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Molecular Endocrinology, doi:10.1210/me.2004-0256
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Molecular Endocrinology 19 (1): 25-34
Copyright © 2005 by The Endocrine Society

Analysis of Ligand-Dependent Recruitment of Coactivator Peptides to Estrogen Receptor Using Fluorescence Polarization

Mary Szatkowski Ozers, Kerry M. Ervin, Corrine L. Steffen, Jennifer A. Fronczak, Connie S. Lebakken, Kimberly A. Carnahan, Robert G. Lowery and Thomas J. Burke

Invitrogen Corporation, Madison, Wisconsin 53719

Address all correspondence and requests for reprints to: Mary Ozers, Ph.D., Invitrogen Corporation, 501 Charmany Drive, Madison, Wisconsin 53719. E-mail: Mary.Ozers{at}invitrogen.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ER{alpha} or ERß in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ER{alpha} and ERß with affinity ranging from 20–500 nM. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17ß-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17ß-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERß was observed in the presence of the ERß-selective agonist diarylpropionitrile, but not the ER{alpha}-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ESTROGEN RECEPTOR (ER) is a transcription factor that belongs to the nuclear receptor superfamily and mediates activation of estrogen-responsive genes. In the current model of ER action, ligand binding induces a conformational change in ER leading to a cascade of events including dissociation of bound repressor proteins, recruitment of coactivator proteins possessing histone acetylase activity, local unwinding of chromatin, and subsequent binding of Pol II and other transcriptional factors (1). Both ER{alpha} and ERß have an N-terminal transactivation domain, a highly homologous DNA-binding domain, and a C-terminal ligand-binding domain (LBD) containing a second transactivation activity. The two ER isoforms possess 58% amino acid identity in their LBDs with some differences in their ligand binding specificities (2, 3). The sites needed for interaction with coactivator proteins have been localized to the transactivation region of the ER LBD. Coactivator proteins such as SRC-1/NCoA-1, SRC-2/GRIP1/TIF2/NCoA-2, and SRC-3/ACTR/AIB1/p/CIP/RAC3/TRAM1 of the p160 family of coactivators interact with steroid hormone receptors via an {alpha}-helical LXXLL motif of the coactivator protein, where L is leucine and X is any amino acid (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). Crystal structure studies have indicated that helix 12 of the LBD plays an integral role in interaction with coactivator proteins (17). When agonist is bound to ER, helix 12 adopts a conformation forming a hydrophobic pocket around the bound ligand and a surface for interaction with coactivator proteins. Helix 12 in antagonist-bound ER repositions to occupy the coactivator binding sites, precluding interaction with coactivator proteins (17, 18).

Several studies have used coactivator proteins, peptides, or LXXLL motifs to explore ligand-dependent conformational changes in steroid hormone receptors and the binding characteristics between receptors and coactivators (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29). Experiments conducted with LXXLL motifs isolated by random phage display via agonist-dependent interaction with ER{alpha} or ERß indicated that these short peptides were able to differentiate between ligand-induced conformational changes in the receptor (20, 21, 22). Sequences flanking the LXXLL motif were shown to play a role in specificity of binding to steroid hormone receptors (20). LXXLL peptides that were recruited to ERß, but not ER{alpha}, were also identified (24). Several non-LXXLL peptides have been isolated by random phage display and shown to interact with full-length ER or the ER LBD occupied with 17ß-estradiol or antagonists such as tamoxifen (28, 30). Recently, a fluorescence polarization competition assay using purified ER LBD and a fluorescently labeled, eight-amino acid LXXLL peptide was employed by Rodriguez and co-workers (29) to identify pyrimidine-based small molecule inhibitors of the coactivator-ER interaction.

