Agonist and Antagonists Induce Homodimerization and Mixed Ligand Heterodimerization of Human Progesterone Receptors in Vivo by a Mammalian Two-Hybrid Assay

Susan A. Leonhardt, Magda Altmann and Dean P. Edwards

Department of Pathology and Program in Molecular Biology University of Colorado Health Sciences Center Denver, Colorado 80262


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
This study utilizes the mammalian two-hybrid system to examine the role of ligand in the dimerization of human progesterone receptor (hPR). The GAL4 DNA-binding domain and the herpes simplex virus VP16 transactivation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) to produce chimeric proteins. PR dimerization was detected by the ability of cotransfected GAL4/PR and VP16/PR chimeras in COS cells to induce expression of a reporter gene under the control of GAL4-binding sites (pG5CAT). Hormone agonist-dependent interactions were observed between the two like isoforms of PR (A-A and B-B) and between PR-A and PR-B (A-B), indicating that hormone can stimulate the formation of the three possible dimeric forms of PR within cells. In contrast, neither type I (ZK98299) nor type II (RU486, ZK112993) progestin antagonists stimulated interaction between these same hybrid PR proteins. However, activation of the VP16/PR chimera by antagonists on a progesterone response element-controlled reporter gene (DHRE-E1b-CAT) was only a fraction (4–13%) of that stimulated by agonist R5020. One possibility for the failure to detect an induction in the two-hybrid assay is antagonist-induced repression of the activity of the VP16/PR fusion protein rather than a failure of antagonists to stimulate interaction between the hybrid proteins. To test this idea, an UP-1 carboxyl-terminal truncation mutant of PR was used to construct the two-hybrid proteins. PR-UP-1 selectively binds antagonists, but not agonists, and is fully activated in response to antagonists. Both types of progestin antagonists stimulated interactions between GAL4/PR(UP-1) and VP16/PR(UP-1) hybrid proteins, indicating that antagonists are capable of stimulating PR dimerization in cells and do not function by disrupting or preventing dimerization. To determine whether PR bound to an antagonist can dimerize in whole cells with PR bound to agonist, GAL4/PR(UP-1) was paired in the two- hybrid assay with a VP16/PR fusion protein harboring a point mutation in PR at amino acid 722 (Gly-Cys) that specifically binds progestin agonist but not antagonist. Neither R5020 nor RU486 alone stimulated interaction between these ligand-specific PR hybrid proteins. However, strong interaction was detected by addition of both agonist and antagonists, indicating the formation of mixed ligand heterodimers and that both PR partners require ligand for dimerization to occur. Based on electrophoretic gel mobility shift assays (EMSAs), these heterodimers appear to have substantially reduced DNA binding activity. Progestin antagonists inhibit agonist activation of PR at concentrations that are too low to be accounted for by a simple competition mechanism for binding to PR. We propose that antiprogestin inactivation of PR in trans by heterodimerization contributes to the biological potency of these compounds.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human progesterone receptor (hPR) is a ligand-activated transcription factor that mediates the actions of the sex steroid progesterone in target cells. A member of the steroid receptor superfamily, hPR is expressed as two distinct isoforms of 94 kDa, PR-A, and 120 kDa, PR-B (1). The two isoforms have identical sequence in their carboxyl-terminal ligand-binding domain (LBD) and the centrally located DNA-binding domain (DBD). PR-A differs from PR-B only by lacking 164 amino acids (aa) of the extreme N terminus (1). In the absence of hormone, PR is found in oligomeric complexes with several other proteins, including heat shock protein 90 (hsp90), hsp70, p59, and immunophilins (2). Ligand binding dissociates receptors from this inactive complex to allow receptor dimerization and binding to hormone-response elements (HREs) that are typically located in the 5'-regulatory region of steroid-responsive genes (3). After binding DNA, PR activates or represses transcription of target genes by yet incompletely understood mechanisms. This process appears to involve direct interaction with the general transcription machinery (4) and or association with coactivators/corepressors that link the receptors with the general transcriptional machinery (5, 6, 7).

For the sex steroid receptors and glucocorticoid receptor (GR), dimerization appears to be a critical factor controlling receptor binding to palindromic HREs (8, 9). Steroid receptors have been shown to be able to form dimers in solution, and as full-length proteins they bind preferentially to HREs as preformed dimers. In general, steroid receptors interact with target palindromic HREs as a single type of homodimer. Exceptions to this are hPR, which binds to target DNA as three dimeric species composed of AA, AB, and BB subunits (10, 11, 12, 13) and the more recent demonstration that GR can form a heterodimer complex on DNA with mineralocorticoid receptor (MR) (14, 15). In addition, the newly discovered estrogen receptor-ß (ERß) can heterodimerize with ER{alpha} (16). In contrast, receptors for nonsteroid hormones, such as vitamin D, thyroid hormone, and retinoids, preferentially bind DNA as heterodimers with retinoic acid X receptor (17).

Two dimerization domains have been described within steroid receptors. A DNA-dependent dimerization interface located within the DBD is important for restricting steroid receptor recognition of palindromic HREs separated by three intervening nucleotides and for stabilization of receptor dimers on DNA (18). A second dimerization function resides in a region(s) outside the DBD that is involved in solution dimerization in the absence of DNA (18, 19, 20). The LBD of ER contains a strong hormone-dependent dimerization function that is required for solution dimerization of ER (8, 21, 22). Other studies with GR, androgen receptor, and PR have suggested that the LBD alone is not sufficient for stable solution dimerization and that amino-terminal sequences are also involved (23, 24, 25, 26, 27). Recently, we have analyzed the sequence regions involved in solution dimerization and have found that both the hinge and amino-terminal sequences contribute to PR dimerization (28).

Several synthetic ligands have been developed that compete effectively for binding of progestins to PR and are capable of inhibiting receptor activation. The mechanism by which antiprogestins inhibit activation remains incompletely understood. Original studies based on in vitro electrophoretic gel mobility shift assays (EMSA) classified ZK98299 (Onapristone) as a type I compound that failed to promote PR binding to DNA. Other antiprogestins such as RU486 (Mifepristone) were classified as type II compounds that efficiently enhance PR binding to DNA (13) and thus impair receptor transactivation at step(s) downstream of DNA binding. However, assays based on detection of PR-DNA binding in whole cells (29, 30) and our more recent results with altered EMSA conditions (30) indicate that ZK98299 does enhance PR binding to DNA. It has been well documented that antiprogestins induce conformational changes within the LBD of PR that are distinct from that induced by agonists (11, 12, 31, 32, 33, 34). Similar results have been shown with ER and estrogen antagonists (35). One consequence of the different conformation is to prevent the interaction of specific steroid receptor coactivators with the hormone agonist-dependent transcription activation function 2 (AF-2) (5, 6, 7). ZK98299 and RU486 have been shown to have different effects on PR conformation, suggesting that they may represent different classes of progestin antagonists based on inducing distinct conformational changes within PR (30, 36, 37).

The biological activity of antiprogestins as assessed by transfection experiments indicates that these compounds inhibit transcriptional activation of PR at substoichiometric concentrations with hormone. This suggests that antiprogestins do not act by simple competition with hormone for binding to PR (38). This unusually high potency of antiprogestins could, in part, be explained by the PR-antiprogestin complex functioning to repress the PR-agonist complex in trans. Since PR requires dimerization for function, this could be achieved by heterodimerization between a PR bound to agonist with a PR bound to antagonist.

Numerous studies have recently taken advantage of the two-hybrid protein-protein interaction assay to examine dimerization of eukaryotic transcription factors (39). Originally developed in yeast (40) and later adapted to mammalian cells (41), the assay involves coexpression of two proteins of interest fused to an autonomous DBD (from the GAL4 or LEXA transcription factors) and a strong transcriptional activation domain (from GAL4 or VP16). Stable protein-protein interactions can functionally reconstitute the separate DBD and activation domains of the transcription factor, resulting in activation of a reporter gene bearing DNA-binding sites for the DBD fusion protein. Previously, a yeast two-hybrid assay was used to show ligand-inducible dimerization of ER in vivo (42) and, more recently, a mammalian based two-hybrid assay was used to demonstrate estrogen-dependent heterodimerization between ERß and ER{alpha} in whole cells (16). In the present study, we used a mammalian two-hybrid assay to investigate whether the three detected PR dimer forms complexed to DNA in vitro can be formed in whole cells and whether or not this is ligand dependent. In addition, we have used this system to examine the effect of progesterone antagonists on receptor dimerization and whether PR bound to agonist can heterodimerize in the cell with PR bound to antagonist.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of GAL4/PR and VP16/PR Fusion Proteins
To examine PR dimerization in whole cells, we have used a mammalian based two-hybrid protein-protein interaction assay. Either the GAL4 DBD or the VP16 heterologous transcriptional activation domain were fused to the amino terminus of full-length hPR (both the A and B isoforms) (see Fig. 1AGo). The chimeric constructs were transiently transfected into PR- negative COS-1 cells along with a reporter plasmid, pG5CAT, that contains five GAL4 consensus binding sites and the minimal E1b promoter driving the chloramphenicol acetyl transferase (CAT) gene. Interaction between two PR molecules was assessed by the ability of cells transfected with GAL4/PR and VP16/PR constructs to activate the pG5CAT reporter gene as compared with cells transfected with either PR chimera alone.



