD are presented in parenthesis and are expressed relative to that of estradiol, which is set at 100.

 
The helix-loop transition at C530 in ER{alpha} is most dramatically observed between the wrapped-back position of helix 12 in the fully activated ER{alpha}-agonist complexes and the extended position of helix 12 in the partially inactivated ER{alpha}-SERM complexes (10) (Fig. 1Go). There are no published crystallographic structures of ER{alpha}-pure antagonist complexes; however, the ERß-pure antagonist and peroxisome proliferator-activated receptor {alpha} (PPAR{alpha})-corepressor structures suggest that there might be yet another mechanism of NR inactivation that is elicited through an increase in the dynamic nature of helix 12, thus compromising the function of the coactivator binding groove in the LBD (5, 19). Because the mode of receptor inactivation with pure antagonist ligands (fully inactivated state) does not appear to place helix 12 in the extended ER-SERM conformation, the degree of helicity near the C-terminal end of helix 11 (near C530) is between the fully activated and partially inactivated receptor conformations (5).

The ligand-induced changes in the position of helix 12 (wrapped back or extended), which is transmitted to the position of C530 (helical or loose), can be probed by several fluorescent methods. Anisotropy can detect conformational changes representing alterations in fluorophore rotational flexibility associated with going from a rigid (helix) to a loose (loop) receptor environment, as well as size changes resulting from proteolysis, through which the protease digestion can be monitored in real time. The polarity-sensitive fluorophore, acrylodan (6-acryloyl-2-dimethylaminonaphthalene), can be used to measure polarity shifts in the receptor upon binding of different ligands. The combination of these fluorescent techniques, when interpreted in the context of the known x-ray structures of the ER-LBD, enables direct monitoring of the extent of receptor activation—from the fully activated state (agonist-bound) to the fully inactivated state (pure antagonist-bound)—along with the status of receptor structural features in the unliganded ER (apo-ER) form.

Fluorescence Anisotropy Reveals an Increase in Regional Dynamics at the End of Helix 11 Induced by SERMs
Fluorescence anisotropy (also referred to as fluorescence polarization), in addition to monitoring changes in molecular size, can also detect changes in local rotational flexibility. The anisotropy of emission from a fluorophore, site-specifically attached to a protein, provides quantitative information regarding the local rotational motion of the fluorophore and can be used to distinguish between rigid (helical) or fluid like (loop) environments near its point of attachment to the protein of interest (11, 20, 21, 22). The fluorophore size, fluorescence lifetime, and length of the linker between the fluorophore and point of attachment to the protein are critical factors that determine the degree to which one can monitor changes in regional dynamics using fluorescence anisotropy. Our experience with site-specific fluorescent-labeled ER{alpha}-LBDs suggest that tetramethylrhodamine-5-maleimide (MTMR) has structural features (a short linker with the maleimide group directly attached to the fluorophore) and a fluorescence lifetime (~2 nsec) that are appropriate for monitoring ligand-regulated changes in the degree of rotational dynamics in the molecular region of the ER near the fluorophore. When the fluorophore is attached at C530, the rotational dynamics of the fluorophore are affected by ligand binding. The fluorescence signal shows higher anisotropy values when the local protein environment is rigid (e.g. lower regional mobility or more helical) and lower values when it is more fluid (e.g. higher regional mobility or less helical) (20, 21).

Using the fluorescence anisotropy of C530-(MTMR) ER{alpha}-LBD, we followed ligand-induced helix-loop transitions at position 530 in the apo and various ligand-bound forms (Fig. 3AGo). The ER-agonist complexes showed higher anisotropy (consistent with greater helical character at C530) than did ER-SERM complexes, whereas the anisotropy values for the pure antagonist-receptor complexes (ICI compounds) are clearly different from SERMs and more similar to those of the agonists (see Discussion). We found that the range of fluorescence anisotropy values was large, spanning 70 mA units or approximately 20% of the theoretically possible range (22). The different degrees of local rotational motions detected by the fluorophore at C530 suggest that there are distinctive positions for helix 12 in the SERMs vs. pure antagonist receptor complexes.



