help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2006-0203
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koide, A.
Right arrow Articles by Koide, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koide, A.
Right arrow Articles by Koide, S.
Molecular Endocrinology 21 (4): 829-842
Copyright © 2007 by The Endocrine Society

Identification of Regions within the F Domain of the Human Estrogen Receptor {alpha} that Are Important for Modulating Transactivation and Protein-Protein Interactions

Akiko Koide1, Changqing Zhao1, Misuzu Naganuma, Judith Abrams, Sarah Deighton-Collins, Debra F. Skafar1 and Shohei Koide1

Department of Biochemistry and Molecular Biology (A.K., M.N., S.K.), University of Chicago, Chicago, Illinois 60637; and Department of Physiology (C.Z., S.D.-C., D.F.S.), Wayne State University School of Medicine, and the Barbara Ann Karmanos Cancer Institute (J.A., D.F.S.), Detroit, Michigan 48201

Address all correspondence and requests for reprints to: Debra F. Skafar, Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, Michigan 48201. E-mail: dskafar{at}med.wayne.edu; or Shohei Koide, Department of Biochemistry and Molecular Biology, University of Chicago, 929 E. 57th, Chicago, Illinois 60637. E-mail: skoide{at}uchicago.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The estrogen receptor (ER){alpha} is a biologically and clinically important ligand-modulated transcription factor. The F domain of the ER{alpha} modulates its functions in a ligand-, promoter-, and cell-specific manner. To identify the region(s) responsible for these functions, we characterized the effects of serial truncations within the F domain. We found that truncating the last 16 residues of the F domain altered the activity of the human ER{alpha} (hER{alpha}) on an estrogen response element-driven promoter in response to estradiol or 4-hydroxytamoxifen (4-OHT), its sensitivity to overexpression of the coactivator steroid receptor coactivator-1 in mammalian cells, and its interaction with a receptor-interacting domain of the coactivator steroid receptor coactivator-1 or engineered proteins ("monobodies") that specifically bind to ER{alpha}/ligand complexes in a yeast two-hybrid system. Most importantly, the ability of the ER to induce pS2 was reduced in MDA-MB-231 cells stably expressing this truncated ER vs. the wild-type ER. The region includes a distinctive segment (residues 579–584; LQKYYIT) having a high content of bulky and/or hydrophobic amino acids that was previously predicted to adopt a ß-strand-like structure. As previously reported, removal of the entire F domain was necessary to eliminate the agonist activity of 4-OHT. In addition, mutation of the vicinal glycine residues between the ligand-binding domain and F domains specifically reduced the 4-OHT-dependent interactions of the hER{alpha} ligand-binding domain and F domains with monobodies. These results show that regions within the F domain of the hER{alpha} selectively modulate its activity and its interactions with other proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ESTROGEN RECEPTOR (ER){alpha} is a member of the steroid hormone receptor family. It mediates many of the actions of estrogens and plays important roles in the normal development and malignant growth of organs such as the uterus, breast, bone, brain, and cardiovascular tissues (1). It is also an important drug target for the prevention and treatment of breast cancer (2).

The ER{alpha} has a domain structure that is well conserved within the nuclear receptor superfamily (1, 3). The A/B domain, which is located at the N terminus, has a ligand-independent transcription activation function called activation function (AF)-1. The highly conserved C domain is a DNA-binding domain (DBD), whereas the D domain is a hinge between the C and E domains. The E domain (ligand-binding domain, LBD) is a dimerization, transactivation, and ligand binding domain containing transcription AF-2. The F domain is located at the C terminus of the receptor. The DBD and LBDs have well-defined structures, whereas the A/B, D, and F domains are generally considered to be flexible (4, 5, 6, 7, 8).

The human (h)ER{alpha} has a particularly large F domain (41 residues; residues 555–595) among nuclear receptors. The F domain of the ER{alpha} does not have a high degree of sequence homology with other steroid hormone receptors, including another subtype of ER, ERß (9, 10). Although the F domain is not required for ligand binding or transcriptional activation on an estrogen response element (ERE)-driven promoter, results from several laboratories show that the F domain exerts a complex modulatory role on the activity of the receptor (11, 12, 13, 14, 15, 16, 17, 18). The F domain is located immediately C terminal to the most C-terminal helix (helix 12; H12) of the LBD that is critical for AF-2 (3). Thus, one can envision that changes in the F domain conformation and/or interactions may lead to significant effects on the LBD function.

Deletion of the entire F domain from the full-length ER{alpha} eliminates the agonist activity of certain antiestrogens and reduces the agonist activity of estradiol in a cell-specific manner (11). For example, upon deletion of the F domain, 4-hydroxytamoxifen (4-OHT) loses its agonistic activity in both MDA-MB-231 human breast cancer cells and Chinese hamster ovary cells, whereas ICI 164,384 loses agonist activity only in Chinese hamster ovary cells (11). Furthermore, when part of the F domain, residues 579–595, is deleted, the ER{alpha} loses its ability to activate transcription via interactions with promoter selective transcription factor 1 (Sp1) (14). In addition, we have shown that the agonist activity of 4-OHT is reduced or eliminated not only by deleting the F domain, but by introducing point mutations within it (15). Of note, the F domain can promote the interaction between the ER{alpha} and the corepressor TATA-binding protein-associated factor 1ß (TAF-Iß) (16). It is tempting to speculate that this may be related to the observation that deletion (18) or mutation (15) of the F domain modulates the antagonist activity of 4-OHT. Using a yeast two-hybrid system, Peters and Khan (18) showed that there was an inhibitory activity in the F domain in the presence of estradiol (E2). The full-length ER{alpha} interacted with its LBD, but not its LBD F domain, in the presence of E2 (18). They also demonstrated that the F domain inhibited the interaction of the LBD with the coregulator receptor interacting protein 140 (RIP140) in the presence of E2 (18). Because the ligand-bound ER is dimeric, the interaction tested by a yeast two-hybrid system between a dimeric ER "bait" and a dimeric ER "prey" is complex, and interpretation of such interaction in terms of the underlying molecular mechanism is difficult.

We have established a system to engineer binding proteins, "monobodies," using a scaffold of the fibronectin type III domain (19, 20, 21, 22). Previously, we obtained a series of monobodies that binds to the LBD of hER{alpha} in a ligand-specific manner. We also established a yeast two-hybrid system to investigate conformational changes of the hER{alpha} LBD using these monobodies and a steroid receptor coactivator-1 (SRC-1) fragment as probes (20). An advantage of such a yeast system is the absence of endogenous coregulators of the ER that may complicate analysis (23). Using this system, we demonstrated that the deletion of the F domain increases the interaction of the hER-LBD with SRC-1 receptor interaction domain (RID) and monobodies in the absence of ligand. These results suggest that 1) the hER{alpha} LBD exists in a conformational ensemble; 2) it samples the "activated" state in the absence of an agonist albeit infrequently; and 3) in the absence of a bound ligand the removal of the F domain shifts this conformational equilibrium toward the activated state.