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Figure 1. Schematic and Expression of GAL4 DBD and VP16 PR Chimeras

A, PR chimeric constructs used in the mammalian two-hybrid assay. PR-B (aa 1–933) and PR-A (aa 165–933) were fused at their amino terminus to the GAL4 DBD (aa 1–147) and the VP16 activation domain (aa 411–455). DNA binding (cross-hatched boxes) and ligand binding (dark solid boxes) domains of the receptors are also shown. B, The expression level of the GAL4/PR and VP16/PR fusion proteins was analyzed by transient transfection into COS-1 cells, followed by immunoblot analysis with the PR-specific MAb, 1294/H9. For comparison, equal amounts of proteins from T47D breast cancer cells and COS-1 cells that had been transfected with the plasmid pSVhPR-B, to express wild-type PR-B, were also analyzed. The sizes of the molecular mass markers in kilodaltons are indicated by line markings, and the sizes of native PR-A and PR-B in T47D cells are indicated by arrows.

 
We first analyzed transfected COS-1 cells for expression levels and the ability of the receptor fusion proteins to bind hormone. As shown by immunoblot assay of COS-1 cell lysates with a PR-specific monoclonal antibody (MAb), the chimeric PR constructs produced immunoreactive proteins of larger apparent mol wt than native PR-A and PR-B from T47D breast cancer cells and full-length PR-B (transfected with pSVhPR-B) expressed in COS-1 cells as a nonfusion protein (Fig. 1BGo). In addition, each of the PR fusion proteins was expressed at approximately the same level as native PR in T47D cells (Fig. 1BGo). It should be noted that even though the VP16 domain is of smaller mol wt than the GAL4 DBD, the VP16 fusion proteins exhibit slower mobility than the GAL4 fusion proteins. A similar phenomenon was reported for androgen receptor two-hybrid proteins suggesting that the highly acidic nature of VP16 causes anomalous electrophoretic mobility (43). The immunoreactive doublets observed in the PR-A and PR-B GAL4 lysates may be a result of proteolytic cleavage of the fusion proteins; however, these lower mol wt species do not seem to interfere with our ability to detect interactions with the VP16 fusion proteins (see below). The expressed PR fusion proteins were also characterized for steroid binding activity by a whole cell-binding assay. The results summarized in Table 1Go show that all PR fusion proteins exhibit similar specific binding to the synthetic progestin [3H]R5020 with equal capacity, again approaching the level of native PR in T47D cells. Thus, fusion of PR to either the GAL4 DBD or VP16 did not adversely affect the expression or the steroid binding activity of PR.


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Table 1. GAL4 DBD and VP16 Fusion Proteins Bind Hormone

 
Hormone-Dependent Dimerization of the A and B Isoforms of hPR
The A and B forms of PR have been shown to bind in vitro, in a hormone-dependent manner, to target progesterone response elements (PREs) in three dimeric forms: AA, AB, and BB dimers (11, 12, 44, 45). PR dimerization in solution has also been demonstrated in vitro by coimmunoprecipitation (45, 46, 47), nondenaturing gel electrophoresis (48), and pull-down assay with his tagged PR (28). However, results are variable as to whether solution dimerization in vitro is hormone dependent, and these in vitro assays have not been able to distinguish between the different possible isoform compositions of the dimers. Therefore, we have used a mammalian two-hybrid assay to ask whether the three possible dimers of PR (AA, AB, and BB) are able to form in whole cells and to what extent these dimerization interactions are hormone dependent.

When COS-1 cells were cotransfected with either, or both, parental vectors that express GAL4 DBD or VP16 without PR sequences, activation of the GAL4- controlled pG5CAT reporter gene was negligible (Fig. 2Go). Also, little or no activation of pG5CAT was observed when cells were cotransfected with a single VP16/PR-B chimeric vector cotransfected with a GAL4 DBD empty vector control (Fig. 2Go). The low CAT activity observed under these conditions was considered as the basal activity of the pG5CAT promoter in COS-1 cells. However, the GAL4/PR-B chimeric construct as a single hybrid (cotransfected with VP16 empty control vector) did activate pG5CAT 2- to 4-fold above the basal level in response to R5020 (Fig. 2Go). Activation of the pG5CAT reporter by the single GAL4/PR-B chimeric receptor is not entirely unexpected. Full-length PR-B harbors multiple transactivation domains (49, 50) and has the potential, through direct utilization of these domains, to mediate transcription when tethered to the pG5CAT through its GAL4 DBD fusion sequences. In contrast, PR-A, which is generally a weaker transcriptional activator than PR-B (51, 52), mediates no induction of the pG5CAT reporter as a single hybrid in response to hormone (not shown and Fig. 3Go). The data in Fig. 2Go illustrate the importance of factoring CAT expression mediated by the single GAL4/PR hybrid to reliably detect induction of PR dimerization as reflected by interaction of the two PR fusion proteins. In later two-hybrid experiments, the GAL4/PR-B chimeric construct as a single hybrid mediated a higher activation of the pG5CAT reporter in response to R5020 (see below) than shown in Fig. 2Go, further emphasizing the need to factor in the contribution of this single hybrid when calculating CAT expression that represents PR dimerization.



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Figure 2. Background Activity of PR-B Single Hybrids in Mammalian Cells

COS-1 cells were transiently transfected with the indicated constructs and the pG5CAT reporter. Twenty four hours posttransfection, cells were treated for another 24 h with either vehicle (ethanol) or 100 nM R5020. CAT activity was measured as described in Materials and Methods and was calculated as counts per min per µg of protein from duplicate treatment wells. The results are from a single representative experiment of three independent determinations.

 


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Figure 3. Hormone-Dependent Dimerization of Different PR Isoforms In Vivo

COS-1 cells were cotransfected with GAL4/PR chimeric vectors and VP16 (empty vector) construct or with GAL4/PR chimeric vectors and VP16/PR chimeric vectors and the pG5CAT reporter plasmid. Twenty four hours posttransfection, cells were treated with either vehicle or 100 nM R5020 for 24 h. CAT activity was determined and calculated as counts per min per µg of protein from duplicate treatment wells. Within each group of PR hybrid interactions (A-A, A-B, or B-B), relative CAT activity was determined as described in Materials and Methods. Values are averages from multiple independent experiments (± SEM; n = 7). The fold hormone stimulation of dimerization was calculated as the difference in relative CAT activity mediated by cells expressing two hybrid proteins plus ligand and the relative CAT activity mediated by the GAL4 single hybrid plus ligand (see Materials and Methods). The fold stimulations were: 3.44 for PR-A-PR-A, 7.16 for PR-A-PR-B, and 3.20 for PR-B-PR-B interactions. Statistical analysis using a t test revealed a significant difference between the relative CAT activity mediated by two hybrid proteins plus R5020 and the activity mediated by one hybrid protein plus R5020 for all three types of PR-PR interactions (P < 0.05). No significant difference was measured between two hybrid and one hybrid mediated CAT activity in the absence of ligand. The schematic underneath the graph illustrates CAT activity mediated by the one hybrid protein in response to R5020 and CAT activity mediated by two hybrid proteins in response to R5020 treatment.