To further investigate the affinity and dose dependency of the ER-coactivator interaction, this report describes the development of a fluorescence polarization assay to examine the ligand-dependent recruitment of a series of coactivator-like peptides to ER. Fluorescence polarization assays offer a number of advantages for studying biomolecular interactions at equilibrium in a homogeneous, high-throughput format (31). This technique was used to detect ligand-dependent recruitment of short LXXLL-containing peptides to ER{alpha} or ERß in either an equilibrium-binding or dose dependency format and was able to predict the agonist/antagonist character of the ligands tested.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Binding of ER to the fluorescently labeled LXXLL peptide was detected using fluorescence polarization (Fig. 1Go). Light emitted from plane-polarized excitation of a fluorescent label on a molecule of interest provides an indication of the molecular volume of the complex (for review, see Ref. 32). Larger complexes rotate more slowly in solution, resulting in more of the emitted light in the same plane as the original excitation light and a larger polarization value. Uncomplexed reporter molecules rotate more quickly in solution, yielding a smaller polarization value, because more of the emitted light is depolarized. When agonist-occupied ER binds to the fluorescently labeled LXXLL peptide, the complex formed has a higher polarization value than the unbound LXXLL peptide. The altered position of helix 12 in the antagonist-occupied ER conformation results in a lower affinity for the LXXLL peptide and a larger fraction of unbound LXXLL peptide, yielding a lower polarization value.



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  		Fig. 1. Principle of the ER-Coactivator Peptide Assay

Fluorescence polarization is proportional to the molecular volume of the fluor molecule. Agonist-bound ER adopts a favorable conformation for interaction with the fluorescently labeled LXXLL peptide, and the resulting larger complex has a higher polarization value. Antagonist-bound ER has a lower affinity for the fluorescent LXXLL peptide, yielding a larger fraction of unbound LXXLL peptide and thus a smaller polarization value. Only one bound LXXLL peptide/ER is depicted for simplicity in the diagram. E2, 17ß-estradiol; ICI, ICI-182,780.

 
The peptides (Table 1Go) tested contain an LXXLL motif (underlined) flanked by either natural coactivator sequences from the p160 class of coactivators (second LXXLL motif) or random sequences isolated by phage display (4, 10, 20, 24). The peptides were labeled at the N terminus with either fluorescein (F) or rhodamine (Rh). The phage-display sequences were grouped into three classes based on the resemblance of their LXXLL flanking sequences to known coactivator sequences (in parentheses). A fourth group of ERß-specific peptides was also tested. The affinity of the LXXLL peptide for the ligand-occupied ER was measured using fluorescence polarization by varying the concentration of ER in the presence of 1 nM fluorescein-labeled LXXLL peptide and saturating amounts of ligand. A fluorescein-labeled random phage display peptide, F-D22, had a dissociation constant (Kd) of 46 nM for 17ß-estradiol-occupied ERß (Fig. 2Go). The rank order of Kd values for the ERß-F-D22 interaction from highest affinity to lowest, based on ligand occupancy, was: 17ß-estradiol, genistein, estrone. Unliganded ERß in buffer with dimethylsulfoxide (DMSO) solvent did have measurable affinity for the LXXLL peptide, suggesting that either a population of the purified protein was in an active conformation or that interaction with the LXXLL peptide induced a conformational change that stabilized peptide binding. Similar ligand-independent binding of ER to coactivator peptides/proteins has also been observed in other studies using purified ER{alpha} and/or ERß in a time-resolved fluorescence in vitro assay with coactivator peptides (25), with real-time interaction analysis using surface plasmon resonance involving full-length coactivator proteins (33), and in a flow cytometric multiplexed binding assay using both random phage display peptides and peptide sequences from cofactor proteins (28). Binding of F-D22 to ERß occupied with ICI-182,780 (full antagonist) or 4-hydroxytamoxifen (partial antagonist) was not detected under the conditions tested. The affinity of F-C33, another random phage display peptide resembling TRAP220 or RIP140, for ligand-occupied ERß was also measured (Fig. 3Go). The rank order of affinity of ligand-occupied ERß for F-C33, based on ligand bound to ERß, was: estrone, 17ß-estradiol, genistein. Similar to F-D22, there was low level affinity of unliganded ERß for F-C33 and no detectable binding of F-C33 to ERß in the presence of ICI-182,780 and 4-hydroxytamoxifen. D22 and C33 were able to compete with one another for binding to ERß (data not shown), indicating that these coactivator-like peptides bind to the same interaction pocket on ERß.


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  		Table 1. Fluorescently Labeled LXXLL Peptides

 


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  		Fig. 2. Affinity of ERß for F-D22 in the Presence of Various Ligands

Purified ERß was serially diluted into ERß coactivator equilibrium binding assay buffer with final reaction conditions containing 5 µM ligand, 1 nM F-D22 fluorescein-labeled peptide, and 2% DMSO. The samples were incubated at room temperature for 1 h. Fluorescence polarization was measured in the TECAN Polarion instrument. A representative experiment of five replicates is shown. Values reported are Kd values for the interaction of ligand-occupied ERß (ligand listed) and F-D22. The sequence of F-D22 is shown on the graph. ND, Not determined; DMSO, unliganded ER in buffer plus DMSO solvent.