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  		Fig. 3. The Regional Dynamics of ER{alpha}-LBD

Ligand-induced modulation in the regional dynamics of C530-MTMR (panel A) and C417-MTMR (panel B) detected through fluorescence anisotropy (mA = anistropy x 1000). Values are the mean ± range or the SEM of two to five experiments.

 
With this technique, we can also detect subtle differences in regional flexibility among members of the SERM category. The anisotropy value of raloxifene (RAL)-bound receptor is markedly higher than that of the other two SERMs [trans-4-hydroxytamoxifen (TOT) and pyrazole ethyl antagonist (PEAn) (23)] assayed (compound structures shown in Fig. 2Go). Biologically, RAL is the most antagonistic of the SERMs, and in our assay it appears to produce a conformation closer to the pure antagonists than to the other SERMs (Fig. 3AGo) (see Discussion). We also monitored the fluorescence anisotropy of C417-MTMR ER{alpha}-LBD and found, as expected, minimal ligand pharmacology-dependent changes in regional dynamics at this conformational control site (Fig. 3BGo).

The Extent of ER Tryspin Proteolysis Near Cysteine 530 Is a Sensor of the Helix 12 Position and Discriminates ER Agonists from Antagonists
Putting a new twist on an old technique, fluorescence anisotropy can provide an exquisitely sensitive and easily quantifiable means for measuring the progress of trypsin proteolysis by detecting the decrease in the size of the labeled receptor fragment and thereby distinguish subtle differences in ligand-induced changes going from a helix to a loop conformation (20, 21). Trypsin will cleave proteins more readily at Arg or Lys sites that are exposed in a loop region than those in a more structured, helical region (24). The MTMR fluorophore at C530-ER, positioned between K529 and K531, is ideally situated to monitor how ligand pharmacological character affects the progress of trypsin cleavage at these sites. Cleavage at K529 produces a small peptide fragment (<3 kDa, amino acids 530–554), containing helix 12 and the fluorophore-labeled C530 (25, 26, 27, 28, 29); thus, cleavage here represents a great reduction in the size of the fluorophore-labeled component from the initial 60-kDa LBD dimer. Because there is an inverse relationship between fluorescence anisotropy and the size of the fluorophore-labeled receptor fragment, we can use fluorescence anisotropy to monitor the progress of trypsin proteolytic release of helix 12 with the fluorescent-labeled cysteine 530 from the receptor in real time, and thereby identify with high sensitivity subtle ligand-induced changes in receptor conformation.

We find that the progress of trypsin proteolysis of helix 12 is remarkably sensitive to ligand binding and ligand pharmacological class. Agonist-bound complexes [E2, diethylstilbestrol, estriol, and pyrazole ethyl agonist (30)] are strongly protected against helix 12 cleavage (presumed to be at K529), whereas apo receptor, and complexes bound with SERMs TOT and PEAn are the least protected against helix 12 cleavage (Fig. 4Go).



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  		Fig. 4. The Extent of Apo and Ligand-Bound ER{alpha}-LBD Helix 12 Trypsin Proteolysis

A, Fluorescence anisotropy-based C530-MTMR ER trypsin proteolysis with a final trypsin concentration of 1 µg/ml. B, Comparison of mA (anisotropy x 1000) values of C530-MTMR ER after 7 h of limited trypsin proteolysis. Values are the mean ± SEM of four to eight experiments.

 
Because trypsin cleaves more slowly at sites in helical than in loop regions, these results are consistent with the higher degree of helicity of helix 11 near K529/K531 seen in the crystal structures of ER agonist complexes (Fig. 1Go), and with earlier analyses by SDS-PAGE (25, 29). The fluorescence anisotropy assay, however, can distinguish more subtle differences in helix 12 positions between the SERM and pure antagonist ER complexes, and even among different SERMs, a task not effectively done by SDS-PAGE nor clearly evident from x-ray analyses (25, 29).