 
We next compared the ability of the different isoforms of hPR to interact with each other in the two-hybrid assay in the presence and absence of R5020. Reporter gene induction in the two-hybrid interaction experiments was determined as relative CAT activity by arbitrarily setting the CAT enzyme activity obtained with the single GAL4/PR hybrid in the absence of ligand (ethanol) to one (see Materials and Methods). GAL4/PR single hybrids were cotransfected with empty VP16 vector controls (see Fig. 3Go). As shown in Fig. 3Go, hormone (R5020) treatment of cells transfected with the GAL4/PR-A single hybrid did not stimulate CAT activity above the ethanol control, whereas R5020 stimulated a significant (6-fold) increase of CAT expression in cells transfected with the GAL4/PR-B single hybrid. When cells were cotransfected with both GAL4/PR and VP16/PR fusion proteins, no significant stimulation of CAT activity above the single hybrid controls was obtained in the absence of ligand. Hormone (R5020) addition to cells cotransfected with the different two hybrid combinations in each case stimulated a significant increase of CAT activity as compared with ethanol controls and, more importantly, hormone also stimulated a significant increase of CAT activity over the single hybrid controls treated with R5020 (Fig. 3Go). The two-hybrid combinations included GAL4/PR-A plus VP16/PR-A (A-A interaction), GAL4/PR-A plus VP16/PR-B (A-B interaction), and GAL4/PR-B plus VP16/PR-B (B-B interaction). To determine the fold hormone stimulation due to two-hybrid protein interaction (i.e. dimerization), CAT activity contributed by the single GAL4/PR hybrid in the presence of R5020 was factored out by calculating the relative CAT activity obtained from the two-hybrid plus hormone divided by the relative CAT activity obtained by the single hybrid plus hormone (see Materials and Methods). By this analysis, hormone stimulated a 3.44-fold increase in CAT activity mediated by PR-A/PR-A interaction, a 7.16-fold increase mediated by PR-A/PR-B interaction, and 3.2-fold increase mediated by PR-B-/PR-B interactions. Hormone-dependent interaction between the A and B form of PR was also observed when PR-B was fused to GAL4 DBD and PR-A was fused to VP16. However, in this orientation the fold hormone (3-fold) stimulation was less than that obtained with the fusion proteins in the opposite orientation in Fig. 3Go (data not shown). This result, taken together with the fact that PR-B has a higher intrinsic activity than PR-A, makes it difficult to interpret whether the higher 7-fold stimulation mediated by GAL4/PR-A and VP16/PR-B in Fig. 3Go actually reflects a stronger dimerization between PR-A and PR-B as compared with A-A and B-B interactions. Nonetheless, the results in Fig. 3Go strongly support the conclusion that formation of the three possible forms of PR dimers (AA, AB, and BB) occurs in the cell in a hormone-dependent manner.

Antagonist-Stimulated PR Dimerization in Whole Cells
Using the same PR chimeric constructs, we next asked how different progesterone antagonists would effect PR-PR interactions in the intact cell. The antagonists included RU486 and closely related ZK112993 (type II), which behave as strong antagonists but display partial agonist activity under certain conditions, and the more complete antagonist ZK98299 (type I). None of these compounds stimulated pG5CAT activity above basal levels in cells transfected with both GAL4/PR-B and VP16/PR-B chimeras (data not shown). This was surprising since these same antagonists have been shown to promote efficient binding of PR to target DNA in whole cells (29, 30). We therefore considered the possibility that antagonists may reduce the transcriptional activity of the VP16 fusion proteins in such a manner that interaction between VP16/PR and GAL4/PR chimeras is undetected, due to the failure of the VP16 fusion protein recruited to the promoter to mediate a strong transcriptional response. To test this idea, we analyzed the ability of the VP16/PR-B chimera to function as a positive transactivator of a PRE-controlled reporter gene (DHRE-E1b-CAT) in response to agonists and antagonists. As shown in Fig. 4Go, R5020 stimulated a substantial 125-fold induction of DHRE-E1b-CAT reporter activity in cells transfected with VP16/PR-B. VP16/PR-B transactivation of the DHRE-E1b-CAT gene was also stimulated by all antagonists examined, including ZK98299. However, the fold induction was only 4–13% (range is from three independent experiments) of that mediated by hormone agonist (Fig. 4Go). The reduced ability of antagonists to stimulate CAT activity does not appear to be due to promoting a less efficient binding of the VP16/PR fusion protein to DNA than hormone agonist. We have performed a control experiment by cotransfecting VP16/PR-B with a carboxyl-terminal truncation UP-1 mutant of PR that binds and responds to RU486 as an agonist (Ref. 33 and see below). Cotransfected VP16/PR-B inhibited the transcriptional activity of VP16/PR-B(UP-1) in a dose-dependent manner in cells treated with RU486 (not shown). This inhibitory effect suggests that the VP16/PR-B chimeric receptor dimerizes and efficiently binds to DNA in the cell in response to RU486. In further support of antagonists reducing the transcriptional activity of the VP16/PR-B fusion protein as opposed to reducing its binding efficiency for DNA, we recently showed by promoter interference assays that all progestin antagonists used in this study stimulate binding of PR to PREs in vivo (whole mammalian cells) with the same efficiency as the agonist R5020 (30). How antagonists reduce the transcriptional activity of VP16/PR-B fusion protein is not known. A possible mechanism is suggested by recent studies showing that PR and ER bound to antagonists strongly associates with corepressors that can functionally silence partial agonist effects of antagonists (53, 54, 55).



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Figure 4. Effects of PR Antagonists on Transactivation Mediated by VP16/PR-B and VP16/PR-B(UP-1) Chimeric Receptors

COS-1 cells were transiently cotransfected with either VP16/PR-B (10 ng) or VP16/PR-B(UP-1) (10 ng), the progestin responsive DHRE-E1b-CAT reporter (500 ng), and the CMV-ß-gal expression vector used as an internal control for transfection efficiency. At 24 h posttransfection, the cells were treated with vehicle (ETOH), R5020 (100 nM), RU486 (100 mM), ZK112993 (100 nM) or ZK98299 (500 nM), incubated for another 24 h, and then harvested. CAT activity was normalized to the ß-gal activity, and the data are represented as fold hormone induction over the response of cells containing VP16/PR-B treated with vehicle only. The data are from a representative experiment of n = 3 independent experiments.

 
Because of the substantially weakened transcriptional activity of the VP16/PR chimera bound to antagonists, we turned to the use of a carboxyl-terminal deletion UP-1 mutant of hPR for assessing the effects of antagonists on PR dimerization in the two-hybrid assay. Deletion of 42 aa from the carboxyl terminus of human PR results in a receptor that is unable to bind hormone agonists but is capable of binding and being activated by antagonist to a level that approaches agonist activation of wild-type PR (33). These properties of PR-UP-1 suggest that agonist and antagonists contact different aa residues within the LBD of hPR and that the carboxyl-terminal tail contains a repressive function that antagonists are unable to overcome. For purposes of our study, the functional behavior of PR-UP-1 also predicts, although not formally demonstrated by in vitro assays, that antagonists stimulate dimerization and DNA binding of PR-UP-1. Therefore, we reasoned that use of PR-UP-1 mutants may be one way to detect the effects of antagonists on PR dimerization in two-hybrid assays. The UP-1 mutant of PR-B was inserted into the GAL4 DBD fusion vector (GAL4/PR-B(UP-1)) and into the VP16 fusion vector (VP16/PR-B(UP-1)). We first compared VP16/PR-B(UP-1) with VP16/PR-B as transactivators to determine whether VP16/PR-B(UP-1) exhibits better activation potential than VP16/PR-B in response to antagonists. As expected, VP16/PR-B(UP-1) is not capable of being activated by hormone agonist (Fig. 4Go). In contrast, when COS-1 cells were transfected with VP16/PR-B(UP-1), all antagonists tested stimulated a substantial induction of DHRE-E1b-CAT. The fold induction stimulated by RU486 and ZK112933 was equal to that of R5020 induction mediated by the VP16/PR-B construct (Fig. 4Go). VP16/PR-B(UP-1) transactivation induced by ZK98299 was approximately 50% lower than other antagonists but was still substantially greater than the level of transactivation mediated by wild-type PR-B fused to VP16 in response to ZK98299 (Fig. 4Go).