 


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  		Fig. 3. Affinity of ERß for F-C33 in the Presence of Various Ligands

These experiments were performed using the same conditions as described in Fig. 2Go, except that F-C33 was used as the LXXLL peptide. The sequence of F-C33 is shown on the graph. A representative experiment of two replicates is shown.

 
Several of the random phage display peptides with the highest affinity for ERß and the best dynamic range were selected for further analysis (Table 2Go). Under the buffer conditions tested, the random phage display LXXLL peptides bound with higher affinity than the peptides derived from the second LXXLL motif of the natural coactivators, SRC-1 and AIB1. The affinity of the natural coactivator sequences for ER could not be accurately measured because the concentration of purified ER required to detect this low-affinity interaction was too high to achieve in the experimental conditions tested (data not shown). These data indicated that ER had a different affinity for the fluorescently labeled LXXLL peptides depending on its ligand occupancy, indicating some selectivity between ligand-induced receptor conformations. For example, ERß had the highest affinity for F-D22 when bound with 17ß-estradiol whereas estrone-occupied ERß had the highest affinity for F-C33.


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  		Table 2. Affinity of Ligand-Occupied ERß or ER{alpha} for LXXLL Peptide

 
After buffer optimization, similar equilibrium binding experiments were conducted using purified ER{alpha} or ERß and rhodamine-labeled D22 peptide (Rh-D22). Use of a fluorescent tracer in the red region is often desirable to eliminate background fluorescence in the green/fluorescein region found in compound libraries. The affinity of 17ß-estradiol-occupied ER{alpha} for Rh-D22 was measured as 31 ± 2 nM (Table 2Go).

Using the affinity measurements for the LXXLL peptide-ER interaction, dose dependency experiments were designed to determine the amount of ligand necessary to promote or disrupt the binding of the LXXLL peptide to ligand-occupied ER. The concentration of ER and fluorescent LXXLL peptide tracer in the dose dependency experiments were chosen based on the amount of ER from the equilibrium binding experiments that resulted in 75–90% of tracer bound, yielding higher polarization values for agonist-bound ER-tracer complexes and lower polarization values for unbound tracer in the presence of antagonist-occupied ER. Accordingly, ligand was serially diluted into reaction buffer containing 75 nM ER and 1 nM fluorescent LXXLL peptide. The addition of ligand results in a population of conformationally altered ER-ligand complexes with different affinity for the LXXLL peptide than the unliganded receptor. The EC50 values represent the point at which this population is sufficient to cause an increase (for agonists) or decrease (for antagonists) of 50% in the amount of LXXLL tracer bound. Thus, these EC50 values reflect both the affinity of ligands for ER and the affinity of liganded receptor for the LXXLL peptide. Because the tracer is the limiting factor at 1 nM, this assay provides a means to measure even very potent (low nanomolar EC50) ligands. ERß occupied with agonists such as 17ß-estradiol, estriol, genistein, and estrone formed a complex with the LXXLL peptide, yielding a larger fraction of bound LXXLL peptide and a higher polarization value compared with unliganded ERß (Fig. 4AGo). The LXXLL peptide was not recruited to ERß occupied with antagonists such as ICI-182,780, 4-hydroxytamoxifen, tamoxifen, and clomiphene, resulting in unbound LXXLL peptide and a lower polarization value compared with unliganded ERß. The dose dependency experiments provided distinct EC50 values for each ligand and correlated well with the known biological function of the ligands as an agonist or antagonist. The predictive value of this assay for discriminating between agonists and antagonists was also demonstrated for ER{alpha} (Fig. 4BGo). The low level of binding of unliganded ER to Rh-D22 in buffer plus ligand solvent (DMSO or methanol) was detected as an intermediate constant polarization value between the ligand-occupied ER-Rh-D22 complex (high polarization) and unbound Rh-D22 (low polarization). Compounds that had little affinity for ER{alpha}, such as testosterone, produced similar results to the unliganded receptor.