The pure antagonist (ICI 182,780 and ICI 164,384)-bound ER complexes show an intermediate extent of helix 12 cleavage compared with E2 and TOT (Fig. 4Go), which suggests that there are distinctive conformations of helix 12 in solution for the fully inactivated (ER-pure antagonist) vs. partially inactivated (ER-TOT) states of receptor, respectively (5). The level of anisotropy for these intermediate states could reflect a variety of mechanistic possibilities, including cleavage occurring at K531 in some cases. In this case, the fluorophore at cysteine 530 would remain attached to the truncated, ligand-bound LBD, which is larger than the fluorescent-peptide resulting from cleavage at K529 thus resulting in anisotropy levels intermediate between those of the full-length LBD and of the fluorescent-peptide released from cleavage at K529. Interestingly, according to this assay, the SERM RAL appears to induce an ER{alpha}-LBD conformation in solution with topological characteristics quite similar to those of the pure antagonist ER complexes and different from TOT-bound receptor. This is consistent with the results of the previous rotational flexibility experiments and with the more complete pharmacological antagonist activity of RAL compared with TOT (31). It is of note that higher trypsin concentrations are required to cause a change in fluorescence anisotropy of C417 ER{alpha}-LBD, consistent with the fluorophore in this location being far from any conformationally sensitive trypsin cleavage sites (data not shown).

Apo ER{alpha}-LBD has Unique Loosely Structured Conformations that Can Be Sensed with a Polarity-Sensitive Fluorophore, Acrylodan
The spectral characteristics of acrylodan, a cysteine-specific fluorophore, are known to be sensitive to regional dielectric near the site of attachment to target proteins (32, 33), with the emission maximum shifting from 400 nm in highly hydrophobic to 570 nm in hydrophilic aqueous environments (32, 33, 34, 35, 36). We have used the spectral characteristics of our acrylodan-labeled C530-ER{alpha}-LBD construct to monitor ligand-induced shifts in polarity near this residue (Fig. 5Go).



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  		Fig. 5. Regional Polarity Detected near Residue 530 of ER{alpha}-LBD

A, Fluorescence emission spectra of C530-acrylodan ER in the apo and ligand-bound complexes. B, Relative C530-acrylodan fluorescence emission intensities at 442 nm. Denatured, Temperature denatured receptor (80 C for 15 min). Values are the mean ± range of two experiments.

 
The apo C530-acrylodan ER shows a unique emission spectrum composed of two distinct peaks, suggestive of a mix of a highly hydrophobic (442 nm) and a more hydrophilic (486 nm) environment (Fig. 5AGo). These two emission peaks have distinct excitation spectra, suggesting that the fluorophore in apo-ER has multiple conformations (data not shown). Interestingly, when ligands of any pharmacological character bind to the receptor, emission from C530-acrylodan ER shifts to the longer wavelength position (498–514 nm), characteristic of a more hydrophilic environment (Fig. 5BGo), with the highly hydrophobic environment (442 nm) seen in apo-ER being completely abrogated.