We next analyzed the effect of antagonists on interaction between the two PR-UP-1 chimeras. As expected R5020, which does not bind the UP-1 PR mutant, did not stimulate CAT activity above the ethanol controls in cells transfected with either the single hybrid GAL4/PR-B(UP-1) control or with both GAL4 and VP16 fusions of PR-B(UP-1) (Fig. 5Go). In contrast, the antagonists RU486 and ZK98299 each stimulated a significant increase (3.2-fold and 2.8-fold, respectively) of CAT activity in cells cotransfected with both fusion proteins over the single hybrid GAL4/PR-B(UP-1) control treated with the same antagonist (Fig. 5Go). Although ZK112993 also stimulated CAT activity (2.6-fold) in cells transfected with both hybrid PR-B(UP-1) proteins over the single-hybrid control (Fig. 5Go), the increase was statistically significant only at a P value of 0.08 suggesting a trend toward stimulating PR-PR interaction.



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Figure 5. Antagonists Induce PR Homodimerization in Whole Cells

COS-1 cells were transiently cotransfected with GAL4/PR-B(UP-1) and VP16 (empty vector control) or with GAL4/PR-B(UP-1) and VP16/PR-B(UP-1), the pG5CAT reporter, and the CMV-ß-gal expression internal control for correcting differences in transfection efficiency. At 24 h posttransfection, the cells were treated with vehicle (ETOH), R5020 (100 nM), RU486 (100 nM), ZK112993 (100 nM), or ZK98299 (500 nM), incubated for another 24 h, and then harvested. Relative CAT activity was determined, and the data were represented as described in Materials and Methods. The values for relative CAT activity are averages (±SEM) from n = 6 independent experiments. The fold ligand induction of relative CAT activity due to dimerization was calculated as described in Materials and Methods and was as follows: 3.2-fold for RU486, 2.62-fold for ZK112993, and 2.82-fold for ZK98299. Statistical analysis using a t test revealed that there were no significant differences in CAT activity between the one hybrid control and two hybrid proteins in the absence of ligand and with R5020 treatment. RU486 and ZK98299 produced a significant increase in CAT activity mediated by two-hybrid interaction compared with the one-hybrid control (P < 0.05), while the stimulation observed with ZK112993 was significant only at a P value of 0.08.

 
Because ZK98299 has a 10-fold lower affinity for PR than RU486 (29), whole cell binding experiments were performed with [3H]ZK98299 to determine whether it can stably bind and fully occupy PR-B(UP-1) in cells. Both PR-B(UP-1) fusion proteins expressed in COS-1 cells bound to ZK98299 with the same capacity as [3H]RU486 under conditions of the two-hybrid experiments (ZK98299 added at 500 nM and other ligands at 100 nM). Whole-cell binding experiments also confirmed that PR(UP-1) fusion proteins, as expected, failed to bind [3H]R5020 but did bind to [3H]RU486 and [3H]ZK98299 (data not shown). These results strongly suggest that progestin antagonists are capable of stimulating PR homodimerization within cells.

Heterodimerization between PR Bound to Agonist and PR Bound to Antagonist in Whole Cells
An interesting and unexplained property of these progestin antagonists is their ability to inhibit activation of PR at concentrations well below that of the hormone, indicating that antagonists do not act by simple competition with hormone for PR binding sites (11, 38). Despite the fact that RU486, ZK112993, and ZK98734 all have the same binding affinity for PR as the agonist R5020 (Ref. 29 and unpublished observations), these compounds inhibit activation of PR at substoichiometric concentrations with R5020. This is illustrated in Fig. 6Go with ZK112993 and ZK98299 in a breast cancer cell line containing a stably integrated mouse mammary tumor virus (MMTV)-CAT, a progestin-inducible reporter gene (11). Cells were treated with a saturating amount of R5020 (5 nM) or were treated with 5 nM R5020 and increasing amounts of ZK112993 (Fig. 6AGo) or ZK98299 (Fig. 6BGo). Reporter gene activity stimulated by 5 nM R5020 was inhibited by 50% at a 10-fold lower concentration of ZK112993 (average IC50 = 0.4 nM, n = 5), while maximal inhibition was obtained at approximately 3 nM ZK112993 (Fig. 6AGo). The IC50 for the antagonist ZK98299 was between 3–10 nM (average 7 nM, n = 5), and maximal inhibition was obtained between 100 and 500 nM of ZK98299 (Fig. 6BGo). When the 10-fold lower affinity for PR as compared with R5020 is corrected for, ZK98299 also antagonizes at substoichiometric concentrations with R5020. Thus, both antagonists have an observed IC50 that is approximately 10-fold lower than the expected.



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Figure 6. Inactivation of PR by Antiprogestins Occurs at Substoichiometric Concentrations with Progestin Agonists

T47D cells containing a stably integrated MMTV-CAT reporter gene (11 ) were treated for 24 h with 5 nM R5020 alone or with 5 nM R5020 and increasing amounts as indicated of ZK112993 (A) or ZK98299 (B). CAT enzyme activity was measured as described in Materials and Methods. CAT activity obtained with R5020 treatment alone was set as 100%, and activity in the presence of antagonists was calculated as relative to the 100% control. For each antagonist, the data are from a single representative experiment of multiple (n = 5) independent determinations.

 
One possible mechanism contributing to the biological potency of these progestin antagonists is repression of PR bound to agonist in trans by heterodimerization with PR bound to antagonist. To test whether such mixed ligand agonist/antagonist heterodimers can form in whole cells, we have adapted the mammalian two-hybrid assay to direct the binding of agonist and antagonist to one or the other receptor chimera. To do this, we have taken advantage of the PR-UP-1 mutant that binds antagonists selectively and a previously described mutation at aa position 722 (Gly->Cys) of hPR, which abolishes binding of RU486 but retains normal progestin agonist binding affinity and functional response to progestins (56). In the context of full-length PR-B, we inserted the 722 (Gly->Cys) mutant into the GAL4 or VP16 fusion vectors and used these fusion proteins partnered with PR-B(UP-1) fusion proteins to create a mammalian cell two-hybrid system that expresses receptor hybrids with the desired agonist/antagonist ligand specificities (see Fig. 7AGo). To confirm that these mutant PRs as fusion proteins possess the ligand specificities expected, a steroid binding assay in whole cells was performed with [3H]R5020 and [3H]RU486 in COS-1 cells transfected with each receptor GAL4 and VP16 chimeric construct. The VP16/PR-B(722m)-fusion protein bound [3H]R5020 specifically and exhibited no specific binding to [3H]RU486. Conversely, the GAL4/PR-B(UP-1) fusion protein bound [3H]RU486 in whole cells but not [3H]R5020. As expected, native PR in T47D cells as a control bound R5020 and RU486 equally (Fig. 7AGo). It should be noted that whole-cell binding assays used a 50 nM concentration of R5020 and RU486, which is approximately equal to the concentrations used in two-hybrid assays (see Fig. 7BGo). Even at these high concentrations, the mutant PRs show complete agonist (PR722m) and antagonist (PR(UP-1)) specificity. The whole-cell steroid binding assays also show (Fig. 7AGo) that PR(UP-1) and PR722m fusion proteins are expressed at approximately equal levels in transfected COS-1 cells, which was also confirmed by immunoblot analysis of the cell lysates (not shown).



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Figure 7. Heterodimerization between PR Bound to Agonist and PR Bound to Antagonists

A, Schematic of the mutant chimeric receptors and their ligand specificities. The GAL4 DBD was fused to the amino terminus of PR-B containing deletion of 42 aa from the carboxyl terminus to yield GAL4/PR-B(UP-1). VP16 was fused to the amino terminus of PR-B with a change of aa 722 from Gly to Cys to yield VP16/PR-B(722 m). Whole-cell steroid binding assays were performed by incubating transfected COS-1 or T47D cells with 50 nM [3H]R5020 or 50 nM [3H]RU486 as described in Materials and Methods. The specific binding was calculated as picomoles of steroid binding/mg protein. Data from a representative experiment are shown. B, Mammalian two-hybrid assay using PR ligand specificity mutants. COS-1 cells were transiently transfected with single hybrid GAL4/PR-B(UP-1) plus empty VP16 vector control (one hybrid) or GAL4/PR-B(UP-1) and VP16/PR-B(722m) (two hybrid), along with the pG5CAT reporter and the CMV-ß-gal plasmid for determining transfection efficiency. At 24 h posttransfection, the cells were treated with either vehicle (ETOH), 100 nM R5020, 100 nM RU486, or cotreated with 100 nM R5020 and 50 nM RU486. Cells were incubated for another 24 h and then harvested. Relative CAT activity was calculated by normalizing the single hybrid in the absence of ligand to 1, and the data are represented as in Figs. 3Go and 5Go and as described in Materials and Methods. The values are averages (±SEM) from four independent experiments. Statistical analysis using a t test revealed that were no significant differences in CAT activity between the one hybrid control and two hybrids for ethanol, R5020, and RU486 treatment groups; a significant increase in CAT activity was observed with the two hybrids cotreated with R5020 and RU486 compared with the single hybrid treated with both ligands. The increase in relative CAT activity due to dimerization was determined to be 4.2-fold. The schematic underneath the graph illustrates mixed ligand heterodimer formation between antagonist-bound PR-B and agonist-bound PR-B.