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  		Fig. 4. Dose Dependency of the ER/Rh-D22 Interaction

Ligand was serially diluted in ERß coactivator dose dependence assay buffer in a 96-well plate with final reaction conditions containing 75 nM ERß and 1 nM Rh-D22 in a 100 µl volume (A) or in complete ER{alpha} coactivator assay buffer in a 384-well plate with the final reaction conditions containing 75 nM ER{alpha} and 1 nM Rh-D22 in a 40 µl volume (B). The samples were incubated for 1 h at room temperature. Fluorescence polarization was measured in the TECAN Ultra instrument. Values reported are ligand EC50 values for the ER/Rh-D22 interaction. ND, Not determined; DMSO, unliganded ERß in buffer plus DMSO solvent; MeOH, unliganded ER{alpha} in buffer plus methanol solvent.

 
The ERß-selective agonist, diarylpropionitrile (DPN), was compared with propyl pyrazole triol (PPT), an ER{alpha}-selective agonist, in the ERß coactivator assay with 17ß-estradiol and 4-hydroxytamoxifen included as a control agonist and antagonist, respectively (Fig. 5AGo) (23, 34, 35). As expected, DPN binding to ERß resulted in recruitment of Rh-D22 to ERß, whereas addition of PPT to the assay resulted in unliganded receptor and no detectable binding of the LXXLL peptide to ERß. In the complementary experiment using DPN and PPT in the ER{alpha} coactivator assay, both DPN and PPT behaved as agonists (Fig. 5BGo). Meyers and co-workers (34) have shown that DPN can act as an agonist on both ER{alpha} and ERß, but DPN has a 70-fold higher relative binding affinity for ERß over ER{alpha} in radioactive binding assays using full-length receptor. DPN was also reported to be 170-fold more potent with ERß than ER{alpha} in transcriptional assays (34). In three independent ER{alpha} coactivator dose dependency experiments, PPT was more potent than DPN in eliciting recruitment of the Rh-D22 peptide to ER{alpha}, but DPN consistently behaved as an agonist of ER{alpha} (Fig. 5BGo and data not shown).



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  		Fig. 5. Dose Dependency of ERß-Selective and ER{alpha}-Selective Agonists

Ligands were serially diluted in ERß coactivator dose dependence assay buffer with final reaction conditions containing 75 nM ERß and 1 nM Rh-D22 (A) or complete ER{alpha} coactivator assay buffer with final reaction conditions containing 75 nM ER{alpha} and 1 nM Rh-D22 (B) in a 384-well plate. The samples were incubated for 1 h at room temperature. Fluorescence polarization was measured in a TECAN Ultra instrument. Values reported are ligand EC50 values for the ER/Rh-D22 interaction. A representative example of three to four independent experiments is shown. DMSO, Unliganded ER in buffer plus DMSO solvent (2% DMSO in most concentrated well).

 
Z'-factor values are a statistical parameter of the robustness of an assay (36). As an example, the Z' values were calculated for the ER{alpha}/Rh-D22 dose dependency format in the presence of 17ß-estradiol (agonist), 4-hydroxytamoxifen (antagonist), or no ligand (buffer plus solvent) in either a 96-well plate using 30 data points or a 384-well plate using 50 data points (Fig. 6Go). The assay was considered robust with Z' values greater than 0.5 for antagonist vs. agonist for up to 8 h in a 384-well format.



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  		Fig. 6. ER{alpha} Assay Robustness and Z'-Factor Values

The Z' values (A) for the ER{alpha} coactivator assay were calculated using the dose dependency format, in the presence of 17ß-estradiol (agonist), 4-hydroxytamoxifen (antagonist), or no ligand in a 384-well plate using 50 data points per condition (B) or a 96-well plate using 30 data points per condition (C) for hours (listed) of incubation. The final concentration of solvent in each experiment was 2% ethanol. Z' values were determined from two experiments (agonist vs. no ligand; antagonist vs. no ligand) or three experiments (agonist vs. antagonist). The Z' values were generated from two to three independent determinations of standard deviation ({sigma}) values as noted above, and the Z' value listed is an average of these independent determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In these assays, ER occupied with agonists formed a complex with the LXXLL peptides, resulting in a higher polarization value compared with unliganded receptor. Our results also indicated that the LXXLL peptides were not recruited to antagonist-occupied ER. In equilibrium binding studies, several LXXLL peptides had measurable affinity for purified ER{alpha} or ERß in the nanomolar range (Kd or EC50 of 20–500 nM). These affinities were similar to or better than previous reports of coactivator peptide-steroid hormone receptor interactions and highlighted the utility of these LXXLL peptides as tools to probe ligand-dependent recruitment of coactivators to receptors (25, 37). The affinity values we obtained for the interaction of ER with the LXXLL peptides were within the same order of magnitude as the values found for the interaction of ER with SRC/p160 coactivator nuclear receptor interaction domains (33).