The studies described above have established that the ER F domain modulates LBD function. However, it is not known which region(s) within the F domain is (are) important for its functions. To address this question, we analyzed the response of a series of truncation mutants of the hER{alpha} to E2 and 4-OHT on an ERE-driven promoter, the sensitivity of the hER{alpha} to coexpression of the coactivator SRC-1, and the interaction of a series of hER{alpha} F domain truncation and point mutants with an SRC-1 RID fragment and a monobody. In addition, we compared the ability of a truncated ER (Q580stop) and the wild-type (wt) ER{alpha} to induce the endogenous pS2 gene in clones of the MDA-MB-231 cell line stably expressing either protein.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
F Domain Truncation and the Response of the hER{alpha} to E2 and 4-OHT
We have previously shown that point mutations in the region from residue 559 to 565 alter the ER’s response to E2 and 4-OHT (15). To determine the role of other regions within the F domain in the response of the ER{alpha} to E2 and 4-OHT on an ERE-driven promoter, we constructed additional truncation mutants, Q580stop (containing residues 1–579) and G572stop (containing residues 1–571) (Fig. 1Go). These positions were chosen based on secondary structure predictions (15) that suggested an {alpha}-helical structure in residues 559–570 and an extended (ß-strand-like) structure in residues 580–585. Note that the secondary structure predictions serve primarily as guides to efficiently identifying regions of interest in the ER{alpha}: the results of these functional studies do not require that the predicted structure correspond with the actual structure. The effects of these truncations on the responses to E2 and 4-OHT were assessed in transient cotransfection assays using an ERE-driven reporter in HeLa cells (22).


Figure 1
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 1. Sequence of the hER{alpha} F Domain and the Mutants Used in These Studies

 
E2 increased the activity of the wt and all truncated proteins (Fig. 2Go; P = 0.007 or less). However, the activity of the ER{alpha} in the presence of E2 was strikingly reduced in the truncated receptors compared with the wt protein (P < 0.001). There were no differences among the truncated receptors in their activity in the presence of E2.


Figure 2
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 2. Response of the hER{alpha} Truncation Mutants to E2

The activity of the wt hER{alpha} and F domain truncation mutants in the absence of ligand and in response to E2 (100 nM) was measured after transient transfection into HeLa cells as described in Materials and Methods. The results are shown as RLUs, determined as the ratio of firefly luciferase activity to Renilla luciferase activity. The fold stimulation was calculated by dividing the RLU in the presence of E2 by the RLU in the absence of ligand for each corresponding receptor. These data are the mean ± SEM of five independent experiments, each conducted in triplicate. Inset, Immunoblot of wt and F domain truncation mutants. Western immunoblotting was carried out using extracts from MCF-7 cells and from HeLa cells expressing the wt and mutated ERs as described in Materials and Methods. From left to right: Lane 1, MCF-7 cells; lane 2, wt hER{alpha} (M1-V595); lane 3, G572stop (M1-A571); lane 4, Q580stop (M1-L579); lane 5, S554stop (M1-T553). This immunoblot is representative of three independent experiments.

 
Because deletion of the F domain does not alter the half-life of the ER (24), down-regulation of the ER{alpha} by E2, or the effect of the proteasome inhibitor MG132 (25), deleting the F domain does not alter the stability, turnover, and/or degradation of the hER{alpha}. Western immunoblotting shows that the mutated receptors were expressed at levels at least as great as the expression level of the wt protein (Fig. 2Go, inset). Note that each truncation mutant can be distinguished from the endogenous ER in MCF-7 cell extracts, from the transfected wt ER{alpha}, and from the other truncation mutants, by migration on SDS-PAGE; the observed differences in migration are consistent with the predicted molecular weights of the truncated receptors. Also note that the reduced response of the truncation mutants is not due to differences in the receptor expression (Fig. 2Go, inset) and it is unlikely to result from decreased binding of E2, because deletion of the entire F domain (S554stop, containing residues 1–553) increases, rather than decreases, the affinity of the ER{alpha} for E2 (15). Thus, these results support the idea that there is a functional element within the C-terminal 15 residues of the ER{alpha} that increases the activity of the ER{alpha} in the presence of E2 on an ERE-driven promoter.

In the absence of E2, the activity of the G572stop and S554stop mutants was less than the activity of the wt ER{alpha} (P ≤ 0.02). Because of this reduced basal activity, the fold stimulation by E2 is not different between the wt ER{alpha} and the two mutants (wt, 6.4 ± 1.7; G572stop, 3.9 ± 0.6; S554stop, 3.8 ± 1.2). The fold stimulation by E2 of the Q580stop mutant is substantially reduced, 1.8 ± 0.2.

Next, we tested the effect of the same serial truncations on the weak agonist activity of 4-OHT (10–8 M, 10–6 M) on an ERE-driven reporter in HeLa cells (Fig. 3Go). All truncated hERs exhibited less activity than the wt in the presence of either concentration of 4-OHT (P < 0.001). Furthermore, 4-OHT increased the activity of the wt hER{alpha}, the Q580stop, and the G572stop mutants (P ≤ 0.01) but not the S554stop mutant. There was no difference in the 4-OHT-stimulated activity (10–6 M) of the Q580stop and G572stop mutants, but the activity of each of these mutants was different from the activity of the wt hER{alpha} and the S554stop mutant (P ≤ 0.007). These results show that deletion of two regions within the F domain, the C-terminal 15 residues and the region between S554 and G572, reduces the agonist activity of 4-OHT on an ERE-driven promoter. We have previously reported that point mutations within the region between S554 and G572 impair (S559A/E562A) or eliminate (Q565P) 4-OHT agonism (15). Taken together, these results show that the two regions within the F domain of the hER{alpha} selectively modulate its response to ligands. One region, from Q580 to V595, increases the activity of the ER{alpha} in the presence of either E2 or 4-OHT on an ERE-driven reporter. A different region, between S554 and G572, is essential for the agonist activity of 4-OHT on an ERE, yet is dispensable for the agonist activity of E2 on this same promoter.


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 3. Response of the hER{alpha} Truncation Mutants to 4-OHT

The activity of the wt hER{alpha} and F domain truncation mutants in the absence of ligand and in response to 4-OHT (10 nM, 1000 nM) was measured after transient transfection into HeLa cells as described in Materials and Methods and the legend to Fig. 2Go. The fold stimulation was calculated by dividing the RLU in the presence of 4-OHT by the RLU in the absence of ligand for each corresponding receptor. These data are the mean ± SEM of five independent experiments, each conducted in triplicate.

 
Sensitivity of F Domain Truncation Mutants to SRC-1 Overexpression
We then tested the sensitivity of receptors having mutations in the F domain to overexpressed SRC-1 using a transient cotransfection assay in mammalian cells (Fig. 4Go). These experiments were carried out by cotransfecting an expression plasmid containing the cDNA for SRC-1 into HeLa cells, along with vectors for the hER{alpha}, ERE-driven luciferase reporter, Renilla luciferase transfection control, and empty vector to equalize the amount of DNA transfected (26). Transcriptional activity was measured after the addition of medium containing 100 nM E2 or vehicle. Preliminary experiments were carried out to determine the amounts of hER{alpha}-encoding and SRC-1-encoding plasmids that would yield significant stimulation of transcription upon addition of SRC-1 to the wt hER{alpha} in the presence, but not the absence, of E2 (data not shown). Note that the optimal amount of hER{alpha}-encoding plasmid for these experiments, 200 ng, is substantially less than that used in the previous experiments, 800 ng.


Figure 4
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 4. Response of the hER{alpha} Truncation Mutants to Overexpressed SRC-1

The activity of the wt hER{alpha} and F domain truncation mutants in the presence of either plasmid expressing SRC-1 (pBK-CMV-SRC-1) or empty vector and the absence (open bars) and presence (filled bars) of 100 nM E2 was measured after transient transfection into HeLa cells as described in Materials and Methods and the legend to Fig. 2Go. These data are the mean ± SEM of three independent experiments, each conducted in triplicate.

 
In the presence of both SRC-1 and E2, each truncated protein exhibited less activity than the wt (P < 0.0001) (Fig. 4Go). Furthermore, the Q580stop mutant also exhibits even less activity than the G572stop and S554stop mutants (P < 0.001). The activity of the G572stop mutant and the S554stop mutant is not different (P = 0.97). There were no differences among the proteins in the absence of E2 and SRC-1 (P = 0.83), in the absence of E2 and presence of SRC-1 (P = 0.92), or in the presence of E2 and absence of SRC-1 (P = 0.39).