 
We next assessed the ability of the GAL4/PR-B(UP-1) fusion protein to interact with VP16/PR-B(722m) in a two-hybrid setting in response to treatment with agonist, antagonist, or both compounds. When cells were transfected with GAL4/PR-B(UP-1) alone as a single hybrid, RU486 stimulated a 6-fold increase of the pG5CAT reporter gene expression over the no-ligand (ethanol) control. As expected, no induction of CAT expression was mediated by GAL4/PR-B(UP-1) as a single hybrid in response to R5020, and the stimulation obtained by cotreatment with R5020 and RU486 was no different from RU486 alone (Fig. 7BGo). In COS-1 cells cotransfected with both GAL4/PR-B(UP-1) and VP16/PR-B(722m) hybrid constructs, no further increase in pG5CAT activity over the single hybrid control (GAL4/PR-B(UP-1) + VP16) was observed by addition of RU486 alone. Addition of R5020 alone also did not stimulate a significant increase in CAT activity over the single hybrid also treated with R5020. A significant 4.2-fold stimulation of CAT activity over the single hybrid control was observed only when cells were cotreated with R5020 and RU486 (Fig. 7BGo). Thus, both agonist and antagonist were required for interaction between GAL4/PR-B(UP-1) and VP16/PR-B (722m). These results provide strong evidence that PR bound to an antagonist is capable of heterodimerization in the cell with PR bound to antagonist. These results also underscore that dimerization in whole cells requires ligand binding to both PR partners.

Mixed Agonist/Antagonist PR Heterodimers in Vitro Exhibit Reduced Ability to Bind to DNA
The two-hybrid assay results indicate that PR bound to an antagonist can heterodimerize with PR bound to an agonist but does not determine whether these mixed ligand heterodimers are functional. As one means to examine this question, we have performed EMSAs of separately expressed A and B receptors bound to either agonist or antagonist. Because of the molecular size difference of the A (90 kDa) and B receptors (120 kDa), mixing of the two PR isoforms results in the formation of an intermediate mobility PR-A/PR-B heterodimer complex that we and others have shown previously to be easily discernible by EMSA from the faster mobility AA and slower mobility BB dimers (11, 12, 13, 38, 44, 45, 46). The A and B receptors were expressed in the baculovirus insect cell system as previously described, and ligands were added to Sf9 insect cell cultures for the last 6–8 h of expression to allow binding to PR in the cell before extraction of receptor from nuclei with 0.4 M NaCl. Free ligand was removed by treatment of Sf9 cell extracts with dextran-coated charcoal as previously described, and assays were performed at 0–4 C under conditions that keep ligand dissociation to a minimum (45). When a constant amount of PR-B bound to R5020 was mixed with increasing amounts of PR-A bound to R5020 in a DNA binding reaction with a 32P-labeled PRE oligonucleotide probe, a single PR-B-PRE complex was detected at the lowest amount of PR-A, while two additional mobility complexes were detected at higher amounts of PR-A (Fig. 8AGo, lanes 1–6). As shown previously by supershifts with appropriate PR antibodies (11, 12, 44, 45), the intermediate mobility complex is composed of PR-A-PR-B heterodimers, while the fastest mobility complex contains PR-A homodimers. In contrast, little or no intermediate mobility complexes were detected when PR-B bound to R5020 was mixed with increasing amounts of PR-A bound to the antagonist ZK112993 (Fig. 8AGo, lanes 7–12) or with increasing amounts of PR-A bound to ZK98734 (Fig. 8AGo, lanes 13–18). We and others previously observed similar results when one PR isoform bound to RU486 was mixed with another bound to R5020. The amount of intermediate mobility heterodimer complex was minimal compared with the intermediate mobility heterocomplexes obtained when PR-A and PR-B were bound to the same ligand (either R5020 or RU486) (12, 45). It should be noted that the amount of PR-B (R5020) DNA complex decreased as increasing amounts of PR-A antagonist were added to the EMSA assays, albeit the decrease is more predominant with PR-A-ZK98734 than with PR-A-ZK112993 (Fig. 8AGo). This decrease of the PR-B complex at the expense of the PR-A complex, without the appearance of a third intermediate mobility complex [which does occur when mixing PR isoform bound to the same ligand (R5020)], is consistent with the formation of mixed ligand heterodimers in solution that exhibit reduced DNA binding affinity.



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Figure 8. Agonist/Antagonist PR Heterodimers Formed In Vitro have Impaired DNA Binding Activity

A, EMSA. PR-A and PR-B were separately expressed in Sf9 insect cells by the baculovirus system as previously described (44 ). PR-B was bound to R5020 (100 nM) and a separately expressed preparation of PR-A was bound to either R5020, ZK112993, or ZK98734. PR in whole cell extracts was incubated with a [32P]PRE oligonucleotide in a DNA binding reaction previously described (11 ), and binding was detected by EMSA. Lanes 1–6 shows PR-B bound to R5020 mixed with increasing amounts of PR-A bound to R5020; lanes 7–12 show PR-B bound to R5020 with increasing amounts of PR-A bound to ZK112993; lanes 13–18 show PR-B bound to R5020 with increasing amounts of PR-A bound to ZK98734. AA, AB, and BB indicate the position of DNA complexes containing PR-A homodimers, PR-A-PR-B heterodimers, and PR-B-PR-B homodimers, respectively. B, Heterodimerization detected in vitro by coimmunoprecipitation assay. PR-A expressed in Sf9 insect cells was bound to the different ligands indicated while expressed PR-B was bound to R5020. Whole-cell extracts were prepared and PR levels were estimated by immunoblot blot. Equal amounts of PR-B and PR-A were mixed and immunoprecipitated with the PR-B specific MAb, B-30, under conditions at 0–4 C that minimize exchange of ligands (45 ). The immunoprecipitates were analyzed by immunoblot with the PR-A and PR-B reactive MAb, AB-52. Coimmunoprecipitation of PR-A in the presence of PR-B (+PR-B•R5020, lanes 1–5) indicates the formation of PR-A-PR-B dimers in solution. As controls for nonspecific binding of PR-A to the B-30 MAb protein A-Sepharose resins, each PR-A preparation was subjected to immunoprecipitation with B-30 in the absence of PR-B (lanes 6–10).

 
We next considered the possibility that failure to detect agonist/antagonist PR heterodimers bound to DNA may be due to an inability to form solution dimers in vitro despite the fact that heterodimerization was detected in whole cells by the two-hybrid assay. To determine whether agonist/antagonist PR heterodimers can form in solution in vitro, coimmunoprecepitation assays were performed with the baculovirus-expressed PR-B bound to R5020 and PR-A bound to either R5020 or different antagonists including RU486, ZK98299, ZK98734, and ZK112993. A and B receptors were mixed together for 30 min at 0–4 C and immunoprecipitated with a MAb (B-30) to the unique amino terminus of PR-B. The immunoprecipitates were analyzed by immunoblot with a MAb (AB-52) that reacts with both PR-A and PR-B. As shown in Fig. 8BGo, PR-A was efficiently coimmunoprecipitated with the PR-B-R5020 complex whether it was bound to R5020 or any of the antagonists (lanes 1–5). Specificity of PR-A interaction with PR-B is shown by the lack of PR-A coimmunoprecipitation with the B-30 MAb in the absence of added PR-B (Fig. 8BGo, lanes 6–10). These results suggest that PR bound to hormone agonist is capable of forming a solution heterodimer with PR bound to antagonist in the absence of DNA. These in vitro results, taken together with the results of the two-hybrid assay with the ligand specificity mutant PRs, strongly support the conclusion that PR bound to antagonist is capable of forming a heterodimer with PR bound to agonist and that these heterodimers have impaired ability to bind to target DNA sequences.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The A and B isoforms of PR have been shown to bind in vitro, in a hormone-dependent manner, to target PREs as three dimeric forms: AA, AB, and BB dimers (11, 12, 44, 45, 46). The present study using the mammalian two-hybrid system demonstrates that the three possible forms of PR dimers can also form in whole cells in a hormone-dependent manner.