Ligand-occupied ER had different affinities for the LXXLL-peptides from the various classes of randomphage display peptides tested. Others have reported that ER{alpha} preferentially interacts with full-length SRC-3 and ERß binds with highest affinity to full-length SRC-1 (33). Liu and co-workers (27) reported that agonist-occupied ER{alpha} and ERß bound with preference to coactivator peptides corresponding to SRC-1, with intermediate affinity for peptides derived from PGC-1 and lower affinity for peptides from cAMP response element binding protein-binding protein (CBP) and the PPAR interacting protein (PRIP). Whereas interactions between fluorescently tagged D11 or D30, corresponding to the random phage display class I peptides with similarity to the p160 class of coactivators, and ER were detected (data not shown), the highest affinity interaction under the buffer conditions tested was between ER{alpha} or ERß and the fluorescent D22 peptide of the class III random phage display peptides. These findings can be explained, in part, by the fact that the peptides used in these studies resembled, but were not exact replicas of, the LXXLL motifs of actual coactivator proteins, and therefore the affinities of these LXXLL peptides for ER may not be the same as those observed for actual coactivator sequences.

Our results also indicated that the affinity of the fluorescently labeled LXXLL peptide for ER was affected by ligand occupancy. Different LXXLL peptides elicited different profiles of affinity for ER depending on which ligand was bound to the receptor. This was best demonstrated by the example of LXXLL peptides D22 and C33. Competition experiments (data not shown) indicated that D22 can effectively compete for binding of C33 to ERß, suggesting that C33 and D22 are binding to the same site(s) on ERß. These results strongly suggest that the different LXXLL peptides are recognizing unique ligand-induced conformations of ER. The overall increase in polarization values [also known as {Delta}mP (millipolarization)] from unbound LXXLL peptide to ER-complexed LXXLL peptide also varied depending on which ligand was bound to ER. Estrone-occupied ER complexed to the fluorescent D22 or C33 peptides consistently had a smaller {Delta}mP shift compared with 17ß-estradiol-occupied ER complexed with fluorescent D22 or C33, suggestive of a difference in ER conformation. It is also possible that a slight difference in D22 and C33 peptide binding to ER is causing a localized effect on the fluorescent tracer molecule or its linkage, resulting in a change in the {Delta}mP shift.

The results from this assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators based on either receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known ligands. The equilibrium binding format offered a larger dynamic range, as indicated by the larger {Delta}mP between unbound LXXLL peptide (in the presence of antagonist-occupied ER) and peptide bound to agonist-occupied ER. In the dose dependency assays, the potencies of the various ligands in the recruitment of Rh-D22 to ER mimicked the biological activity of the different ligands. For example, estriol and estrone are known to be weaker physiological estrogens than 17ß-estradiol, and their potencies in the dose dependency format correlated well (diethylstilbestrol > 17ß-estradiol > estrone/genistein for ER{alpha}; 17ß-estradiol > estriol/genistein > estrone for ERß) with their biological activity (3). The EC50 values determined in these assays can be thought of as composite measurements of the ligand concentration required for the receptor-ligand and receptor-peptide interaction, and thus the potency (EC50) for each ligand tested did not equal the reported ligand affinity value for the ligand-ER interactions (3). However, the ligand EC50 values for interaction of Rh-D22 with ERß in the presence of 17ß-estradiol, tamoxifen, and 4-hydroxytamoxifen were similar to reported IC50 values for these ligands binding to ERß in a fluorescence polarization competitive ligand affinity assay (38). Interestingly, Gee et al. (39) has shown that coactivator peptides and larger coactivator domains can stabilize agonist, but not antagonist, binding to the ER LBD by a reduction in the ligand dissociation rate from the receptor. To further examine how ligand binding and coactivator recruitment might be thermodynamically linked, additional experiments using the full-length ER and the phage display LXXLL peptides would help elucidate how LXXLL peptide-induced stabilization of ligand binding and the rate of LXXLL tracer dissociation from ER in the presence of different ligands might affect the ligand dose required to recruit LXXLL peptide to the receptor in our system.