These results show that eliminating the C-terminal 15 residues of the ER{alpha} reduced its activity in the presence of E2 and overexpressed SRC-1. These results are consistent with the observation that removal of the same C-terminal 15 residues reduced the activity of the hER{alpha} in the presence of E2 (Fig. 2Go) and suggest that the C-terminal 15 residues of the hER{alpha} contain a region that increases sensitivity to SRC-1 overexpression. Surprisingly, successive truncation of the F domain then increased the activity of the ER{alpha} in the presence of E2 and overexpressed SRC-1, although activity was still less than that of the wt protein. This shows that multiple regions within the F domain modulate the sensitivity of the hER{alpha} to SRC-1 overexpression.

Effect of the F Domain on Protein-Protein Interactions Mediated by the LBD
The previous functional studies show that truncation of the F domain modulates the activity of the hER{alpha}, but they do not show whether these truncations directly alter the interaction of the ER with other proteins. We therefore constructed and tested the interactions of truncation mutants of the segment corresponding to the LBD and F domains of hER{alpha} (hER{alpha}-LBD-F) with either the SRC-1 RID or the monobody E3#6 using the yeast two-hybrid system. Like a coactivator, the E3#6 monobody is specific to the agonist-bound form of hER-LBD, but it contains a single LXXLL motif, whereas the SRC-1 fragment contains multiple LXXLL motifs (27).

Figure 5Go shows the interactions of the ER{alpha}-LBD-F truncation mutants with SRC-1 RID or with the E3#6 monobody. In the presence of E2, all the truncation mutants interacted with both the E3#6 monobody and the SRC-1 RID, indicating that the F domain is not necessary for the interaction between the E2-bound LBD and a coactivator or a coactivator-like protein. However, the truncation mutants showed substantial differences in the interactions in the absence of a ligand. As previously reported, there was little interaction of the full-length ER{alpha}-LBD-F (residues 297–595) with the SRC-1 RID or monobody E3#6 (21). Although residues 297–585 and larger fragments showed little interaction with these probes, residues 297–579 and shorter fragments exhibited significant interaction, suggesting that the inhibitory activity of the F domain requires residues 580–585.


Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 5. Yeast Two-Hybrid Assays for the Interactions of ER{alpha}-LBD-F Truncation Mutants with Monobody E3#6 (A–C) or the SRC-1 RID (D–F)

Monobody E3#6 is specific to the hER/agonist complex. A and B, Levels of the ß-galactosidase reporter activity for the interaction between monobody E3#6 and a series of ER{alpha} fragments (horizontal axis) in the presence (A) and absence (B) of 1 µM E2. C, The ratios of ß-galactosidase activity in the absence of E2 (panel B) over that in the presence of E2 (panel A). D and E, Levels of the ß-galactosidase reporter activity for the interaction between SRC-1 RID and a series of ER{alpha} fragments in the presence (D) and absence (E) of 1 µM E2. F, The activity ratios for panels D and E. The error bars show the SD values determined from triplicates.

 
To more finely map residues responsible for the inhibitory activity observed in yeast, we tested all the possible truncation mutants between Q580 and T585 for their interactions with the SRC-1 RID or monobody E3#6 (Fig. 6Go). In the presence of E2, all the truncation mutants that have a C terminus between Q580 and T585 interacted with the monobody E3#6 as strongly as full-length ER{alpha}-LBD-F, as expected (Fig. 6AGo). In the absence of E2, residues 297–582 and longer fragments showed very weak interaction with the monobody (Fig. 6BGo). Significant increases in interaction were observed when Y582 and Q580 were respectively truncated (Fig. 6BGo), suggesting that these residues are important for the modulatory function of the F-domain.


Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Effects of C-Terminal Truncations of ER{alpha}-LBD-F Ranging from Residue 579 to 585 on Its Interactions with Monobody E3#6 (A–C), the SRC-1 RID (D–F), and Single RID motifs of SRC-1 (G)

The data for monobody E3#6 and SRC-1 RID are shown in the same manner as in Fig. 5Go. A and D, Levels of ß-galactosidase activity in the presence of E2 (1 µM); B and E, data without a ligand. C and F, Activity ratios (activity in the absence of E2 over that in the presence of E2). G, The levels of ß-galactosidase activity in the presence of E2 (1 µM) for the interactions of SRC-1 RID2 (black bars) and SRC-1 RID3 (gray bars) with ER{alpha} fragments.

 
We found significant variations in the interaction between the E2-bound receptor fragments and the SRC-1 RID (Fig. 6DGo), a phenomenon that we did not observe when probed with monobody E3#6 (Fig. 6AGo). A 3-fold difference in reporter activity was observed, with the least activity for residues 297–581 and the activity gradually increasing as more residues were included. Because the ER{alpha}-LBD-F truncation mutants were expressed at similar levels as determined by Western blotting (data not shown), the differences were not simply due to different levels of expression. Interestingly, this profile is similar to that for the sensitivity of the hER{alpha} to overexpressed full-length SRC-1 in mammalian cells (Fig. 4Go).

In the absence of E2, the fragment 297–579 interacted with the SRC-1 RID strongly, but the longer mutants showed only weak interaction (Fig. 6EGo). When the reporter activity in the absence of E2 is normalized to that in the presence of E2 (Fig. 6FGo), gradual decreases in the interaction were seen as residues 297–581 to 297–583 were included, suggesting that residues 580–583 are important for the inhibitory activity.

We then examined whether the variation of the interaction of the receptor fragments with SRC-1 in the presence of E2 (Fig. 6DGo) is due to the presence of three LXXLL motifs in SRC-1 RID. The monobody, for which no such variant was observed (Fig. 6AGo), contains only one LXXLL motif. We tested interactions of the receptor fragments with shorter segments containing a single LXXLL motif, SRC-RID2 and SRC-RID3 (TARHKILHRLLQEGSPS and SKDHQLLRYLLDKDEKD, respectively; the LXXLL motifs are underlined). SRC-RID2 binds the strongest to ER{alpha} among the three LXXLL motifs in SRC-1 (28, 29).

The reporter activities were much weaker with these single RIDs (Fig. 6GGo) than the larger fragment used in the previous experiments (Fig. 6DGo), indicating that simultaneous interactions of multiple RIDs in the longer SRC-1 segment with the receptor strengthen the binding between these molecules. Interestingly, SRC-RID2 gave the same activity profile (Fig. 6GGo) as the original SRC-1 fragment (Fig. 6DGo). By contrast, the activity of SRC-RID3 was too weak to be quantitatively measured. At present, we do not have a mechanistic explanation for this modulation of ER{alpha}-LBD/SRC-1 interaction by the F domain, but it is clear that RID2 is responsible for this phenomenon and that the presence of multiple RIDs is not the cause of this activity difference.

To test whether residues 580–583 are necessary and sufficient for the observed inhibitory activity of the F domain in the yeast assay, the effects of alanine substitution mutations, rather than truncation mutations, were characterized. We selected Y582 and Y583 for mutation because the truncation studies described above indicated that these residues are centrally located in the region that altered the inhibitory activity of the F domain, and they have the largest side chains among the residues in the region. We constructed two single mutants, Y582A and Y583A, and a double mutant, Y582A:Y583A, in the ER{alpha}-LBD-F and examined their interactions with monobody E3#6 or the SRC-1 fragment (Fig. 7Go). The mutations increased the interaction between ER{alpha}-LBD-F and these probes in the absence of a ligand. The levels of increase were smaller than the truncation mutants (compare Fig. 7CGo with Fig. 6CGo, and Fig. 7FGo with Fig. 6FGo). Furthermore, the double mutant exhibited no effect on its response to either E2 or 4-OHT on an ERE-driven promoter in HeLa cells (data not shown). Thus, perturbations greater than the removal of the phenolic side chains of Y582 and Y583 are required for abolishing the observed modulatory activity of the F domain.