The two-hybrid system has been used previously to examine dimerization of steroid receptors. Using a yeast two-hybrid assay, Wang et al. (42) showed that dimerization of human ER{alpha} in vivo is estrogen dependent. A mammalian-based two-hybrid system was used to show ligand-dependent interactions between the {alpha}- and ß-subtypes of mouse ER (16). Similar to our results with PR in a mammalian-based system, a estrogen-dependent rise in reporter gene activity was observed with a single GAL4/mER{alpha} hybrid (and VP16 empty vector). This activity was attributed to the ligand-inducible transactivation function of ER{alpha} itself, when directed to the promoter of the GAL4 reporter construct by binding via the GAL4 DBD (16). Reporter gene activity induced by the single GAL4/ER{alpha} hybrid was approximately 5-fold, which is similar to the induction level observed to be mediated by the GAL4/PR-B single hybrid treated with hormone (6-fold) (Fig. 3Go). Interestingly, we did not observe an R5020 induction of reporter gene activity (pG5CAT) with the A isoform as a single GAL4 fusion protein. Both the A isoform of PR and ER{alpha} contain two transactivation functions, whereas the B isoform of PR contains an additional third activation domain (49, 50). It would, therefore, appear that the two activation functions (AF-1 and AF-2) of ER{alpha} are stronger than those of PR, at least in the context of the two-hybrid assay. In our initial two-hybrid experiments, we attempted to reduce the assay background activity of the single GAL4/PR-B hybrid protein by taking advantage of known mutations in AF-2 of PR. Replacement of conserved glutamate residues at positions 907 and 911 with alanine results in a mutant receptor that is able to bind progesterone but has greatly reduced AF-2 hormone-dependent activity (51). By using PR-B(907, 911m) to construct the GAL4 DBD fusion, we observed a reduction in hormone-dependent activity of the single hybrid protein. However, the fold hormone induction of pG5CAT dependent on coexpression of the second VP16/PR-B hybrid protein was no greater than that obtained with wild-type PR-B as the GAL4 DBD fusion construct (not shown). In the study of ER dimerization by the yeast two-hybrid assay, no assay background activity was detected with the single GAL4/hER{alpha} hybrid (42). This clearly contrasts with our data with PR and with the reported assay background detected with the GAL4 DBD/mER{alpha} as a single hybrid protein in mammalian-based two-hybrid assays (16). This apparent discrepancy between yeast- and mammalian-based two-hybrid assays is likely due to receptors in yeast possessing lower intrinsic transcriptional activity due to the absence of steroid receptor coactivators that are normally present in mammalian cells (6, 7, 57).

Our initial two-hybrid experiments using wild-type PR-B to analyze the effects of antagonists on PR dimerization suggested that these compounds do not promote dimerization in vivo. Recent results showing that PR and ER bound to antagonists inappropriately forms a strong association with nuclear receptor corepressors (NCOR or SMRT) provides a likely explanation for the lack of antagonist-induced two-hybrid dependent activation of the pG5CAT reporter (53, 54, 55). In the context of the two-hybrid assay, we would argue that recruitment of corepressors suppresses the transcriptional activity of the VP16/PR fusion protein even though interactions may occur efficiently between GAL4/PR and VP16/PR hybrids in the presence of antagonists. In support of this idea we observed that the ability of the VP16/PR fusion protein to transactivate a PRE-controlled reporter gene in response to antagonists was reduced as compared with agonist (Fig. 4Go). To resolve this issue we turned to the PR-UP-1 mutants in the two-hybrid assay. The VP16/PR-B(UP-1) fusion protein, in response to antagonist, mediated transactivation of the PRE-controlled reporter gene to a similar extent as agonist-induced transactivation mediated by the VP16 fusion protein constructed with wild-type PR-B (VP16/PR-B). This suggested to us that the PR-UP-1 mutant would be useful to detect antagonist-dependent PR dimerization in whole cells by two-hybrid assay. This indeed was the case. The progestin antagonists RU486 and ZK98299 both stimulated a significant interaction between GAL4/PR-B(UP-1) and VP16/PR-B(UP-1) as detected by the induction of CAT reporter gene activity over that obtained by the single GAL4/PR-B(UP-1) hybrid in response to antagonists. Although statistical analysis did not show a significant stimulation, the antagonist ZK112993 also exhibited a trend of stimulating interaction between the GAL4/PR-B(UP-1) and VP16/PR-B(UP-1) fusion proteins (Fig. 5Go). These two-hybrid results support the conclusion that antagonists induce PR dimerization in whole cells and do not appear to act by impairing receptor dimerization.

Antiestrogens were reported to induce dimerization of human ER{alpha} when measured in a yeast two-hybrid assay (42). Both tamoxifen and ICI 182,780 were observed to induce ER-ER interactions. However, the level of reporter ß-galactosidase (ß-gal) activity induced by tamoxifen and ICI 182,780 was only 15% and 20%, respectively, of the activity induced by estradiol-17ß (42). Additionally, it was shown that dimerization is perturbed when yeast cells were cotreated with estrogen and antiestrogens. Our data with PR antagonists would suggest that the lower induction of ß-gal observed by antiestrogens is due to suppression of the heterologous transcriptional activation domain of the GAL4 AAD/ER construct as opposed to a reduction of ER-ER interaction per se. It is interesting that antagonist-induced ER dimerization was detected using wild-type ER as the fusion protein constructs, whereas we observed no dimerization using wild-type PR to construct hybrid proteins. Antiprogestin-induced PR-PR interactions were detected only with PR-UP-1 mutants. This apparent difference could be explained as a difference between yeast- and mammalian-based two-hybrid assays since it appears that yeast does not express an NCOR- or SMRT-like molecule (53, 57).

It is now well established that binding of PR to antagonists induces a conformational change in the LBD of the receptor that is different from that induced by agonist (31, 32, 33, 34, 35, 36, 37). These conformational differences were initially suggested to prevent the formation of heterodimers between receptors bound to agonist and receptors bound to antagonist. This was based on EMSA experiments in which mixing of one hPR isoform liganded with RU486 and another liganded with R5020 failed to produce an intermediate heterodimer complex bound to a PRE (12). However, the ability of RU486- and R5020-liganded receptors to form heterodimers in solution in the absence of DNA was not examined (12). In a subsequent study, we examined whether mixed agonist/antagonist heterodimers could form in solution by a coimmunoprecipitation assay. We observed that PR-A bound to RU486 can heterodimerize as efficiently with PR-B bound to R5020 as the two PR isoforms bound to the same ligand: either R5020 or RU486 (45). Similar to results of Meyer et al. (12), we also observed a dramatically reduced ability of mixed R5020/RU486 heterodimers to bind to PREs by EMSA (45). By use of ligand specificity PR mutants, we show here that mixed R5020/RU486 PR heterodimers can form in whole cells as detected by the mammalian two-hybrid assay (Fig. 7BGo). This suggests that antagonists do not alter the dimer interface sufficiently to prevent dimerization with PR bound to agonist. In addition, the fact that both R5020 and RU486 were needed to form these heterodimers provides new information that both PR partners must be bound to ligand in order for dimerization to occur; ligand binding to one partner is not sufficient. In further support of the compatibility of dimer interfaces is the crystal structure of the ER LBD bound to estradiol and the antiestrogen raloxifene (58). The dimerization interfaces appear to be similar for the LBD bound to estradiol and raloxifene; the major conformational difference induced by agonist and antagonist is the position of helix 12, which contains the conserved sequences in AF-2 (58). The recently published crystal structure of PR LBD bound to progesterone, and modeling with the antagonist RU486, also suggest that dimerization interfaces are similar for the LBD bound to progesterone and RU486 (59). Similar to ER, it was predicted that the major difference between the PR LBD bound to progesterone and RU486 is the displacement of helix 12 and the C-terminal tail.