In our assay, the agonists and antagonists performed as expected, with agonists inducing peptide recruitment to ER and antagonists disrupting this interaction. The ERß-selective ligand, DPN, functioned as an agonist for peptide recruitment to ERß, whereas the ER{alpha}-selective ligand, PPT, behaved as expected resulting in unliganded ERß under dose dependency conditions. In the ER{alpha} dose dependency experiment using PPT and DPN, PPT behaved as an agonist as expected. DPN also resulted in LXXLL peptide recruitment to ER{alpha}, although it is considered an ERß-selective agonist due to its 70-fold higher relative binding affinity and 170-fold higher relative transcriptional activity with ERß over ER{alpha} (34). In our assay system with three independent experiments (each done in triplicate), the relative potency (EC50) of DPN was 6-fold higher for ERß over ER{alpha} (Fig. 5Go and data not shown). Sun and co-workers (35) have shown that only a limited number of residues in the ERß LBD are responsible for the ERß-selective nature of DPN binding and that conversion of a single amino acid in ER{alpha} can cause DPN binding similar to that observed with ERß. Additional experiments will be needed to determine whether DPN can recruit other LXXLL peptides to ER{alpha} or if this is a D22-specific observation. The lack of correlation with transcriptional assays may reflect a limitation of using nonphysiological LXXLL peptides for making in vivo predictions about ligand-coregulator effects.

The equilibrium binding and dose dependency formats described herein to evaluate the ligand-induced binding characteristics of LXXLL peptides to ER have many advantages as a high-throughput plate assay. These assays are able to differentiate agonist vs. antagonist ligands for the ER isoforms. The nanomolar affinity interaction between Rh-D22 and ER helped to reduce the amount of ER protein per well, allowing for the assay to be used as a primary or secondary screen for test ligands. Unlike fluorescence resonance energy transfer-based assays, which require labeling multiple interaction partners with fluorescent tags, this assay utilizes a single fluorescent label on the LXXLL peptide, providing a simple means to synthesize and label other similar LXXLL peptides for comparison. With a fluorescence polarization-based assay, the interaction reaction is carried out entirely in solution without the need to bind one of the interaction partners to a solid surface, as in surface plasmon resonance techniques. Whereas these other techniques offer their unique advantages and disadvantages, the factors above offer an element of simplicity, high throughput convenience, and assay robustness (Z' values) to examining ER-coactivator peptide interactions by fluorescence polarization. Moreover, the equilibrium binding and dose dependency formats were highly tolerant of solvents such as DMSO, methanol, and ethanol, commonly found in compound libraries (data not shown).

Our results highlight the usefulness of an assay to examine the ER interaction with LXXLL coactivator-like peptides for elucidating the biological function of unknown ligands as agonists or antagonists. Recently, a unique coactivator interaction surface within the ER LBD was discovered using non-LXXLL containing peptides that selectively bound to tamoxifen-occupied ER{alpha} and ERß (30). Such non-LXXLL peptides as well as other LXXLL-containing coactivator-like peptides would be useful in a panel of peptides to probe different ligand-induced ER conformations and coactivator-ER interaction binding characteristics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Human ER{alpha} or ERß was isolated from recombinant baculovirus-infected insect cells to a physical purity of approximately 80% (Invitrogen, Madison, WI). Purified ER{alpha} was stored in 10% glycerol, 50 mM Tris-HCl (pH 8.0), 500 mM KCl, 2 mM dithiothreitol (DTT), 1 mM EDTA, and 1 mM orthovanadate. Purified ERß was stabilized in storage buffer containing 10% glycerol, 50 mM bis-Tris-propane (pH 9.0), 400 mM KCl, 2 mM DTT, and 1 mM EDTA. The concentration of active ER was measured by a [3H]17ß-estradiol ligand-binding assay.

The LXXLL peptides were synthesized and labeled with fluorescein (F) at the N terminus by the University of Wisconsin Biotechnology Center (Madison, WI). Rhodamine (Rh)-bearing LXXLL peptides were prepared and labeled by the Keck Institute (Yale University; New Haven, CT) and further purified by HPLC.