Figure 7
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 7. Effects of Y582A and Y583A Mutations of ER{alpha}-LBD-F on Its Interactions with the Monobody E3#6 (A–C) or the SRC-1 RID (D–F), as Measured Using a Yeast Two-Hybrid ß-Galactosidase Assay

The data are presented in the same manner as in Figs. 5Go and 6Go. A and D, Data in the presence of E2; B and E, data without a ligand. C and F, Ratio of activity in the absence of E2 to that in the presence of E2.

 
Selective Effects of F Domain Point Mutations on Reaction with Probes for the 4-OHT-Bound Conformation of the ER
Point mutations within the hER{alpha} F domain have been previously shown to alter its response to E2 as well as the agonist and antagonist activity of 4-OHT (15). We investigated the effect of these mutations (G556L/G557L, S559A/E562A, and Q565P) on the interaction of monobodies with ER{alpha}-LBD-F in the presence of 4-OHT or E2. We used three types of monobodies with different binding specificity for ER{alpha}-LBD-F/ligand complexes. The monobodies E3#6 and E2#23 are ER{alpha}-LBD-F/E2 specific, monobodies OHT#1 and OHT#33 are ER{alpha}-LBD-F/OHT specific, and monobody OHT#6 interacts with both ER{alpha}-LBD-F/E2 and ER{alpha}-LBD-F/4-OHT complexes. Surprisingly, only the G556L/G557L double mutation, which is located near the junction of the LBD and F domains and has minimal effects on the agonist activity of both E2 and 4-OHT on an ERE-driven promoter in HeLa cells (15), exhibited different reactivity with monobody probes than the wt receptor. This mutation eliminated the OHT-dependent binding of the ER{alpha}-LBD-F/4-OHT-specific monobodies (OHT#1 and OHT#33) to ER{alpha}-LBD-F (Fig. 8Go, D and E, respectively). However, the G556L/G557L mutation had no measurable effect on the E2-dependent interaction of the ER{alpha}-LBD-F/E2-specific monobodies (E3#6 and E2#23), as well as the SRC-1 fragment, with the receptor (Fig. 8Go, A–C). This distinct effect of the G556L/G557L mutation on 4-OHT-dependent receptor-monobody interactions is best illustrated by monobody OHT#6, which interacts with both ER{alpha}-LBD-F/E2 and ER{alpha}-LBD-F/4-OHT complexes. We found that the G556L/G557L mutation selectively eliminated the 4-OHT-dependent interaction of this monobody with ER{alpha}-LBD-F, while having no effect on its E2-dependent interaction with the receptor (Fig. 8FGo).


Figure 8
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 8. Effects of the G556L:G557L Double Mutation on the Interactions of ER{alpha}-LBD-F with Various hER{alpha}-Interacting Proteins, as Measured Using a Yeast Two-Hybrid ß-Galactosidase Assay

The identity of the interacting protein used is shown in the figure. For each panel, the leftmost three lanes indicate levels of ß-galactosidase activity for the full-length hER{alpha}-EF, and the rightmost three lanes indicate data for the G556L:G557L mutant (abbreviated as GG/LL). Ligands added to the media are indicated at the bottom (1 µM E2, 1 µM 4-OHT, and no added ligand, respectively).

 
The F Domain and Expression of the Endogenous pS2 Gene
The previous studies, as well as earlier studies from our and other laboratories, show that truncation or mutation of the F domain alters the expression of exogenous reporter genes, as well as the interaction of the hER{alpha} with the coactivator SRC-1 and other, engineered, proteins. To test the role of the F domain in the expression of an endogenous gene, we constructed lines of MDA-MB-231 cells that stably express comparable levels of the wt or Q580stop ER, and we measured the induction of the pS2 gene by E2 using quantitative real-time PCR.

In two independent experiments using three cell lines each, truncation of the ER greatly reduced, but did not eliminate, the ability of the ER{alpha} to respond to E2. In each experiment, the wt ER exhibited an approximately 15-fold greater induction of pS2 mRNA by E2 than did the Q580stop mutant (Fig. 9Go). This is consistent with the reduced induction of an exogenous reporter and reduced interaction with SRC-1 exhibited by the Q580stop mutant (Figs. 2Go and 4Go). Thus, truncation of the F domain alters the activity of the hER{alpha} not only on exogenous reporter constructs, but on an endogenous gene in a breast cancer cell line.


Figure 9
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 9. Induction of pS2 mRNA by E2 in Cell Lines Stably Expressing the wt or the Q580stop ER

MDA-MB-231 cell lines stably expressing the wt or truncated ER were isolated as described in Materials and Methods and seeded in dextran-coated charcoal-stripped medium. After 25 h, cells were washed and medium containing either vehicle alone (ethanol) or 10–7 M E2 was added. After an additional 48 h, total RNA was isolated as described in Materials and Methods. The level of ps2 transcripts in each sample was analyzed by real-time PCR using Brilliant SYBR Green and primers designed to amplify a pS2 cDNA fragment of 157 bp between exon 2 and exon3 (pS2 forward, ATA CCA TCG ACG TCC CTC CAG; ps2 reverse. gac acc ctc cag gaa gcg a). The results shown are the mean values of pS2 mRNA levels of three clones each of wt and Q580stop ER in response to E2, in two separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Our results show that specific regions in the F domain of the hER{alpha} alter its response to E2 and 4-OHT as well as its interaction with the coactivator SRC-1 and monobodies. In mammalian cells, eliminating residues Q580–M595 reduced ligand-stimulated transcription activation on an ERE-driven promoter and the endogenous pS2 gene, as well as sensitivity to overexpressed SRC-1. In transiently transfected mammalian cells, truncation of additional residues partly restored activity in response to E2 on an exogenous reporter and interaction with SRC-1; removal of the entire F domain was required to eliminate the agonist activity of 4-OHT. In yeast, residues Q580–Y583 contain an inhibitory function that reduces the interaction of ER{alpha} with the monobody E3#6 and the SRC-1 RID in the absence of a ligand. Both assays reveal a previously unsuspected functional complexity within this relatively small region of the protein.

Both the mammalian cotransfection assay and the yeast two-hybrid assay show that the C-terminal residues of the F domain alter the activity of the ER and its interaction with coactivators. However, in the mammalian system basal activity is decreased, whereas in the yeast system basal activity is increased. The difference between the mammalian and yeast systems may be attributed to several factors. The mammalian system uses the full-length receptor and an ERE-based reporter, whereas the yeast system uses a LexA fusion protein of the ER{alpha}-LBD-F. Similarly, the mammalian system uses endogenous coactivators or full-length SRC-1, whereas the yeast system uses the SRC-1 RID or an engineered monobody probe. In addition, the complement of other cellular proteins, particularly coactivators and bridging factors, is different between mammalian cells and yeast (30). Even so, it is remarkable that both assays show that the extreme C-terminal region of the F domain modulates the activity of the ER{alpha}.

We have proposed that the LBD is in an equilibrium among multiple conformations, and removing the F domain shifts the equilibrium toward the active state in the absence of a ligand (21). Crystal structures of the hER LBD support the presence of such a conformational ensemble. In the crystal structure of the LBD of the ß-subtype of ER (hERß-LBD) bound to an ERß-specific antagonist, 5,11-cis-diethyl-5,6,11,12-tetra-hydrochrysene-2,8-diol (THC) (31), H12 took on a position that was "in between" those in agonist-bound LDB, where H12 forms a part of a coactivator binding groove, and those in LBD bound to 4-OHT and raloxifene, where H12 occupies the coactivator binding groove. This conformation resembles that of the hERß-LBD/genistein complex, where genistein is a partial agonist (32). Furthermore, the apo ERß-LBD binds to a nuclear receptor-box peptide more strongly than the ERß-LBD/THC complex, suggesting that a small fraction of apo ERß-LBD exists in a conformation capable of coactivator binding (31).