The fact that PRs bound to RU486 and R5020 are capable of forming heterodimers in vitro (in solution) and in whole cells may aid in the interpretation of the mechanistic basis for the biological potency of RU486 and other structurally related progestin antagonists. All antagonists that we have tested, including ZK98299 after correction for its 10-fold lower affinity than R5020 for PR, inhibit activation of PR at substoichiometric concentrations with R5020 (Refs. 11, 38 and Fig. 6Go). This suggests that these compounds do not work solely by a simple mechanism of competing with agonist for binding to PR. One possible mechanism to explain these biological results is that receptors bound to antagonists can inactivate receptors bound to agonist in trans by heterodimerization. The implication is that all receptors do not need to be occupied by antagonist for complete antagonism to occur. This is diagrammed schematically in Fig. 9Go, where the agonist/antagonist heterodimer forms in solution and then is unable to bind efficiently to a PRE.



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Figure 9. Progestin Antagonist Transrepression by Heterodimerization

Treatment of cells with progestin agonist or antagonist stimulates PR homodimerization and binding to progesterone response elements (PREs). Antagonists fail to stimulate transcription because they induce an altered conformational change in the LBD (black filled symbols) that does not permit PR interaction with coactivators required to transmit the receptor signal into a transcriptional response. When cells are treated with both agonist and antagonists, heterodimers can be formed between PR bound to antagonist and PR bound to agonist. Based on EMSA results, these heterodimers do not efficiently bind to PREs and thus appear to be inactive. The basis for the inability of these heterodimers to bind to DNA is not known. Although differences in conformation in the LBD does not prevent heterodimerization, the mixed ligand heterodimers may be asymmetric such that the DBDs (hatched symbols) are not oriented correctly over the inverted repeat palindromic PRE. The amino-terminal domain of PR is represented by open symbols. Thus we propose that progestin antagonists can act in trans to inhibit hormone agonists by heterodimerization and that this accounts in part for the ability of these compounds to inhibit PR activation at concentrations that are substoichiometric with progestin agonists.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[3H]R5020 (promegestone: [17{alpha}-methyl-3H]17{alpha}, 21-dimethyl-19-norpregna-4,9-diene-3, 20-one; 87 Ci/mmol) and unlabeled R5020 were obtained from Dupont/NEN Research Products (Boston, MA). [3H]RU486 (Mifapristone: [17{alpha}- hydroxy-11[4-dimethyl-aminophenyl]17-propenyl-estra-4,5- diene-3-one) and unlabeled RU486 were gifts from R. Daraedt (Roussel UCLAF, Romanville, France). ZK98299 (Onapristone), ZK112993, ZK98734, and [3H]ZK98299 were provided by David Henderson (Schering, Berlin, Germany). 1294/H9 is an unpublished MAb that recognizes the A and B forms of hPR. MAbs B-30 and AB-52 to PR have been described previously (60).

Construction of Two-Hybrid Fusion Plasmids for PR
The Mammalian MATCHMAKER Two-Hybrid Assay Kit was obtained from CLONTECH (Palo Alto, CA). Included within the kit is the pM cloning vector for expressing GAL4 DBD (aa 1–147) fusion proteins and pVP16 (aa 411–455) for expressing the VP16 transcriptional activation domain. Also included is pG5CAT, a reporter vector that contains the CAT gene downstream of five consensus GAL4 binding sites and the minimal promoter of the adenovirus E1b gene. Human PR-B was cloned into the pM and pVP16 vectors in the following manner. A 2.93-kb AflII-Asp718 fragment of YEphPR-B (33) was filled in with Klenow and inserted into the unique BamHI restriction site of pGAD424 (CLONTECH) that had been blunt ended with Klenow. This plasmid, pGAD424.PR-B, was used as the source of hPR-B cDNA for fusion with the GAL4 DBD and the VP16 protein of the herpes simplex virus. A 2.9-kb EcoRI-PstI fragment from pGAD424.hPR-B was ligated into the EcoRI-PstI restriction sites in the multiple cloning cassette of both the pM and pVP16 vectors to yield, respectively, pM.PR-B (referred to subsequently as GAL4/PR-B) and pVP16.PR-B (referred to subsequently as VP16/PR-B). This places the GAL4 DBD and the activation domain of VP16 immediately amino-terminal to hPR cDNA. Cloning junctures were sequenced (Sequenase 2.0, US Biochemical, Cleveland, OH) and PR-B was determined to be correctly oriented and in-frame with GAL4 DBD or VP16. For PR-A constructs, a 2.4 kb BspHI-Asp718 fragment of YEphPR-B (33) was filled-in with Klenow and inserted into the unique SmaI restriction site of pGBT9 (CLONTECH). This plasmid was used as the source of hPR-A cDNA for fusion with the GAL4 DBD and the VP16. A 2.4 kb EcoRI-PstI fragment from pGBT9.hPR-A was cloned into the EcoRI-PstI of pM and pVP16 to yield respectively pM.PR-A (referred to subsequently as GAL4/PR-A) and pVP16.PR-A (referred to subsequently as VP16/PR-A). Cloning junctures were sequenced (Sequenase 2.0, US Biochemical) and hPR-A was determined to be correctly oriented and in-frame with the GAL4 DBD or VP16.

A C-terminal truncation mutant of hPR-B (pSVhPR-B 891) was provided by Donald McDonnell (Duke Medical Center, Durham, NC). To construct fusions with hPR-B 891, a 2.8-kb BamHI fragment from pSVhPR-B 891 was inserted into GAL4/PR-B and VP16/PR-B vectors that had been previously digested with BamHI to drop out the 2.8-kb PR-B fragment. This hPR cDNA contains a reading-frame mutation that generates a stop codon at aa 892, resulting in a carboxyl-terminal truncated receptor by 42 aa (33). The resulting plasmids, GAL4/PR-B(UP-1) and VP16/PR-B(UP-1), were sequenced to confirm the presence of the nucleotide deletion at base 2849.

A two-hybrid vector expressing PR-B containing a point mutation at aa 722 was constructed by digesting VP16/PR-B with the restriction enzymes StuI and EcoN I and replacing the resulting 1.3-kb fragment with a 1.3-kb StuI-EcoN I fragment from pBK·CMVhPR-BRU (provided by Dawn Wen, Ligand Pharmaceutical, San Diego, CA). pBK·CMVhPR-BRU contains full-length hPR-B cDNA with a G to T substitution at nucleotide position 2339. This mutation results in a Gly->Cys change at aa 722 in the human PR-B amino acid sequence. This generates a receptor that has lost detectable RU486 binding, but retains wild-type binding affinity for progestin agonists and is fully activated by progestin agonists (56). The resulting plasmid, VP16/PR-B(722m), was sequenced to confirm the presence of the G to T substitution.

For PR transactivation experiments, the progesterone responsive reporter, DHRE-E1b-CAT, was used. This contains two synthetic PREs linked to the TATA box of the adenovirus E1b (61) and CAT (30).

Transient Transfections
Monkey kidney COS-1 cells were plated in DMEM (GIBCO-BRL) with 10% FBS at a density of 1.75 x 105 to 2.0 x 105 cells per well in six-well dishes (Falcon, Franklin Lakes, NJ) at 37 C. After 24 h the cells were transfected by an nonrecombinant adenovirus-mediated DNA transfer technique as previously described (62). This method involves the use of a replication-defective adenovirus that is coupled with poly-L-lysine to bind the plasmid DNA noncovalently. This process facilitates cellular uptake of the plasmid DNA by receptor-mediated endocytosis. For mammalian two-hybrid experiments, 50 ng/well of plasmids encoding either GAL4 DBD or VP16 fusion proteins were used and 500 ng/well of the pG5CAT reporter plasmid. In early two-hybrid experiments, we used charcoal-treated serum and found that it made no difference compared with whole FBS, so we used whole FBS for our studies. For experiments to measure PR as a transactivator, the DHRE-E1b-CAT was added at 500 ng/well, and the PR expression plasmid was added at 10 ng/well. To treat cells with various PR-ligands, 24 h after transfection, the medium was replaced with DMEM + 10% FBS containing vehicle (0.01% ethanol) or ligand at the concentration indicated in the figure legends, followed by incubation at 37 C. After an additional 24 h, the cell monolayers were rinsed with CAT wash buffer (40 mM Tris-Cl, pH 7.4, 150 mM NaCl, and 1 mM EDTA) and lysed in the well by the addition of 300 µl of cell lysis buffer (20 mM potassium phosphate, pH 7.4, 5 mM MgCl2, and 0.5% Triton X-100). To control for variation in transfection efficiency, cells were cotransfected with a CMV-ß-gal reporter plasmid, and ß-gal was measured in a luminometer (Monolight 2001) with the Galacto-Light Plus kit (Tropix, Bedford, MA) according to manufacturer’s instructions.