Black 96-well and 384-well plates were purchased from Costar (Corning, NY) or LabSystems (Franklin, MA). ICI-182,780, 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), and 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) were obtained from Tocris Cookson (Ellisville, MO). All other ligands were purchased from Sigma Chemical Co. (St. Louis, MO). The following reaction buffers were from Invitrogen: complete ER{alpha} coactivator assay buffer (20 mM HEPES, pH 7.5; 0.005% Nonidet P-40; 10 mM DTT); ERß coactivator equilibrium binding assay buffer (100 mM potassium phosphate, pH 7.4; 100 µg/ml acetylated bovine {gamma}-globulin; 0.02% sodium azide); ERß coactivator dose dependence assay buffer (20 mM Tris, pH 7.5; 0.02% sodium azide; 0.01% Nonidet P-40).

Methods
Equilibrium Binding Assay.
Purified ER{alpha} or ERß was serially diluted into reaction buffer with final concentrations of 5 µM ligand and 1 nM fluorescent peptide in a black 96-well or 384-well plate. Final volumes were 100 µl or 40 µl for a 96-well and 384-well plate, respectively. Each plate also included the following controls: 1 nM fluorescent peptide in reaction buffer; fluorescein or rhodamine polarization reference; and reaction buffer containing the same final concentration of ligand solvent. The samples were mixed by gentle agitation of the plate and protected from light. The samples were incubated at room temperature 1–2 h before measurement of fluorescence polarization (described below).

Dose Dependency Assay.
Ligand was serially diluted into reaction buffer in a 96-well or 384-well plate with final reaction conditions containing 75 nM ER{alpha} or ERß and 1 nM fluorescently tagged LXXLL peptide. Final volumes were 100 µl or 40 µl for a 96-well and 384-well plate, respectively. The plates were gently agitated, protected from light, and incubated for 1–8 h, before measurement of fluorescence polarization (described below).

Fluorescence Polarization.
Fluorescence polarization was measured using either the Beacon 2000 system (Invitrogen) or the Polarion (TECAN; Research Triangle Park, NC) with excitation at 485 nm and emission at 535 nm for fluorescein or the Ultra (TECAN) with excitation at 535 nm and emission at 590 nm for rhodamine. The Polarion and Ultra instruments are microplate detection systems with flash lamps capable of measuring fluorescence polarization in high-throughput top-read mode with the appropriate filter sets to detect either fluorescein (Polarion) or rhodamine (Ultra), which were the tracers used in the experiments described. Fluorescence polarization (mP = P x 1000) is the ratio of light intensities consisting of the difference between parallel (Int||) and perpendicular light (Int{perp}) relative to the total light intensity (Int|| + Int{perp}) (for review, see Ref. 32). Data were fit with Prism (GraphPad Software, Inc., San Diego, CA) using nonlinear regression analysis with a sigmoidal dose-response (variable slope) equation.


   ACKNOWLEDGMENTS
 
We acknowledge Tonia Buchholz, Hildegard Eliason, Heidi Braun, Heather Lee-Reppen, David Lasky, Andy Kopp, and the laboratory support staff of Invitrogen for their technical support. We also thank Donald P. McDonnell for useful discussions.


   FOOTNOTES
 
Present addresses are as follows. C.L.S.: Wisconsin Alumni Research Foundation, 4350 La Jolla Village Drive, 7th Floor, San Diego, California 92122; R.G.L.: BellBrook Labs, University Research Park, 505 South Rosa Road, Madison, Wisconsin 53719; T.J.B.: 5 Oakwood Circle, Madison, Wisconsin 53719.

First Published Online September 16, 2004

Abbreviations: DMSO, Dimethylsulfoxide; DPN, diarylpropionitrile or 2,3-bis(4-hydroxyphenyl)propionitrile; DTT, dithiothreitol; ER, estrogen receptor; F, fluorescein; LBD, ligand-binding domain; mP, millipolarization; NCoA, nuclear receptor coactivator; PPT, propyl pyrazole triol or 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol; Rh, rhodamine; SRC, steroid receptor coactivator.

Received for publication June 24, 2004. Accepted for publication September 10, 2004.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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NURSA Molecule Pages Link:

Nuclear Receptors:   ERα  |  ERβ
Coregulators:   RIP140  |  TRAP220
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



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