Our results in this work suggest that, like the effects of ligands, mutations within the ER{alpha} LBD-F domain affect its conformation equilibrium, or energy landscape. The F domain is located immediately C terminal to H12, which is central to the conformational changes of the ER{alpha} LBD. From this architecture, it is not surprising that mutations in the F domain affect the structure and dynamics of the LBD, presumably through the positioning of H12. Indeed, early studies on the F domain suggested that it stabilizes the conformation of the ER{alpha} in the agonist-bound as well as the antagonist-bound state (11). Note that, in other steroid hormone receptors including androgen receptor, glucocorticoid receptor, and progesterone receptor, an 11-residue F domain interacts with the LBD by forming an antiparallel ß-sheet with a region between helices 8 and 9 of the LBD (33, 34, 35). More recent studies show the F domain of the retinoic acid receptor {alpha} stabilizes this receptor in a repressed state (36). One major question to address is the mechanism through which mutations in the F domain affect the conformational equilibrium of the ER{alpha} LBD.

Because the constructs used in our yeast two-hybrid studies contained only the LBD and F domains, it is reasonable to postulate that part of the F domain interacts with the LBD to modulate its conformational dynamics. Because the amino acid sequence of this segment, LQKYY, is distinctly different from the consensus LXXLL sequence of the nuclear receptor box, it is unlikely that the LQKYY segment directly competes against coactivator binding. However, the {alpha}II peptide antagonist identified by Norris et al. (37) as interacting with the E2-bound and the 4-OHT-bound ER{alpha} contains a sequence, FGSWY, reminiscent of the LQKYY sequence within the ER{alpha} F domain. Indeed, recent crystallographic studies show that the {alpha}II peptide binds to a surface of the ER{alpha} LBD that is opposite to the coactivator-binding site (38). Thus, it is possible that the LQKYY segment binds to a surface of the LBD (see below).

The functional data are also best supported by an interaction between the F domain and the LBD and/or other receptor domains. Although the F domain of the hER{alpha} contains only approximately 42 residues, because of its flexibility it could potentially be extended to as long as approximately 100 Å and, thus, it has ample length to interact with the LBD, other receptor domains, and/or other proteins. The agonist-bound LBD monomer is roughly 30 Å x 40 Å x 50 Å and has different surfaces involved in dimerization, ligand association and dissociation, and the binding of coregulatory proteins (39, 40). Because of the length of the F domain, different regions within it could interact with different surfaces of the LBD and, hence, selectively influence the multiple activities of the hER{alpha}. Although we detected no interaction between the hER{alpha}-LBD and the hER{alpha}-F using the yeast two-hybrid system (data not shown), the interaction could exist but may be too weak to be detected when the LBD and F domains are no longer contiguous in the same polypeptide chain.

It is also possible that the F domain interacts with another protein(s) in such a way that helix 12 cannot take on an active conformation in the absence of an agonist. The ER{alpha} interacts with heat shock proteins and cochaperones such as Hsp90, p23, Hsp70, Hop, and Hsp40 (41, 42, 43, 44). Saccharomyces cerevisiae has homologs of these heat shock proteins (hsp) and cochaperones (Hsp82, SbaI, Ssa, Sti1, and Ydj1, respectively), and steroid hormone receptors expressed in yeast bind to all the proteins mentioned above (43, 44). Hsp90 and cochaperones not only help proper ER{alpha} folding, they also prevent ER{alpha} activity in the absence of hormone. Thus, the increased basal hER{alpha} activity in yeast could be due to removal of an interaction between the hsp protein and the F domain. However, this explanation is unlikely because, in mammalian cells, removal of the F domain reduces, rather than increases, basal activity of the hER{alpha}.

Some of the most interesting results described herein are those with the G556L/G557L double mutant. This double mutation eliminated the OHT-dependent, but not the E2-dependent, interaction of three monobodies with ER{alpha}-LBD-F (Fig. 8Go); it had little effect on 4-OHT-driven or E2-driven transactivation (15). In addition, the effects of the G556L/G557L mutation on the interactions with OHT#6 monobody are especially intriguing. This monobody reacts similarly with the E2- and 4-OHT-bound ER{alpha}-LBD-F domain, yet the G556L/G557L mutation eliminated only the interaction in the presence of 4-OHT. These results indicate that residues 556 and 557 of the 4-OHT-bound, but not the E2-bound, LBD are located within or near the monobody binding site, so that the incorporation of the large Leu side chain at these positions selectively disrupts the binding interface. These results clearly indicate that mutations in the F domain can dramatically and selectively influence protein-protein interaction involving the ER{alpha} LBD. This is consistent with results from our and other laboratories demonstrating the role of the F domain in modulating the function of AF-2 (11, 12, 13, 14, 15, 16, 17, 18, 20).

Studies of the hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) provide additional support for the idea that the F domain is a functionally and biologically important modulatory region of the nuclear receptors (45, 46, 47, 48). Alternative splicing gives rise to two forms of the HNF4{alpha}, {alpha}1 and {alpha}2, that differ in their F domains; the HNF4{alpha}2 contains a 10-amino-acid insertion in the middle of F that is absent in HNF4{alpha}1 (45). In transient transfection assays, the HNF4{alpha}2 variant activates transcription 4-fold better than does the HNF4{alpha}1 variant and is more responsive to stimulation by the coactivators glucocorticoid receptor interacting protein-1 and cAMP response element binding protein-binding protein (CBP) (46). Additional studies show that the F domain of HNF4{alpha}1 can functionally interact with coactivators as well as with corepressors (47). Most interestingly, a mutation in a predicted ß-strand-like region of the F domain of HNF4{alpha}2, V393I, is associated with the development of maturity-onset diabetes of the young and exhibits a 50% reduction in activity when compared with the wt protein in transient transfection assays (48). In the light of our results that a predicted ß-strand-like region is involved in interactions between the hER{alpha} and the coactivator SRC-1, it is tempting to speculate there are some functional parallels between the F domains of the two receptors.

In summary, our results show the F domain of the hER{alpha} contains functional elements that selectively modulate the energy landscape of the protein, its response to ligands, its interaction with the coactivator SRC-1, and the expression of the endogenous pS2 gene. The functional complexity of this region may offer novel opportunities for selective control of the activity of the ER{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture and Reagents
HeLa cells were routinely maintained in a 5% CO2 incubator at 37 C using a mixture of phenol-red-free DMEM/F-12 (Invitrogen Life Technologies, Rockville, MD), 10% dextran-coated charcoal-treated fetal bovine serum (HyClone Laboratories, Logan, UT), and 1% penicillin-streptomycin (Invitrogen), as described by Zhao et al. (22). Yeast strains EGY48, MAT{alpha} his3 trp1 ura3 leu2::6LexAop-LEU2, and RFY206, MATa his3{Delta}200 leu2–3 lys2{Delta}201 trp1{Delta}::hisG ura3–52 have been described (49) and were purchased from Origene (Rockville, MD). Yeast cells were grown in yeast extract/peptine/dextrose media or yeast complete dropout media following instructions from Origene and Invitrogen. E2 and 4-OHT were obtained from Sigma Chemical Co. (St. Louis, MO). Oligonucleotides were obtained from Operon (Alameda, CA).