Whole-Cell Steroid-Binding Assay
COS-1 cells plated in six-well dishes at a density of 1.75 x 105 cells per well were grown overnight at 37 C in DMEM + 10% FBS. Cells were then transfected with a single concentration (25 ng) of the indicated expression plasmids by the adenovirus-mediated DNA transfer technique as described above. PR-rich T47D human breast cancer cells (60) were also plated in six-well dishes at a density of 1.75 x 105 cells per well in MEM + 5% FBS. After 44 h the cells were incubated for an additional 4 h at 37 C with 1–2 nM of [3H]R5020, [3H]RU486, or [3H]ZK98299 ± unlabeled 100 nM homologous ligand. Radioactive steroid was extracted from cells with ethanol and quantitated by liquid scintillation counting. Parallel transfected cells were lysed and assayed for protein concentration. Receptor expression levels were normalized to protein and calculated as picomoles of steroid binding per mg of protein. Nonspecific binding was determined by parallel incubations of mock-transfected COS-1 cells. Data represents specific binding taken as total binding minus nonspecific binding.

Immunoblot Analysis
COS-1 cells were plated in six-well dishes at a density of 2 x 105 cells per well in DMEM + 10% FBS for 24 h. Wells were transiently transfected by the adenovirus method (62) with 25 ng of the appropriate plasmid (indicated in the figure legends). After 44 h, the medium was removed and replaced with medium containing either 100 nM of R5020 or 100 nM of RU486. After an additional 4 h at 37 C, the cell monolayers were washed with CAT wash buffer, and cells from triplicate wells were harvested and combined in 0.4 ml of 0.5% Triton X-100 lysis buffer. The lysates were removed from the wells and centrifuged at 12,000 rpm for 10 min at 4 C. The supernatants were assayed for protein concentration by Bradford assay (63). Total protein in 1% SDS sample buffer (250 µg) was boiled and electrophoresed on 7.5% polyacrylamide SDS gels. Separated proteins were transferred to nitrocellulose and incubated with the receptor-specific (1 µg/ml) MAb 1294/H9. Immune complexes were decorated with rabbit antimouse immunoglobins (N. L. Cappel Laboratories, Cochranville, PA) and [35S] protein A (Amersham) and detected by autoradiography.

T47D human breast cancer cells were plated in six-well dishes at a density of 2 x 105 cells per well in MEM (GIBCO-BRL) with 5% FBS. At 44 h after plating, the cells were treated with 100 nM R5020. Cells from triplicate wells were harvested in a total of 0.4 ml of 0.5% Triton X-100 lysis buffer and prepared as the COS-1 lysates.

CAT Assays
CAT enzyme activity was assayed by a radiometric/organic phase extraction method as previously described (64). Enzyme activity was calculated as counts per min of [3H]acetylcoenzyme A converted per µg of protein in the cell lysate. CAT assays were done in duplicate for each lysate. Cell treatment groups were also performed in duplicate. Protein concentration was measured by the method of Bradford (63), and equal amounts of protein (10–30 µg) were added to the assay. Specific CAT activity was determined by subtraction of assay background obtained from lysates of nontransfected cells. To compensate for variation in transfection efficiency, the cells were also transfected with a CMV-ß-gal reporter plasmid. CAT results were calculated as the ratio of CAT activity per unit of ß-gal activity.

Baculovirus Expression of Human PR-A and PR-B
Human PR-A and PR-B were expressed as nonfusion full-length proteins from baculovirus vectors in Sf9 insect cells as previously described (44). To bind ligands to baculovirus-expressed receptors, they were added at 200 nM to Sf9 cell cultures during the last 6–8 h of expression. To prepare whole-cell extracts, Sf9 cells were lysed in TEDG buffer [10 mM Tris-base, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol], containing 0.5 M NaCl and a mixture of protease inhibitors (60). Cell lysates were centrifuged at 100,000 xg for 30 min to yield a soluble supernatant and then dialyzed against lysis buffer containing no NaCl.

Coimmunoprecipitation Assay
Protein A-Sepharose was prebound noncovalently with receptor-specific MAbs and used as an immunoabsorbent as described previously (28, 45). Resins were prebound to rabbit anti-mouse IgG (Cappel) and used as a bridging antibody. Receptor-specific MAbs were then bound to the immobilized rabbit antimouse IgG. Sf9 cell extracts containing PR-A or PR-B were mixed at 10 µg/100 µl of resin in siliconized microcentrifuge tubes and incubated on ice for 30 min. Equal amounts of receptors were added to each assay as determined by ligand-binding assay and immunoblot analysis. MAb-coated protein A-Sepharose beads (100 µl) were added to each tube and incubated at 4 C for 1 h on an end-over-end rotator. Resins were then washed four times by centrifugation in TEG (TEDG minus dithiothreitol) containing 100 mM NaCl, transferred to a new microcentrifuge tube, and washed twice more. Immobilized proteins were eluted with 2% SDS loading buffer and then analyzed by immunoblot, using [35S]protein A and autoradiography as the detection method.

EMSAs
For EMSA, we used a 28-bp oligonucleotide containing a PRE/glucocorticoid response element derived from the MMTV-long terminal repeat (11, 65). Sf9 insect cell extracts containing PR-A or PR-B were incubated for 1 h at 4 C with 32P-labeled DNA (0.3 ng) in a total reaction volume of 25 µl. Also included was 1 µg of poly (dA-dT) as nonspecific competitor DNA. The DNA binding buffer contained 10 mM Tris-base, pH 7.4, 50 mM NaCl, 5 mM dithiothreitol, 2 mM MgCl2, 10% glycerol, and 50 ng/ml of a carrier protein. Samples (25 µl) were electrophoresed on 5% polyacrylamide gels prepared at 40:1 (wt/wt) acrylamide-bis-acrylamide ratio using 20 mM Tris-acetate, 0.5 mM EDTA as the electrode buffer. To maintain constant temperature during electrophoresis, 4 C water was recirculated through the gel apparatus. Gels were dried and subjected to autoradiography (65).

T47D Breast Cancer Cells Stably Transfected with MMTV-CAT to Measure Biological Activity of Progestin Antagonists
T47D human breast cancer cells were cultured as described (11) in MEM supplemented with 5% FBS. A cloned derivative (B-11) of T47D, stably transfected with a construct containing the CAT gene linked to the MMTV promoter/enhancer, has been previously described (11). Cells plated in six-well dishes at a density of 0.5 x 106 cells per well were grown for 3 days at 37 C and then incubated for another 24 h with medium stripped of steroid hormones by treatment with dextran-coated charcoal (11). This was followed by an additional 24 h incubation with hormone and other compounds in the same medium. Harvested cells were washed twice in 40 mM Tris (pH 7.6), 150 mM NaCl, and 1 mM EDTA and lysed in buffer containing 20 mM potassium phosphate (pH 7.8), 5 mM MgCl2, and 0.5% (vol/vol) Triton X-100. Cell lysates were analyzed for CAT enzyme activity as described above.

Data Analysis
For two-hybrid experiments presented in Figs. 3Go, 5Go, and 7Go, CAT activity mediated by the GAL4/PR construct as a single hybrid in the absence of ligand was normalized to 1.0, and the CAT activity mediated by other constructs and construct pairs was calculated relative to the single-hybrid control. These data are expressed as relative CAT activity. The two-hybrid CAT data represent mean values (±SEM) from four to seven independent experiments. To calculate the fold CAT induction mediated by PR dimerization in response to ligand, we used the following calculation:

The values for fold induction were analyzed by the Student’s t test using Excel 5.0 (Microsoft) to determine whether there was a statistically significant difference between the treatment groups. Results were considered statistically significant at P < 0.05.


    ACKNOWLEDGMENTS
 
The authors thank Lori Sherman for expert technical assistance and the University of Colorado Cancer Center Tissue Culture Core (P30CA46934) for assistance with baculovirus-expressed PR and cell culture media.

This work was supported in part by Public Health Services Grant R01DK-49030 (to D.P.E.) and National Research Service Award fellowship F32DK-09662 (to S.A.L.).


    FOOTNOTES
 
Address requests for reprints to: Dean P. Edwards, Department of Pathology and Program in Molecular Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. E-mail: edwardsd{at}defiance.uchsc.edu

Received for publication December 22, 1997. Revision received August 3, 1998. Accepted for publication September 8, 1998.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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