C530-acrylodan receptor that was heat denatured (80 C for 15 min) and no longer able to bind ER ligands exhibits a hydrophobic environment similar to that seen with the apo receptor (Fig. 5BGo). This suggests that, in the absence of ligand, the receptor has a collapsed or loosely structured (molten globule-like) conformation (37). C417-acrylodan ER showed ligand-induced changes in emission similar to those of C530-acrylodan ER (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The conformation of the ligand-binding domains (LBDs) of nuclear receptors is a critical determinant of the pharmacological response that ultimately results from ligand binding (19, 38, 39, 40, 41). In our approach to study nuclear receptor conformation and dynamics by fluorescence, we have attached the fluorophore directly onto ERs, which is fundamentally different from approaches that use fluorescent-labeled antibodies or peptides to study NR conformations indirectly through coregulator interactions (42). By attaching the fluorophore directly onto the LBD, we have transformed the ER into a molecular sensor through which we can probe directly its conformational and dynamic state with high sensitivity. By proper selection of the fluorophore and site of attachment on the receptor LBD, one can obtain spectroscopic signatures that are diagnostic of the various receptor conformations and dynamics engendered upon the binding of ligands of various structures and predictive of their pharmacological class. We find that the position of helix 12 is dynamic in nature, and we can directly follow the equilibrium of its position from the fully activated to the fully inactivated forms through the effects of this helix on the degree of local rotational freedom and helicity near the C terminus of helix 11.

We have used this approach to measure ligand-regulated regional flexibility, protease digestion, and local polarity of fluorophore-labeled native cysteine residues within the ER{alpha}-LBD. In each case, we have found spectroscopic differences that are characteristic of ligands from distinct pharmacological classes (Table 1Go). Our findings are consistent with the detailed conformations revealed by crystallographic structures of various ER-LBD ligand complexes, when they are available, and they suggest conformations or dynamics when they are not. In some cases, we have noted distinctions between members within the same pharmacological class, such as between the SERMs RAL and TOT, which are reflective of known differences in their biological character (31), but differences that were not readily apparent from coregulator recruitment or receptor structural studies (29, 43). Furthermore, our method is rapid and convenient, and it operates in solution under equilibrium conditions. It is of note that in a recent study by Schwabe and co-workers (11), fluorescence anisotropy with a fluorophore-labeled peroxisome proliferator-activated receptor-{gamma} LBD was used to study the stabilization of domain conformational dynamics by ligand and coactivator binding.


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  		Table 1. Summary of Biophysical-Based Results of Site-Specific Fluorophore-Labeled C530-ER{alpha}-LBD

 
Spectroscopic analysis of apo C530- and C417-acrylodan ER{alpha}-LBD showed unique structural features, different from those of any of the ligand-bound receptor complexes (Fig. 5Go). We are currently developing assays to measure binding affinities of ER ligands through the regional polarity changes observed in the acrylodan-ER spectra from apo to ligand-bound forms. The acrylodan emission spectrum of apo-ER suggests that the lower half of the ligand binding domain (37) of the apo receptor (near residues 417 and 530) is loosely organized, exposing some hydrophobic features that shield the tethered fluorophores from the aqueous environment, at least to some degree. Upon ligand binding, this lower subdomain undergoes substantial structural rearrangement that displaces the tethered fluorophore into a hydrophilic environment, shifting the acrylodan emission peaks to longer wavelength (Fig. 5Go). Consistent with this model, the high fluorescence anisotropy values of MTMR-labeled C530- and C417-ER{alpha}-LBD in the absence of ligand are suggestive of rotationally restricted fluorophores, similar to the values observed with agonist complexes (Fig. 3Go). The combined results of fluorescence anisotropy (probing fluorophore rotational flexibility, Fig. 3Go), trypsin proteolysis (probing degree of helicity, Fig. 4Go), and acrylodan fluorescence (probing regional polarity, Fig. 5Go), however, suggest that the MTMR fluorophore is packed against hydrophobic surfaces of the apo receptor, whereas in the agonist complexes the decreased rotational flexibility of the fluorophore (higher anisotropy) is due to the higher degree of helicity in the molecular region of receptor near C530.

These distinct topographical features of apo receptor provide experimental evidence for our previous proposal that the lower subdomain of apo ER{alpha}-LBD can adopt the characteristics of a protein molten globule (37), which allows for efficient ligand association and dissociation, as well as with other reports that apo-NR-LBDs are loosely folded (11, 44, 45). Heat shock proteins or other cellular factors that recognize the exposed hydrophobic character of the apo-LBD could thus determine the cellular location and activation state of receptor in the absence of ER ligands. In fact, McDonnell and co-workers (9) have identified peptide sequences that recognize only apo-ER.