Plasmids
The construction of p2ERE-luc was described by Zhao et al. (22). The construction of the S554stop mutant of the hER{alpha} was described by Schwartz et al. (15). The pSG5-HEGO was a generous gift of Drs. Pierre Chambon and Hinrich Gronemeyer (Institute of Genetics and Cellular and Molecular Biology, Strasbourg, France). The pcDNA3.1 containing the wt hER{alpha} cDNA was the generous gift of Dr. Benita Katzenellenbogen (University of Illinois, Urbana, IL).

Site-Directed Mutagenesis of the hER{alpha} in pSG5 and pcDNA3.1
The G572stop and Q580stop mutants in the pSG5 and pcDNA3.1 (Q580stop only) expression vectors were constructed by site-directed mutagenesis using the GeneEditor in Vitro Site-Directed Mutagenesis System (Promega, Madison, WI), following procedures suggested by the manufacturer. All mutants were confirmed by DNA sequencing.

Transient Transfection and Dual Luciferase Assay
Transient transfections were carried out as described by Zhao et al. (22). Briefly, HeLa cells were seeded at a density of 3.0 x 105 cells per 35-mm well (six-well cell cluster; Costar, Cambridge, MA) in DMEM/F-12, fetal bovine serum (FBS), and antibiotics as described above. The cells were given 20 h to recover after seeding. Transfection experiments were carried out using the Superfect reagent (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. HeLa cells were transiently transfected with a plasmid expressing wt or mutant hER{alpha} (0.80 µg) along with 2ERE-luciferase reporter plasmid (2.0 µg) and transfection control plasmid Renilla luciferase pSV40-RL (20 ng) in serum-containing media.

The cells were incubated for 4 h with the transfection cocktail before medium containing E2 (100 nM), 4-OHT (10 nM or 1 µM), or ethanol vehicle was added. After 40 h the cells were harvested using Passive Lysis Buffer (Promega). The Dual-Luciferase Reporter Assay system (Promega) was used to determine luciferase activities. Luminescence was measured using a Turner (Palo Alto, CA) 20/20 Luminometer. All transfections were performed in triplicate.

In experiments testing sensitivity to coactivator overexpression, preliminary experiments were carried out to determine the optimal amounts of ER-expressing and SRC-1-expressing plasmids. The amount of the ER-expressing plasmid was reduced to 200 ng, and cells were incubated with 400 ng of either the plasmid expressing SRC-1 (pSRC-1-BK-CMV) or the empty vector (pBK-CMV). All other procedures were performed as described above.

Immunoblotting
HeLa cells were plated at a density of 8 x 105 cells/60-mm dish in DMEM/F-12, FBS, and antibiotics as described above. After 24 h, wt hER{alpha} or mutant expression plasmid (4 µg) was used to transiently transfect HeLa cells using the Superfect reagent (QIAGEN) in serum-containing media. The transfected cells were incubated for 4 h before serum-containing media was added. After 40 h cells were lysed in 1x Laemmli sample buffer containing protease inhibitor cocktail (Sigma). The cell protein extracts were separated on a 10% SDS-PAGE gel with 5% cross-linking and transferred to Hybond-P protein transfer membrane (GE Healthcare, Piscataway, NJ). The membrane was blocked in 5% dry milk in T-TBS (20 mM Tris, pH 7.6; 1.5% NaCl; 0.05% Tween) and incubated with the mouse anti-ER monoclonal antibody Ab-11 (Neomarkers, Fremont, CA) (2.0 µg/ml) for 3 h, washed in T-TBS, incubated with antimouse horseradish peroxidase-conjugated secondary antibody (1:2000) for 1 h, and washed in T-TBS. Signal was detected using the enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech).

Statistical Analysis
To analyze the activity of the wt and mutated ER on the ERE-driven reporter (Figs. 2Go and 3Go), the data were first subjected to a log transformation. Random-effects generalized linear models (50) were used to assess the statistical significance of differences in response; observations from experiments run on the same day were assumed to be correlated. The first model fitted included indicators for mutants, for ligand concentrations where appropriate, and for their interaction. Ligand concentrations were included in the model as indicator variables to avoid constraining the shape of the relationship between concentration and response. If the simultaneous test for statistical interaction terms was not significant, a reduced model was fitted in which the interaction terms were omitted. Finally, an even simpler model was fitted in which ligand was parameterized as presence or absence, collapsed over different concentrations. If there was significant interaction in the full model, separate models were fitted for each mutant in which ligands were compared. In the case of significant interaction, separate models for each ligand were also fitted and the mutants were compared. Holm’s stepdown procedure (51) was used to control type I errors when making multiple tests among coefficients. Model goodness of fit was assessed graphically.

To analyze the sensitivity of the wt and mutated ERs to SRC-1 overexpression (Fig. 4Go), the shape of the empirical distribution of the data (relative luciferase units, RLUs) was assessed graphically and found to be left skewed. Application of a square root transformation resulted in a reasonably symmetric distribution, and the statistical models were built and tested using the transformed data. Data from experiments performed on the same date were assumed to be correlated. Therefore, a mixed-effects analysis of variance was used with date of experiment as a random effect and E2, SRC-1, and protein as fixed effects. The initial model included all two-way interactions. In the reduced model, there is strong evidence (P < 0.001) of statistical interaction between E2 and SRC-1 and, for that reason, we fitted separate models to each combination of E2 and SRC-1 and compared proteins. Holm’s procedure (51) was used to adjust for multiple comparisons. The fit of the model was assessed by examination of residuals and influential points (50).

Yeast Two-Hybrid Vector Construction
General molecular cloning procedures were according to Sambrook et al. (52). The plasmids for B42 activation domain-monobody fusions, B42-SRC-1 fusion, and pEGER{alpha}297–595 have been described (20). The plasmids of the F domain deletion mutants were constructed by subcloning PCR fragments with a termination codon at various positions in the F domain followed by XhoI site. The PCR fragments were digested with NcoI and XhoI and ligated into pEGER{alpha}297–595 that had been cleaved with NcoI and XhoI. Alanine substitution mutants were also constructed using PCR. The plasmid that encodes B42-hER{alpha}-LBD (residue 297–554) fusion was constructed by ligating the SstI-XhoI fragment of pEGER{alpha}297–554 (20) into pYesTrp2 (Invitrogen) that had been digested with SstI and XhoI. A plasmid that encodes LexA-hER{alpha}-F (residue 555–595) fusion was constructed by subcloning PCR fragment containing hER{alpha}-F flanked by EcoRI and XhoI restriction sites between the EcoRI and XhoI sites of pEGER{alpha}297–595.

Yeast Two-Hybrid ß-Galactosidase assay
EGY48 cells that express a "prey" (the B42 activation domain fused to either the SRC-1 RID or monobody E3#6) were mated with RFY206 cells that contain a "bait" (the LexA protein fused to ER{alpha}-LBD-F with or without mutation) and a LacZ reporter gene (20). The mated cells were grown in the presence of 2% galactose and in the presence or absence of E2 or 4-OHT for 6 h at 30 C, and the ß-galactosidase activities of the cells were measured as described previously (20). Western blotting was performed as described previously (20).

Establishment of MDA-MB-231 Cell Lines Stably Expressing wt and Q580stop hER
Twenty-four hours before transfection, 1 x 105 MDA-MB231 cells were seeded in each well of six-well cell culture plates and digested the plasmids (pcDNA3.1 containing either the wt hER{alpha} cDNA, or Q580stop mutant, constructed as described above) with SspI. MDA-MB-231 cells were transfected with 1 µg of either linear plasmid in Superfect Transfection Reagent (QIAGEN). Three hours later, the transfection medium was removed and the cells were cultured with regular medium for 40 h. Thereafter the cells were trypsinized and seeded in 15-cm culture dishes with medium containing geneticin (600 µg/ml) (Invitrogen). Single colonies formed in about 2 wk. Twelve colonies from each plate were picked and cultured until confluent in medium with geneticin (400 µg/ml). In about 4 wk, the expression of either hER{alpha} wt or hER{alpha} Q580stop was detected by estrogen stimulated-transcription of an ERE-driven luciferase reporter construct, followed by Western immunoblotting, and stable cell lines with similar high levels of expression of functional receptors were selected.

Quantitative Analysis of the pS2 Transcript by Quantitative Real-Time PCR
Three x 106 MDA-MB-231 cells stably expressing either wt hER{alpha} or Q580stop mutant were seeded in 60-mm dishes in phenol-red free DMEM/F12 medium (Invitrogen) containing 10% charcoal/dextran-treated FBS (HyClone) and 1% penicillin-streptomycin (Invitrogen). Twenty-four hours later, the cells were washed three times with PBS and culture medium containing either vehicle alone (ethanol) or 10–7 M E2 (Sigma) was added. After another 48 h in culture, total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. During the isolation of total RNA, the RNA samples were digested by RNase-Free DNase (QIAGEN). To compare pS2 mRNA levels in the absence and presence of E2, in lines expressing the wt or Q580stop ER, the QuantiTect Reverse Transcription Kit (QIAGEN) was used to synthesize the first strand of cDNA from 1 µg total RNA samples. The level of pS2 transcripts in each reverse-transcribed cDNA sample was measured by real-time PCR in triplicate using the MX4000 Instrument (Stratagene, La Jolla, CA) and Brilliant SYBR Green QPCR Master Mix (Stratagene). A pair of primers was designed to amplify a human pS2 cDNA fragment of 157 bp between exon 2 and exon 3 (pS2 forward, ATA CCA TCG ACG TCC CTC CAG; pS2 reverse, aag cgt gtc tga ggt gtc cg). The pS2 transcript level was standardized to the level of the 36B4 transcript, a human ribosomal protein (36B4 forward, G GTG TTC GAC AAT GGC AGC ATC; 36B4 reverse, gac acc ctc cag gaa gcg a). The results shown are the mean values of pS2 mRNA levels of three clones each of wt and Q580stop ER in response to E2.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grant R01 DK63090 and the University of Chicago Cancer Research Center (to S.K.), and by NIH Grant R01 DK56934 and DAMD 17-00-1-0498 (to D.F.S.).

First Published Online February 13, 2007

1 A.K. and C.Z., and D.F.S. and S.K., respectively, contributed equally to this work. Back

Abbreviations: AF, Activation function; DBD, DNA-binding domain; E2, estradiol; ER, estrogen receptor; ERE, estrogen response element; FBS, fetal bovine serum; h, human; HNF4{alpha}, hepatocyte nuclear factor 4{alpha}; hsp, heat shock protein; LBD, ligand-binding domain; 4-OHT, 4-hydroxytamoxifen; RID, receptor interaction domain; RLU, relative luciferase unit; SRC-1, steroid receptor coactivator-1; wt, wild type.