Acrylodan-labeled ER, therefore, provides a spectroscopic signature that can be used to distinguish apo-ER{alpha}-LBD from a complex of ER with any ligand. By contrast, fluorescence anisotropy-based regional dynamics and trypsin proteolysis of MTMR-labeled ERs can identify ligand-induced receptor conformations that are characteristic of known ligand pharmacological classes (i.e. agonists, mixed agonist-antagonists/SERMs, and pure antagonists), without having to perform transcription or coactivator recruitment assays (Figs. 3AGo and 4Go). These characteristics are summarized in Table 1Go. Whereas a single fluorophore/fluorescence experiment does not always lead to a specific distinction among ligands from all pharmacological classes, a combination of experiments does provide such distinctions clearly.

The results from our experiments with C530-ER{alpha}-LBD, which probe conformational dynamics (Fig. 3Go) and protease sensitivity (Fig. 4Go), show C530 to be in a helix or loop region, depending on the nature of the bound ligand and the stabilized position of helix 12. These are consistent with the length of helix 11 in x-ray structures of ER{alpha}-ligand complexes: Helix 11 is longest in the E2 structure (ends at C530) and shortest in the TOT structure (ends at Y526), with the RAL structure being intermediate (ends at M528). The only ICI structure is with ERß, but in that structure the end of helix 11 (K480) corresponds to K529 in ER{alpha}, and thus is similar to that of RAL-ER{alpha}.

Comparison of apo and TOT-ER{alpha}-LBD conformations reveal an interesting pattern of similarities and differences (Table 1Go). The trypsin challenge experiments show secondary structural similarities near C530 between apo and TOT-bound receptor, suggesting that helix 12 in the apo receptor might adopt an extended conformation leading to reduced helicity near C530, similar to the ER-TOT complex (Fig. 4Go). The regional polarity (Fig. 5Go) and regional dynamics (Fig. 3AGo) data, however, demonstrate that apo receptor has features that are clearly distinguishable from the ER-TOT complex. The C530-tethered fluorophores pack against the collapsed and loosely structured apo receptor binding pocket but not in the ER-TOT complex, as seen through the decreased rotational freedom of MTMR (higher anisotropy) and the highly hydrophobic environment detected by acrylodan in the apo receptor compared with the ER-TOT complex. The subtle structural distinctions between the apo and the ER-SERM complexes might govern their selective interactions with heat shock proteins, corepressor proteins, or other cellular coregulators in ER-responsive tissues.

Our biophysical approach also allows for discrimination between mixed agonist-antagonist (TOT, PEAn, and RAL) and pure antagonist (ICI 182,780 and ICI 164,384) ligands (Table 1Go), a task fundamentally not feasible through standard coactivator-recruitment assays (43). Regional flexibility (Fig. 3AGo) and the extent of helix 12 proteolysis (Fig. 4Go) are reflective of unique positions of helix 12 in the presence of mixed agonist-antagonist and pure antagonist ligands, suggesting distinct mechanisms of receptor inactivation with these ER antagonist ligands of varying efficacies (5). Incidentally, we find conformational similarities between RAL-bound and ICI-bound receptor complexes, which is concordant with the more complete antagonist character of RAL compared with TOT (31), but which is not clearly apparent from a comparison of RAL, ICI, and TOT crystal structures (5, 10). Thus, it appears that the spectroscopic signatures we can obtain from our fluorescent-labeled ERs should serve as convenient predictors of the pharmacological character of novel synthetic ER ligands and hormonal substances found in the environment.

A striking feature of the physiological responses observed with estrogens and antiestrogens is their tissue-selective activation or inactivation of ER-dependent signaling cascades. Even within specific ER-responsive tissues, different antiestrogens exhibit markedly varying efficacies in terms of receptor inactivation and biological response (8), an aspect that leads to their specific clinical uses in treatment of osteoporosis (raloxifene), and first-line (tamoxifen), or second-line (ICI 182,780) treatment of breast cancer. Our in vitro receptor conformational studies appear to constitute a model system through which subtle differences in the observed in vivo pharmacological nature of ER ligands can be deciphered through their tightly regulated effect on the dynamic and conformational properties of ER-ligand complexes. We monitor these effects through site-specific, fluorophore-labeled ER-LBDs, acting as molecular sensors. Because these receptor conformations are critical determinants of the subsequent array of interactions that ER has with cellular coregulators and promoter elements of ER-response genes and the ultimate transcriptional response (8, 9, 13, 25), the spectroscopic signatures we observe can be related, as well, to the pharmacological characteristics of the ligands. We are also currently using our fluorescent ERs to investigate how different classes of coactivator proteins recognize and further stabilize the conformation and dynamics of the receptor as a possible mechanism of the observed tissue-selective activity of ER signaling pathways. It is anticipated that other NR family members can be site-specifically labeled with fluorophores, as we have done here with ER, to investigate how the structure and function of their LBDs are regulated by their cognate ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Compounds and materials were obtained from the sources indicated: estradiol ([3H]E2, 54 Ci/mmol) (Amersham, Piscataway, NJ), estradiol, estriol, trans-4-hydroxytamoxifen, and TPCK-treated trypsin (Sigma, St. Louis, MO), ICI 182,780 (Tocris Cookson, Ballwin, MO), MTMR, 5-iodoacetamidofluorescein, and acrylodan (Molecular Probes, Eugene, OR), black polypropylene round bottom 96-well microtiter plates and polyolefin clear sealing film (Nalge Nunc, Rochester, NY). Raloxifene, Pyrazole Ethyl Antagonist, Pyrazole Ethyl Agonist, and diethylstilbestrol were synthesized in our laboratory (23, 30). ICI 164,384 was a gift from Dr. Alan Wakeling (Astra-Zeneca, Macclesfield, UK). Fluorescence experiments used a Spex Fluorolog II (model IIIc) cuvette-based fluorometer with Data Max 2.2 software (Spex Industries, Edison, NJ) or a PerkinElmer Life Science Wallac Victor2 V 1420 multilabel HTS counter with Wallac 1420 workstation software (PerkinElmer, Boston, MA). All data were analyzed using Prism 3.00 (GraphPad Software, San Diego, CA). The error bar indicated for each fluorescence value is derived from a number of separate determinations, as specified in the figure legends. In each case, when we have made a comment in the text about differences in fluorescence values, typically between ERs complexed with members from different pharmacological classes, we have determined that these differences are highly significant (P < 0.02), using Student’s t test. The relative binding affinity (RBA) of the ligands was determined using baculovirus expressed full-length human ER{alpha} (PanVera, Madison, WI) or ER{alpha}-LBD, using methods reported earlier (46, 47). Essentially identical RBA values were obtained using either full-length ER{alpha} or ER{alpha}-LBD.

Expression, Purification, and Site-Specific Fluorophore-Labeling of ER{alpha}-LBD Constructs
The expression, purification, and site-specific MTMR, 5-iodoacetamidofluorescein, and acrylodan labeling of single reactive-cysteine ER{alpha}-LBD constructs (C417 and C530, having C381S/C530S and C381S/C417S mutations, respectively) were conducted as described previously (17). The ligand binding characteristics of the unlabeled, MTMR, and 5-iodoacetamidofluorescein-labeled ER constructs were reported previously (17); the estradiol equilibrium dissociation constant of acrylodan-labeled C417- and C530-ER constructs were 1.47 and 1.23 nM, respectively. These constructs are labeled stoichiometrically, and they retain ligand binding and coactivator recruitment activities (17).

Trypsin Challenge Studies with Receptor Preparations
A solution of 5 nM MTMR-ER{alpha}-LBD (C530 or C417), 50 nM unlabeled-ER{alpha}-LBD in Tris-glycerol (pH 8.0) buffer (50 mM Tris-HCl, 10% glycerol) was allowed to undergo monomer exchange (17, 48) for 5–7 h at RT in the dark, so that each ER dimer contained only one fluorescent-labeled monomer, minimizing homofluorescence resonance energy transfer (homoFRET) artifacts (48, 49, 50, 51). Samples (300 µl) were placed in microtiter plate wells and mixed with 4.3 µl of vehicle or 700 µM ligand stock, resulting in 10 µM final ligand concentration. After equilibration for 1 h at room temperature in the dark, 3.75 µl of a freshly prepared stock of trypsin in 1 mM HCl was quickly added using an eight-channel pipette, and the plate was covered with sealing film and placed in the Wallac Victor2 V 1420 fluorometer. Fluorescence anisotropy was followed over time with a 544/15 nm excitation and 590/10 nm emission filter pair at 25 C.

Fluorescence Anisotropy-Based Regional Dynamics Studies
A stock sample of 2 nM MTMR-ER{alpha}-LBD (C530-ER or C417-ER), 100 nM unlabeled-ER{alpha}-LBD, and 0.3 mg/ml chicken ovalbumin in Tris-glycerol (pH 8.0) buffer was allowed to undergo monomer exchange for 5–7 h at room temperature in the dark, to minimize homo-FRET artifacts (48, 49, 50, 51). A 700-µl sample was placed in separate tubes and 5 µl of vehicle or 700 µM ligand stock was added, resulting in 5 µM final ligand concentration. After equilibration for 1 h at room temperature in the dark, a sample was placed in the Spex Fluorolog II fluorometer at 25 C, equipped with an L-configuration polarization unit. Excitation was at 541 nm and MTMR fluorescence anisotropy was monitored at 580 nm.

Acrylodan-ER Regional Polarity Studies
A stock sample of 10 nM acrylodan-ER{alpha}-LBD (C530 or C417), 100 nM unlabeled-ER{alpha}-LBD, and 0.3 mg/ml chicken ovalbumin in Tris-glycerol (pH 8.0) buffer was allowed to undergo monomer exchange for 5–7 h at RT, in the dark. A 700-µl sample of this stock was placed in separate tubes and 5 µl of vehicle or 700 µM ligand stock was added, resulting in 5 µM final ligand concentration. After equilibration for 1 h at room temperature in the dark, a sample was placed in the Spex Fluorolog II fluorometer at 25 C. Excitation was at 391 nm and acrylodan emission monitored at 420–600 nm under magic angle conditions (52). A cysteine-unreactive form of acrylodan (called prodan) shows essentially no specific binding affinity for ER{alpha}-LBD, having a RBA of less than 0.007% (vs. estradiol, RBA 100%).


   FOOTNOTES
 
This work was supported by a grant from the NIH (United States Public Health Service 5R37 DK15556).

Abbreviations: Acrylodan, 6-Acryloyl-2-dimethylaminonaphthalene; apo-ER, unliganded ER; C530-ER, C381S/C417S ER{alpha} construct; C417-ER, C381S/C530S ER{alpha} construct; E2, estradiol; ER, estrogen receptor; LBD, ligand-binding domain; MTMR, tetramethylrhodamine-5-maleimide; mA, anisotropy units x 1000; NR, nuclear receptor; PEAn, pyrazole ethyl antagonist; RAL, raloxifene; SERM, selective ER modulator; RBA, relative binding affinity; TOT, trans-4-hydroxytamoxifen.

Received for publication June 19, 2003. Accepted for publication August 20, 2003.


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 RESULTS
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 MATERIALS AND METHODS
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Nuclear Receptors:   ERα
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen  |  Raloxifene



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