Received for publication May 11, 2006. Accepted for publication December 6, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Nilsson S, Gustafsson JA 2000 Estrogen receptor transcription and transactivation: Basic aspects of estrogen action. Breast Cancer Res 2:360–366[CrossRef][Medline]
  2. Katzenellenbogen BS, Katzenellenbogen JA 2000 Estrogen receptor transcription and transactivation. Estrogen receptor {alpha} and estrogen receptor ß: regulation by selective estrogen receptor modulators and importance in breast cancer. Breast Cancer Res 2:335–344[CrossRef][Medline]
  3. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941–951[CrossRef][Medline]
  4. Wang C, Fu M, Angeletti RH, Siconolfi-Baez L, Reutens AT, Albanese C, Lisanti MP, Katzenellenbogen BS, Kato S, Hopp T, Fuqua SA, Lopez GN, Kushner PJ, Pestell RG 2001 Direct acetylation of the estrogen receptor {alpha} hinge region by p300 regulates transactivation and hormone sensitivity. J Biol Chem 276:18375–18383[Abstract/Free Full Text]
  5. Birnbaumer M, Schrader WT, O’Malley BW 1983 Assessment of structural similarities in chick oviduct progesterone receptor subunits by partial proteolysis of photoaffinity-labeled proteins. J Biol Chem 258:7331–7337[Abstract/Free Full Text]
  6. Dahlman-Wright K, Baumann H, McEwan IJ, Almlof T, Wright AP, Gustafsson JA, Hard T 1995 Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor. Proc Natl Acad Sci USA 92:1699–1703[Abstract/Free Full Text]
  7. Bain DL, Franden MA, McManaman JL, Takimoto GS, Horwitz KB 2000 The N-terminal region of the human progesterone A-receptor. Structural analysis and the influence of the DNA binding domain. J Biol Chem 275:7313–7320[Abstract/Free Full Text]
  8. Warnmark A, Wikstrom A, Wright AP, Gustafsson JA, Hard T 2001 The N-terminal regions of estrogen receptor {alpha} and ß are unstructured in vitro and show different TBP binding properties. J Biol Chem 276:45939–45944[Abstract/Free Full Text]
  9. Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA 1996 Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930[Abstract/Free Full Text]
  10. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  11. Montano MM, Muller V, Trobaugh A, Katzenellenbogen BS 1995 The carboxy-terminal F domain of the human estrogen receptor: role in the transcriptional activity of the receptor and the effectiveness of antiestrogens as estrogen antagonists. Mol Endocrinol 9:814–825[Abstract/Free Full Text]
  12. Weatherman RV, Scanlan TS 2001 Unique protein determinants of the subtype-selective ligand responses of the estrogen receptors (ER{alpha} and ERß) at AP-1 sites. J Biol Chem 276:3827–3832[Abstract/Free Full Text]
  13. Warnmark A, Almlof T, Leers J, Gustafsson JA, Treuter E 2001 Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ER{alpha} and ERß. J Biol Chem 276:23397–23404[Abstract/Free Full Text]
  14. Kim K, Thu N, Saville B, Safe S 2003 Domains of estrogen receptor {alpha} (ER{alpha}) required for ER{alpha}/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells. Mol Endocrinol 17:804–817[Abstract/Free Full Text]
  15. Schwartz JA, Zhong L, Deighton-Collins S, Zhao C, Skafar DF 2002 Mutations targeted to a predicted helix in the extreme carboxyl-terminal region of the human estrogen receptor-{alpha} alter its response to estradiol and 4-hydroxytamoxifen. J Biol Chem 277:13202–13209[Abstract/Free Full Text]
  16. Loven MA, Muster N, Yates JR, Nardulli A 2003 A novel estrogen receptor {alpha}-associated protein, template-activating factor Iß, inhibits acetylation and transactivation. Mol Endocrinol 17:67–78[Abstract/Free Full Text]
  17. Nichols M, Rientjes J M, Stewart AF 1999 Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists. EMBO J 17:765–773
  18. Peters GA, Khan SA 1999 Estrogen receptor domains E and F: role in dimerization and interaction with coactivator RIP-140. Mol Endrocrinol 13:286–296[Abstract/Free Full Text]
  19. Koide A, Bailey CW, Huang X, Koide S 1998 The fibronectin type III domain as a scaffold for novel binding proteins. J Mol Biol 284:1141–1151[CrossRef][Medline]
  20. Koide A, Abbatiello S, Rothgery L, Koide S 2002 Probing protein conformational changes in living cells by using designer binding proteins: application to the estrogen receptor. Proc Natl Acad Sci USA 99:1253–1258[Abstract/Free Full Text]
  21. Richards J, Miller M, Abend J, Koide A, Koide S, Dewhurst S 2003 Engineered fibronectin type III domain with a RGDWXE sequence binds with enhanced affinity and specificity to human {alpha}vß3 integrin. J Mol Biol 326:1475–1488[CrossRef][Medline]
  22. Zhao C, Koide A, Abrams J, Deighton-Collins S, Martinez A, Schwartz JA, Koide S, Skafar DF 2003 Mutation of Leu-536 in human estrogen receptor-{alpha} alters the coupling between ligand binding, transcription activation, and receptor conformation. J Biol Chem 278:27278–27286[Abstract/Free Full Text]
  23. Gyuris J, Golemis E, Chertkov H, Brent R 1993 Cdi1, a human G1 and S phase protein phosphatase that associates with Cdk2. Cell 75:791–803[CrossRef][Medline]
  24. Pakdel F, le Goff P, Katzenellenbogen BS 1993 An assessment of the role of domain F and PEST sequences in estrogen receptor half-life and bioactivity. J Steroid Biochem Mol Biol 46:663–672[CrossRef][Medline]
  25. Lonard DM, Nawaz Z, Smith CL, O’Malley BW 2000 The 26S proteasome is required for estrogen receptor-{alpha} and coactivator turnover and for efficient estrogen receptor-{alpha} transactivation. Mol Cell 5:939–948[CrossRef][Medline]
  26. Onate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  27. Bocquel MT, Kumar V, Chambon P, Gronemeyer H 1989 The contribution of the N- and C-terminal regions of steroid receptors to activation of transcription is both receptor and cell-specific. Nucleic Acids Res 17:2581–2595[Abstract/Free Full Text]
  28. Needham M, Raines S, McPheat J, Stacey C, Ellston J, Hoare S, Parker M 2000 Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif. J Steroid Biochem Mol Biology 72:35–46
  29. Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms of steroid receptor co-activator 1 differ in their ability to potentiate transcription by the oestrogen receptor. EMBO Journal 17:232–243[CrossRef][Medline]
  30. Sheppard HM, Matsuda S, Harries JC, Kindle KB, Heery DM 2003 Transcriptional activation by estrogen receptor (ER{alpha}) and steroid receptor coactivator (SRC1) involves distinct mechanisms in yeast and mammalian cells. J Mol Endocrinol 30:411–422[Abstract]
  31. Shiau AK, Barstad D, Radek JT, Meyers MJ, Nettles KW, Katzenellenbogen BS, Katzenellenbogen JA, Agard DA, Greene GL 2002 Structural characterization of a subtype-selective ligand reveals a novel mode of estrogen receptor antagonism. Nat Struct Biol 9:359–364[Medline]
  32. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstrom O, Ljunggren J, Gustafsson JA, Carlquist M 1999 Structure of the ligand-binding domain of oestrogen receptor ß in the presence of a partial agonist and a full antagonist. EMBO J 18:4608–4618[CrossRef][Medline]
  33. Williams SP, Sigler PB 1998 Atomic structure of progesterone complexed with its receptor. Nature 393:392–396[CrossRef][Medline]
  34. Sack JS, Kish KF, Wang C, Attar RM, Kiefer SE, An Y, Wu GY, Scheffler JE, Salvati ME, Krystek Jr SR, Weinmann R, Einspahr HM 2001 Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone. Proc Natl Acad Sci USA 98:4904–4909[Abstract/Free Full Text]
  35. Kauppi B, Jakob C, Farnegardh M, Yang J, Ahola H, Alarcon M, Calles K, Engstrom O, Harlan, J, Muchmore S, Ramqvist AK, Thorell S, Ohman L, Greer J, Gustafsson JA, Carlstedt-Duke J, Carlquist M 2003 The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism. J Biol Chem 278:22748–22754[Abstract/Free Full Text]
  36. Farboud B, Privalsky M 2004 Retinoic acid receptor-{alpha} is stabilized in a repressive state by its C-terminal, isotype-specific F domain. Mol Endocrinol 18:2839–2853[Abstract/Free Full Text]
  37. Norris JD, Paige LA, Christensen DJ, Chang C-Y, Huacani MR, Fan D, Hamilton PT, Fowlkes DM, McDonnell DP 1999 Peptide antagonists of the human estrogen receptor. Science 285:744–746[Abstract/Free Full Text]
  38. Kong EH, Heldring N, Gustafsson JA, Treuter E, Hubbard RE, Pike ACW 2005 Delineation of a unique protein-protein interaction site on the surface of the estrogen receptor. Proc Natl Acad Sci USA 102:3593–3598[Abstract/Free Full Text]
  39. Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature (Lond) 390:753–758
  40. Shiau AK, Barstad D, Loria, PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
  41. Pratt WB, Toft DO 1997 Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocr Rev 18:306–360[Abstract/Free Full Text]
  42. Johnson JL, Craig EA 1997 Protein folding in vivo: unraveling complex pathways. Cell 90:201–204[CrossRef][Medline]
  43. Fang Y, Fliss AE, Rao J, Caplan AJ 1999 SBA1 encodes a yeast hsp90 cochaperone that is homologous to vertebrate p23 proteins. Mol Cell Biol 18:3727–3734
  44. Fliss AE, Benzeno S, Rao J, Caplan AJ 2000 Control of estrogen receptor ligand binding by Hsp90. J Steroid Biochem Mol Biol 72:223–230[CrossRef][Medline]
  45. Sladek FM, Ruse MD, Nepomuceno L, Huang SH, Stallcup MR 1999 Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4{alpha}1. Mol Cell Biol 19:6509–6522[Abstract/Free Full Text]
  46. Bogan AA, Dallas-Yang Q, Ruse MD, Maeda Y, Jiang G, Nepomuceno L, Scanlan TS, Cohen FE, Sladek FM 2000 Analysis of protein dimerization and ligand binding of orphan receptor HNF4{alpha}. J Mol Biol 302:831–851[CrossRef][Medline]
  47. Ruse MD, Privalsky ML, Sladek FM 2002 Competitive cofactor recruitment by orphan receptor hepatocyte nuclear factor 4{alpha}1: modulation by the F domain. Mol Cell Biol 22:1626–1638[Abstract/Free Full Text]
  48. Hani EH, Suaud L, Boutin P, Chevre JC, Durand E, Philippi A, Demenais F, Vionnet N, Furuta H, Velho G, Gell GI, Laine B, Froguel P 1998 A missense mutation in hepatocyte nuclear factor-4{alpha}, resulting in a reduced transactivation activity, in human late-onset non-insulin-dependent diabetes mellitus. J Clin Invest 101:521–526[Medline]
  49. Finley Jr RL, Brent R 1994 Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc Natl Acad Sci USA 91:12980–12984[Abstract/Free Full Text]
  50. Harrell Jr FE 2001 Regression modeling strategies: with applications to linear models, logistic regression and survival analysis. New York: Springer-Verlag
  51. Holm S 1997 A simple sequentially rejective multiple test procedure. Scand J Stat 6:65–70
  52. Sambrook J, Fritsch EF, Maniatis T 2001 Molecular cloning: A laboratory manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory

NURSA Molecule Pages Link:

Nuclear Receptors:   ERα
Coregulators:   RIP140  |  SPT6  |  p300  |  SRC-1  |  GRIP1  |  AIB1
Ligands:   17β-Estradiol  |  4-Hydroxytamoxifen



This article has been cited by other articles:


Home page
 Endocr Relat CancerHome page
D. J Kojetin, T. P Burris, E. V Jensen, and S. A Khan
Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor
Endocr. Relat. Cancer, December 1, 2008; 15(4): 851 - 870.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. T. Sonoda, L. Martinez, P. Webb, M. S. Skaf, and I. Polikarpov
Ligand Dissociation from Estrogen Receptor Is Mediated by Receptor Dimerization: Evidence from Molecular Dynamics Simulations
Mol. Endocrinol., July 1, 2008; 22(7): 1565 - 1578.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koide, A.
Right arrow Articles by Koide, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koide, A.
Right arrow Articles by Koide, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals