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Molecular Endocrinology, doi:10.1210/me.2004-0534
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Molecular Endocrinology 19 (9): 2258-2272
Copyright © 2005 by The Endocrine Society

Antagonist- and Inverse Agonist-Driven Interactions of the Vitamin D Receptor and the Constitutive Androstane Receptor with Corepressor Protein

Harri Lempiäinen, Ferdinand Molnár, Manuel Macias Gonzalez, Mikael Peräkylä and Carsten Carlberg

Departments of Biochemistry (H.L., F.M., M.M.G., C.C.) and Chemistry (M.P.), University of Kuopio, FIN-70211 Kuopio, Finland

Address all correspondence and requests for reprints to: Professor Carsten Carlberg, Department of Biochemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: carlberg{at}messi.uku.fi.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ligand-dependent signal transduction by nuclear receptors (NRs) includes dynamic exchanges of coactivator (CoA) and corepressor (CoR) proteins. Here we focused on the structural determinants of the antagonist- and inverse agonist-enhanced interaction of the endocrine NR vitamin D receptor (VDR) and the adopted orphan NR constitutive androstane receptor (CAR) from two species with the CoR NR corepressor. We found that the pure VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas ZK168281 resembled more the mouse CAR inverse agonist androstanol in its ability to recruit CoR proteins. Molecular dynamics simulations resulted in comparable models for the CoR receptor interaction domain peptide bound to VDR/antagonist or CAR/inverse agonist complexes. A salt bridge between the CoR and a conserved lysine in helix 4 of the NR is central to this interaction, but also helix 12 was stabilized by direct contacts with residues of the CoR. Fixation of helix 12 in the antagonistic/inverse agonistic conformation prevents an energetically unfavorable free floatation of the C terminus. The comparable molecular mechanisms that explain the similar functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS (NRs) form the largest family of metazoan transcription factors and regulate the expression of target genes that affect processes as diverse as reproduction, development, and metabolism (1). Dietary lipids, such as cholesterol and fatty acids, and their metabolites, such as steroids and oxysterols, act as ligands to many of the 48 human NRs and those of other metazoan species (2). Several NRs are pharmaceutical drug targets for the treatment of diverse diseases, such as type 2 diabetes, atherosclerosis, osteoporosis, and cancer. To aid intervention in these diseases, multiple synthetic NR agonists, antagonists and inverse agonists have been synthesized and characterized (3). The physiology of NRs and their natural bioactive lipid and synthetic analog ligands shows a broad variety, but their actions can be summarized to a couple of common gene regulatory mechanisms.

Basis of the common actions of NRs is their conserved structure. Members of the NR superfamily are identified by the presence of a highly conserved DNA binding domain and a structurally conserved ligand binding domain (LBD) (4). The LBD of most NRs is a characteristic three-layer antiparallel {alpha}-helical sandwich formed by 11–13 {alpha}-helices. In the lower half of the domain, there is no central helical layer but a large nonpolar pocket, to which the various lipophilic ligands bind. One side of this pocket is sealed by the C-terminal helix of the receptor, often called helix 12. This helix serves as a molecular switch by allowing the LBD in its agonistic conformation to interact with coactivator (CoA) proteins, such as steroid receptor coactivator-1, transcription intermediary factor 2 (TIF2), and receptor-associated coactivator 3 (5). In the absence of an agonistic ligand, NRs interact with corepressor (CoR) proteins, such as nuclear receptor corepressor (NCoR), silencing mediator of retinoic acid and thyroid hormone receptor, and Alien (6). CoA and CoR proteins both contain multiple, short receptor interaction domains (RIDs), composed of the sequence LXXLL in case of CoAs (7) and LXXXIXXX[I/L] in case of CoRs (8). Both type of coregulators interact with largely overlapping surfaces on the LBD suggesting that their binding is mutually exclusive (9). The mouse-trap model (10) proposes that helix 12 acts as a lid to the ligand-binding pocket of the LBD, which has to be closed to allow NR interaction with CoAs and open, when the receptor contacts CoRs. This implies that whereas helix 12 takes only one defined position in the agonist-bound receptor, multiple positions of the helix are possible in antagonist-bound or apo-receptor.

The NR for the seco-steroid 1{alpha},25-dihydroxyvitamin D3 (1{alpha},25(OH)2D3), the vitamin D3 receptor (VDR), is one of the 11 classic endocrine members of the NR superfamily that bind their respective ligands with high affinity [dissociation constant (Kd) value of 1 nM or lower] (2). 1{alpha},25(OH)2D3 is a key player in calcium homeostasis and bone mineralization (11) and also has antiproliferative and prodifferentional effects on various cell types (12). Adopted orphan NRs form another subclass within the NR superfamily. These NRs bind a variety of structurally diverse compounds with a relatively low affinity (Kd in the order of 1 µM) (13). Constitutive androstane receptor (CAR) is an interesting adopted orphan NR because it has an exceptionally high constitutive activity (14) and therefore is functionally opposite to the low basal activity of endocrine NRs, such as VDR. CAR plays a key role in the response to chemical stress and regulates an overlapping set of genes, some of which encode proteins, such as P450 cytochrome monooxygenases (CYPs) that are involved in the detoxification of potentially harmful xenobiotics and endobiotics (15). Primary NR target genes are defined through the presence of particular binding sites, referred to as response elements (REs), in their promoter regions (16, 17). Peroxisome proliferator-activated receptors (PPARs), CAR, VDR, and several other members of the NR superfamily form heterodimers with the retinoid X receptor (RXR) on REs that are composed of a direct repeat (DR) of hexameric binding sites (18). Multiple CAR RE clusters are commonly called phenobarbital-responsive enhancer modules (PBREMs). The mouse CYP2B10 (ortholog to human CYP2B6) gene contains two DR4-type REs with an additional binding site for the transcription factor NF-1 (19).

Most natural and synthetic NR ligands are agonists, such 1{alpha},25(OH)2D3 for the VDR, the imidazothiazole derivative 6-(4-chlorophenyl)imidazo[2,1-b] [1, 3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl)oxime (CITCO) for human CAR (20) and the hepato-mitogen 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) (21) for mouse CAR. The antimycotic clotrimazole (13) and the testosterone metabolite androstanol (14) deactivate human and mouse CAR, respectively, and are therefore considered as inverse agonists (22). The two-side chain 1{alpha},25(OH)2D3 analog Gemini can act, at high CoR levels, as inverse agonist of the VDR (23, 24). In contrast, the 25-carboxylic ester 1{alpha},25(OH)2D3 analog ZK168281 (23) is a pure VDR antagonist (25, 26). It is thought that inverse agonists and antagonists stabilize NR-LBDs in different conformations (27), whereas we demonstrate in this study that ZK168281 and clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors. ZK168281 resembled more androstanol in potent CoR recruitment. Molecular dynamics (MD) simulations resulted in comparable models for the LBDs of VDR and both CARs complexed with a CoR-RID peptide and ZK168281, clotrimazole, and androstanol, respectively. We showed that a salt bridge between the CoR and a conserved lysine in helix 4 of the NR-LBDs are important for the stability of the antagonist-/inverse agonist-stabilized NR-LBD-CoR complex. Moreover, the stabilization of helix 12 by direct contacts with residues of the CoR is common to these models. This demonstrates that NR antagonists and inverse agonists resemble each other in their functional profile and molecular mechanisms.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Individual Ligand-Triggered Interaction Profiles of VDR, CARs, and RXR with Coregulators in Solution
The ligand-triggered physical interaction between the NRs human VDR, human and mouse CAR with the CoA TIF2, and the CoR NCoR was assessed by glutathione-S-transferase (GST)-pull-down assays (Fig. 1Go). For this purpose, in vitro-translated, [35S]-labeled wild-type NR proteins were incubated in the presence of their respective agonistic or antagonistic/inverse agonistic ligands (for their structures see Fig. 1DGo) with bacterially produced GST alone, GST-TIF2 or GST-NCoR fusion protein immobilized on Sepharose beads. The GST-TIF2 protein has all three LXXLL RIDs of the CoA, whereas the GST-NCoR protein contains only the second of the two RIDs of the CoR. We tested also the an alternative GST-NCoR fusion protein containing the first RID but did not obtain efficient interaction with the three NRs neither in GST-pull-down nor in supershift assays (data not shown). GST protein alone showed only weak residual association with the three NRs (lane 2 in Fig. 1Go, A–C), which was considered as unspecific background binding. VDR showed reasonable association with TIF2 already in the absence of ligand (Fig. 1AGo, lane 3), which could be increased significantly by the agonist 1{alpha},25(OH)2D3 (lane 4) and decreased by the pure antagonist ZK168281 (lane 5). In addition, VDR displayed ligand-independent binding of NCoR (lane 6), which was decreased by 1{alpha},25(OH)2D3 (lane 7) and increased by ZK168281 (lane 8).



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  		Fig. 1. Individual Ligand-Enhanced Interaction Profiles of VDR and CAR with CoAs and CoRs in Solution

GST-pull-down assays were performed with bacterially expressed wild-type GST-TIF2 or GST-NCoR and full-length in vitro-translated, [35S]-labeled human VDR (A), human CAR (hCAR, B), and mouse CAR (mCAR, C) in the absence and presence of their respective ligands (1 µM for VDR ligands and 10 µM for CAR ligands). GST alone (–) was used as control. After precipitation and washing, the samples were electrophoresed through 15% sodium dodecyl sulfate-polyacrylamide gels and the percentage of precipitated NRs in respect to input was quantified using a Fuji FLA3000 reader. Representative gels are shown. Columns represent the mean values of at least three experiments, and the bars indicate SD. Statistical analysis was performed using a two-tailed, paired Student’s t test, and P values were calculated in reference to respective solvent controls (*, P < 0.05). Two-dimensional structures of VDR and CAR ligands are shown (D).

 
In the GST-pull-down assays, i.e. in solution, human and mouse CAR show a ligand-independent interaction with TIF2 comparable to that of VDR (compare lane 3 in Fig. 1Go, A–C). The species-specific agonists CITCO and TCPOBOP could slightly increase this association between the two CAR orthologs and CoA protein (Fig. 1Go, B and C, lane 4), whereas the human and mouse inverse agonists clotrimazole and androstanol, respectively, both significantly reduced this protein-protein interaction (Fig. 1Go, B and C, lane 5). Human and mouse CAR showed a less prominent basal interaction with NCoR than VDR (compare lane 6 in Fig. 1Go, A–C). The mouse CAR-CoR complex was reduced by the application of the agonist TCPOBOP (Fig. 1CGo, lane 7), whereas the agonist CITCO increased the interaction between human CAR and NCoR (Fig. 1BGo, lane 7). The inverse agonist androstanol behaved as expected and increased the interaction between mouse CAR and NCoR (Fig. 1CGo, lane 8), but its human counterpart clotrimazole (13) reduced the interaction between human CAR and NCoR (lane 8). Taken together, both VDR and the CAR orthologs display individual ligand-enhanced interaction profiles with CoA and CoR proteins in solution, which may be related to the divergent structure of the ligands.

Ligand-Dependent Interaction Profiles of VDR-RXR and CAR-RXR Heterodimers with Coregulators on DNA
To test whether the ligand-enhanced interactions between VDR, both CARs, TIF2, and NCoR were also valid for DNA-bound heterodimeric complexes with RXR supershift assays (Fig. 2Go) were performed with the same panel of ligands as in the GST-pull-down assays (Fig. 1Go). GST alone did not induce any supershift (lane 1 in Fig. 2Go, A–C). Both the VDR ligands induced the known increase in VDR-RXR heterodimer complex formation on DNA (28) (Fig. 2AGo, lanes 2 and 3) and a minority of human CAR molecules displayed DNA binding as a monomer as described previously (29) (Fig. 2BGo, lanes 1, 3, 6, and 9). In the absence of ligand, DNA-complexed VDR did not show any association with TIF2 or NCoR (Fig. 2AGo, lanes 4 and 8), which is in contrast to the interaction profile of VDR in solution (Fig. 1AGo). The high affinity of apo-VDR in solution for CoAs could have technical reasons being related to a significant molar excess of bacterially produced CoA fusion protein. However, more important for the understanding of vitamin D signaling is the demonstration that DNA-bound VDR is able to attract a reasonable amount of CoA proteins after a conformational change induced by 1{alpha},25(OH)2D3 (lane 5). The antagonist ZK168281 did not induce any interaction of VDR with CoA protein (lane 7) and a combination of agonist and antagonist resulted only in a very faint complex of VDR-RXR heterodimers with TIF2 (lane 6). Application of 1{alpha},25(OH)2D3 alone did only induce residual interaction of VDR with NCoR (lane 9), whereas binding of ZK168281 to the VDR resulted in a strong association with NCoR (lane 10). The combined application of agonist and antagonist resulted in the interaction of significant NCoR amounts with DNA-bound VDR-RXR heterodimers (lane 11).



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  		Fig. 2. Ligand-Dependent Interaction Profiles of VDR-RXR and CAR-RXR Heterodimers with CoAs and CoRs on DNA

Combined gel shift and supershift experiments were performed with equal amounts of in vitro-translated wild-type human VDR (A), human CAR (B), or mouse CAR (C), RXR protein and [32P]-labeled DR3- (A) or DR4-type RE (B and C). VDR-RXR and CAR-RXR heterodimers were preincubated with solvent, 100 nM 1{alpha},25(OH)2D3 or 1 µM ZK168281 (A), 10 µM CITCO or clotrimazole (B), and 10 µM TCPOBOP or androstanol (C) as indicated. Equal amounts of bacterially expressed wild-type GST (–), GST-TIF2, or GST-NCoR were then added. Protein-DNA complexes were resolved from free probe through 8% nondenaturing polyacrylamide gels. The relative amounts of supershifted complexes were quantified using a Fuji FLA3000 reader. Representative gels are shown. NS, Nonspecific complexes. Columns represent the mean of at least three experiments, and bars indicate SD. A two-tailed, paired Student’s t test was performed and P values were calculated in reference to the respective solvent control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
In contrast to VDR, DNA-complexed human and mouse CAR showed significant interaction with TIF2 even in the absence of ligand (Fig. 2Go, B and C, lane 4). Interestingly, both receptors interacted also with NCoR in a ligand-independent fashion (lane 7). As expected from an agonist, CITCO increased the interaction of human CAR with CoA, but surprisingly, it also induced a significant interaction of the receptor with NCoR (Fig. 2BGo, lanes 5 and 8). Clotrimazole blunted the constitutive interaction of human CAR with TIF2 and also reduced the interaction of the receptor with NCoR (lanes 6 and 9). Similar to CITCO in case of human CAR, TCPOBOP increased the interaction of mouse CAR with CoA protein (Fig. 2CGo, lane 5), but in contrast to CITCO it decreased the interaction of its receptor with CoR protein (lane 8). Finally, androstanol behaved as expected from an inverse agonist in that it decreased the interaction of mouse CAR with TIF2 (lane 6) and increased the contact with NCoR (lane 9). In summary, as a DNA-bound heterodimer with RXR VDR shows no constitutive interaction with CoA protein and no significant interaction with CoR protein in the presence of agonist. However, in all other aspects the supershift assays confirm the ligand-enhanced interaction profile of VDR and the two CARs with coregulator proteins as observed in the GST-pull-down assays (Fig. 1BGo). This is an indirect proof that RXR has only minor contributions to the ligand profile of VDR and the two CARs. Moreover, it means that the VDR antagonist ZK168281 and the human CAR inverse agonist clotrimazole are both effective inhibitors of the CoA interaction of their respective receptors, whereas concerning potent CoR recruitment ZK168281 resembles more the mouse CAR inverse agonist androstanol.

Coregulator-Triggered Ligand Responsiveness of VDR and CAR in MCF-7 Cells
To compare the functional consequences of agonist and antagonist/inverse agonist application to VDR and the two CARs, we performed reporter gene assays in the transiently transfected model cell line MCF-7 (Fig. 3Go). The transactivation potential of VDR was assessed on four copies of the DR3-type RE of the rat atrial natriuretic factor (ANF) gene (30), whereas the two CARs were tested on the PBREM (which contains two DR4-type REs) of the mouse CYP2B10 gene (31); both REs were fused individually with the thymidine kinase promoter driving the luciferase reporter gene. At endogenous coregulator levels, the very low basal level of VDR on the rat ANF DR3-type RE (Fig. 3AGo, lane 1) was induced nearly 50-fold by 10 nM 1{alpha},25(OH)2D3 (lane 2), whereas 100-times higher concentrations of ZK168281 (1 µM) resulted only in less than a 4-fold induction (lane 4). This very low residual agonistic activity of high concentration of ZK168281 were observed already previously (26). The combined application of agonist and antagonist led to a 16.7-fold induction (lane 3). The overexpression of TIF2 resulted in a significant increase of the basal level (3.8-fold, lane 5) and subsequently a less prominent increase of agonist-stimulated values, so that only an approximately 15-fold induction was observed (lane 6). CoA overexpression increased the response to the antagonist (4.9-fold, lane 8), which is known from a previous report (32). However, the combined application of agonist and antagonist did not provide significantly higher induction (6.5-fold, lane 7) than agonist alone and clearly less than in case of endogenous CoA concentrations. The overexpression of NCoR reduced the basal activity by 40% (lane 9). The effects of agonist and antagonist alone or in combination were also blunted (34.3-, 3.5-, and 2-fold, respectively, lanes 10–12).



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  		Fig. 3. CoA- and CoR-Triggered Ligand Responsiveness of VDR and CAR in MCF-7 Cells

Reporter gene assays were performed with extracts from MCF-7 cells that were transiently transfected with a luciferase reporter construct containing four copies of the DR3-type RE of the rat ANF gene (A) or the PBREM of the mouse CYP2B10 gene (B and C). Wild-type human VDR (A), human CAR (B), and mouse CAR (C) expression vectors as well as plasmids coding for full-length TIF2 or NCoR were also cotransfected as indicated. Cells were treated for 16 h with solvent, 10 nM 1{alpha},25(OH)2D3 or 1 µM ZK168281 (alone and in combination, A), 1 µM CITCO or 1 µM clotrimazole (B), and 1 µM TCPOBOP or 1 µM androstanol (C) as indicated. Relative luciferase activities were measured in reference to agonist-induced values of cells not overexpressing coregulator protein. Fold inductions in relation to solvent are shown on the tip of the columns, and the relative basal activities at the different coregulator levels are indicated below the respective solvent columns. Columns represent the mean of at least three experiments, and bars indicate SD.

 
Human CAR showed a high basal activity on the PBREM (Fig. 3BGo, lane 1), which could only be induced 2.5-fold by CITCO (lane 2) and was reduced by 40% with clotrimazole. TIF2 overexpression had only minor effects on the basal activity and ligand response of human CAR (lanes 4–6). In contrast, NCoR overexpression clearly blunted the responsiveness of the receptor. In this respect, the basal activity was significantly reduced to 30% of the basal activity (lane 7), the induction by CITCO was only 1.6-fold (lane 8) and in the presence of clotrimazole still 90% of basal activity level was observed (lane 9). The response pattern of mouse CAR (Fig. 3CGo) was similar to that of human CAR, but the mouse-specific ligands had more prominent effects than that of human CAR.

Mouse CAR also showed high basal activity on the PBREM (lane 1), which was induced 3-fold by TCBOBOP (lane 2) and reduced by 70% with androstanol (lane 3). CoA protein overexpression slightly increased the basal activity (1.4-fold, lane 4), reduced the response to the agonist (2.6-fold induction, lane 5) and to the antagonist (still 70% of basal activity, lane 6). Also with mouse CAR the overexpression of CoR protein showed more prominent effects than the overexpression of CoA proteins. The basal activity was reduced significantly by 50% (lane 7), the response to TCPOBOP lowered to a 2.2-fold induction (lane 8) and androstanol application reached 40% of the basal activity (lane 9). Taken together, these data indicate that VDR mediates low basal activity and high agonist inducibility, whereas the two CARs show high basal activity and only moderate inducibility. The VDR antagonist showed the known residual agonist activity (32) and could reduce at equimolar concentrations effectively agonist-induced gene activity, whereas the inverse agonists of the two CARs reduced the high basal activity. CoA protein overexpression significantly increased the basal activity and antagonist response of VDR and reduced its agonist inducibility but had only minor effects on the two CARs. In contrast, the basal activity and the ligand responsiveness of all three NRs were significantly reduced by CoR protein overexpression.

Modeling of CoR Interactions of VDR and CAR
Because CoRs seem to have a significant effect on the ligand response and basal activity of both endocrine and adopted orphan NRs, we next investigated in the structural determinants of the interactions of the LBD of VDR and the two CARs with CoR in the presence of antagonist or inverse agonist. The complexes of human VDR-LBD with 1{alpha},25(OH)2D3 and CoA peptide (Fig. 4AGo, top), human CAR-LBD with CITCO and CoA peptide (Fig. 4BGo, top) and mouse CAR-LBD with TCPOBOP and CoA peptide (Fig. 4CGo, top) were modeled on the basis of the crystal structure and our previous MD simulations of the human VDR-LBD (26, 33) and on the recently solved x-ray structures of human and mouse CAR (34, 35, 36). In parallel, we docked the inverse agonists clotrimazole and androstanol to the structures of human and mouse CAR, respectively, whereas for the ZK168281-bound VDR-LBD, we already had a structure from a previous MD simulation study (26). To each of the three LBDs, we docked a peptide representing the amino acids 2275–2291 of the second RID of NCoR and performed MD simulations. The resulting structures (Fig. 4Go) represent the average of the last 50 psec of the MD simulations. The detailed views on these structures indicate interactions of helices 3, 4, and 12 of the NR-LBDs with the CoA- and CoR-RID peptide. The most remarkable and consistent observation of the three CoR-NR model structures was that helix 12 seems to be not flexible but takes a stabilized position. This is also visible in the Protein Data Bank (PDB) file of the cocrystal of PPAR{alpha} with CoR-RID peptide (1KKQ), although this was not discussed in the respective publication (37).



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  		Fig. 4. MD Simulations of Coregulator Interactions of VDR and CAR

On the basis of the crystal structure and our previous MD simulations of the human VDR-LBD (26 33 ) and recently solved x-ray structures of human and mouse CAR (34 35 36 ) supported by information of the rat VDR-CoA peptide complex (55 ) and the human PPAR{alpha}-CoR peptide complex (37 ), MD simulations were performed with the human VDR-LBD in complex with 1{alpha},25(OH)2D3 and CoA peptide (A, top) or with ZK168281 and CoR peptide (A, center), with the human CAR-LBD in complex with CITCO and CoA peptide (B, top) or clotrimazole and CoR peptide (B, center) and mouse CAR-LBD in complex with TCPOBP and CoA peptide (C, top) or androstanol and CoR peptide (C, center). Only helices 3, 4, 11, and 12 (blue) and the side chains of the most important amino acids are shown. The CoA-RID is shown in green and the CoR-RID peptide in red. Dashed lines indicate interactions with a distance below 3.4 Å. The interactions of the CoR-RID peptide are schematically depicted below each structure. Dashed lines with horizontal bars at their end symbolize backbone interaction. The core NR interaction motif is underlayed in dark red; red indicates negatively charged amino acids and blue positively charged residues.

 
In the VDR-LBD-ZK168281 complex, V418 of helix 12 interacts with I2280 of NCoR and S427 of the C terminus makes a backbone contact to F2289 of NCoR (Fig. 4AGo). In a similar way, helix 12 of CAR is stabilized by an interaction between the positively charged K2283 of the CoR-RID peptide and L343 and L353 of human and mouse CAR, respectively (Fig. 5Go, B and C). In addition, K264 of helix 4 in VDR and the homologous residues K195 and K205 in human and mouse CAR, respectively, form a salt bridge with E2278 of NCoR. Moreover, K246 of helix 3 in VDR and its homologous residues K177 and K187 in human and mouse CAR, respectively, contact the CoR-RID peptide at the backbone of L2285. Additionally, K246 also interacts with the backbone of the CoR residue A2284. The antagonist ZK168281 contacts H397 in helix 11, but the side chains of the homologous amino acids Y326 and Y336 in human and mouse CAR, respectively, have shifted their orientation (compare Fig. 4Go, B and C, top and center) and do not contact the two inverse agonists clotrimazole and androstanol. The structures of agonist- and antagonist-bound CAR demonstrate that all four ligands are relatively small and do neither contact helix 12 nor the coregulator RID, i.e. they are not directly involved in the attraction of CoA or CoR protein. In contrast, the long side chain of ZK168281 is directly contacting NCoR at position L2277 (Fig. 4AGo, center). In summary, the models of VDR and both CARs with antagonist or inverse agonist and CoR-RID peptide suggest that both type of ligands stabilize helix 12 in a position that allows several salt bridge and hydrogen bond-based interactions between residues of helices 3, 4, and 12 of the receptors and the backbone and side chains of their partner amino acids in the NCoR RID. However, the antagonist is contacting both helix 11 and the CoR-RID, which is not observed with the smaller inverse agonists.



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  		Fig. 5. Critical Amino Acids of VDR and CAR for Ligand-Triggered Interaction with CoRs

Combined gel shift and supershift experiments were performed with equal amounts of in vitro-translated wild-type or mutant human VDR (A), human CAR (B), or mouse CAR (C), RXR protein, and [32P]-labeled DR3- (A) or DR4-type RE (B and C). VDR-RXR and CAR-RXR heterodimers were preincubated with solvent, 100 nM 1{alpha},25(OH)2D3, or 1 µM ZK168281 (A); 10 µM CITCO or clotrimazole (B); and 10 µM TCPOBOP or androstanol (C) as indicated. Equal amounts of bacterially expressed wild-type GST (–), GST-TIF2 or GST-NCoR were then added. Protein-DNA complexes were resolved from free probe through 8% nondenaturing polyacrylamide gels. The relative amounts of supershifted complexes were quantified using a Fuji FLA3000 reader. Representative gels are shown.

NS, Nonspecific complexes. Columns represent the mean of at least three experiments, and bars indicate standard deviations. Two-tailed, paired Student’s t test was performed, and P values were calculated in reference to the interaction of wild-type receptor (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
Critical Amino Acids of VDR Ligand-Enhanced Interaction with CoRs
To challenge the above-described models, for each of the three receptors a series of point mutants of comparable residues was created and tested by supershift assays. The assays were performed with GST-NCoR in the presence of agonist and antagonist/inverse agonist (Fig. 5Go). GST alone and GST-TIF2 without ligand were used as controls. As already shown in Fig. 2AGo, in the absence of ligand VDR-RXR heterodimers failed to interact with either TIF2 or NCoR (Fig. 5AGo, lanes 2 and 3), whereas 1{alpha},25(OH)2D3 and ZK168281 induced faint and strong interactions with NCoR, respectively (lanes 4 and 5). The mutants K246A, K264A, H397A, V418A, S427A, and N424* (resulting in a truncated helix 12) did not induce any ligand-independent interaction of the receptor with CoA or CoR protein (lanes 7, 8, 12, 13, 17, 18, 22, 23, 27, 28, 32, and 33), but reduced the antagonist-induced interaction with NCoR by approximately 25% in case of K246A, V418A, S427A, and N424* (lanes 10, 25, 30, and 35), 50% in case of H397A (lane 20) and completely (by 100%) in case of K264A (lane 15). The weak, 1{alpha},25(OH)2D3-induced interaction of VDR with NCoR was abolished in case of K246A, K264A, H397A, V418A, and S427A (lanes 9, 14, 19, 24, and 29) and significantly increased in case of N424* (lane 34). Taken together, for the antagonist-triggered interaction of VDR with NCoR K264 is of central importance and also the indirect effect of H397 is critical, whereas the stabilization of the helix 12-NCoR contact via the residues V418 and S427 and the helix 3-NCoR interaction via K246 seem to have minor impact.

Critical Amino Acids of Human CAR for Ligand-Enhanced Interaction with CoR
The ligand-independent coregulator interaction of human CAR (Fig. 5BGo) and mouse CAR (Fig. 5CGo) made the pattern of the homologous mutations more complex. The level of interaction of human CAR with TIF2 in the absence of ligand was not significantly affected by the mutant C347A (Fig. 5BGo, lane 27) and was reduced by approximately 35% with Y326A and the extension of helix 12 by three amino acids (lanes 17 and 32), by 50% with K177A and L343A (lanes 7 and 22) and by 90% with K195A (lane 12). The ligand-independent interaction of human CAR with TIF2 was approximately doubled compared with NCoR (compare lanes 2 and 3), but the latter was slightly increased with K177A (lane 8), not affected with L343A (lane 23) and reduced by approximately 40% with C347A (lane 28) and by more than 70% with K195A, Y326A and the extension of helix 12 (lanes 13, 18, and 33). Interestingly, the CITCO-induced interaction of human CAR with CoR protein was increased by 10–20% with K177A and L343A (lanes 9 and 24) and reduced by approximately 25% with C347A and the extension of helix 12 (lanes 29 and 34) and by more than 80% with K195A and Y326A (lanes 14 and 19). Although CITCO induced the interaction of human CAR with NCoR by 150%, clotrimazole application reduced it by 35% (compare lanes 4 and 5). This ratio between the CITCO and clotrimazole effect remained approximately the same with K177A, L343A, and C347A (compare lanes 9 with 10, 24 with 25, and 29 with 30), whereas, combined with a low basal level, it became nearly equal with K195A and Y326A (compare lanes 14 with 15 and 19 with 20). The only exception was the extension of helix 12, which doubled the ratio of the CITCO- and the clotrimazole-mediated interaction of human CAR with NCoR from approximately 4–8 (compare lanes 34 and 35). In summary, residues K195 and Y326 and a short helix 12 seem to be critical for both the direct and indirect human CAR and NCoR complex stabilization and the effect of the antagonist clotrimazole. Moreover, K195 and Y326, but not the length of helix 12, are important for CoR recruitment via CITCO.

Critical Amino Acids of Mouse CAR for Ligand-Enhanced Interaction with CoR
In the absence of ligand, the interaction of mouse CAR with TIF2 was not significantly affected by the extension of helix 12 by three amino acids (Fig. 5CGo, lane 32). However, it was reduced by 40–60% with K187A, K205A, and Y336A (lanes 7, 12, and 17) and abolished entirely with L353A and C357A (lanes 22 and 27). K187 is part of the charge-clamp and its respective mutant has already been tested in our own (38, 39) and other groups (40). The strength of the effects of this mutant is inversely correlated with the concentration of the bacterially expressed CoA protein, which was relatively high in this study. The ligand-independent interaction of mouse CAR with TIF2 was 30% higher than with NCoR (compare lane 2 and 3). The latter was not affected by L353A and reduced by 10–30% with K187A and C357A (lanes 8 and 28), by approximately 50% with K205A and the extension of helix 12 (lanes 13 and 33) and by 80% with Y336A (lane 18). The agonist TCPOBOP reduced the basal interaction of mouse CAR with NCoR by 35% (compare lanes 3 and 4), but this effect was decreased to approximately 25% with K187A (compare lanes 8 with 9) and blunted with K205A, Y336A, L353A, C357A, and the extension of helix 12 (compare lanes 13 with 14, 18 with 19, 23 with 24, 28 with 29, and 33 with 34). The antagonist androstanol induced the interaction of mouse CAR with NCoR by approximately 80% (compare lanes 3 and 5). This effect was not affected with the mutation K187A (compare lanes 8 and 10), but abolished with the five other mutants (compare lanes 13 with 15, 18 with 20, 23 with 25, 28 with 30, and 33 with 35). Taken together, the lysine in helix 4 and the tyrosine in helix 11, K205 and Y336, are the most important residues for the ligand-enhanced interaction of mouse CAR with CoR protein. This is similar to the findings with human CAR.

Functional Analysis of Critical Amino Acids of VDR and Human and Mouse CAR
To analyze the impact of critical amino acids on the agonist and antagonist/inverse agonist responsiveness of VDR and the two CARs, we performed reporter gene assays in transiently transfected MCF-7 cells (Table 1Go) using the same experimental conditions as in Fig. 3Go. Concerning modulation of basal activities and agonist inducibilities, we obtained essentially the same results as in our previous studies on the critical role of helix 12 on the constitutive activity and CoA recruitment of VDR and human and mouse CAR (38, 39). More interesting is the observation that ZK168281 showed no agonistic potential with the VDR mutants K246A, K264A, H397A, and V418A but lost most of its antagonistic potential (from 66% with wild-type VDR down to 12–25%). In contrast, the mutant S427A showed a profile similar to wild-type VDR. Comparably, with the homologous human CAR mutants K177A, K195A, Y326A, and L343A and their mouse orthologs K187A, K205A, Y336A, and L353A clotrimazole and androstanol lost most (K177A/K187A) or even all of their inverse agonistic potential. In comparison, the inverse agonists were still functional with C347A and C357A. In conclusion, these data confirm the central role of the lysine in helix 4 (K264, K195, and K205). However, most of the tested residues have also an impact on the basal activity and agonist inducibility of the receptors, allowing no trivial functional distinction of the effects of antagonists and inverse agonists.


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  		Table 1. Functional Test of Critical Residues of VDR, Human and Mouse CAR MCF-7 Cells

 
Critical Amino Acids of the CoR NR Interaction Motif
The mutational analysis of all three NRs (Fig. 5Go) and their functional test (Table 1Go) suggested that the conserved lysine in helix 4 (K264, K195, and K205, respectively) is the most important residue in the direct contact of the NR with CoR-RID. Therefore, we mutated in each of the three receptors the positively charged lysine into a negatively charged glutamate. In parallel, we performed similar charge inversion mutagenesis of the CoR-RID, where we mutated E2278 into a lysine and K2283 into a glutamate. Different combinations of these receptor and NCoR mutants were assessed in supershift assays (Fig. 6Go). The ZK168281-induced interaction of VDR-RXR heterodimers with NCoR was reduced by 70% when wild-type CoR protein was replaced with its E2278K mutant (Fig. 6AGo, compare lanes 2 and 3). Although the K2283E mutant resulted in some 40% reduction of CoR interaction (lane 4), this NCoR residue appears not to contact VDR directly (see Fig. 4AGo). As expected, the K264E mutant of VDR completely abolished the CoR contact (lane 6). However, in combination with the NCoR mutant E2278K, 70% of the interaction level of both wild-type proteins was restored (lane 7). Comparable observations were made with human and mouse CAR. The E2278K mutant of NCoR reduced the interaction with human CAR by 50% and that with mouse CAR even by 70% (Figs. 6Go, B and C, lane 3). The K195E and K205E mutants of human and mouse CAR, respectively, blunted the interaction of the receptor with the CoR protein (lane 6) and the combination of mutated receptor with mutated NCoR restored 90% of the interaction between CoR and human CAR. In case of mouse CAR, even a level of 160% was reached (lane 7). A test of the NCoR mutant K2283E with the two CARs showed that it also reduced the receptor-CoR interaction for both receptors by 55–70% (lane 4), but the combination with mutated receptor could not restore the interaction (data not shown). In summary, the salt bridge between E2278 of NCoR and the lysine of helix 4 of VDR and the two CARs seems to be the major direct fixation point between receptor and CoR; however, K2283 is also critical for the stability of helix 12 and the effective interaction of the three NRs with NCoR.



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  		Fig. 6. Critical Amino Acids of the CoR NR Interaction Motif

Supershift experiments were performed with equal amounts of in vitro-translated wild-type or mutant human VDR (A), human CAR (B), or mouse CAR (C), RXR protein, and [32P]-labeled DR3- (A) or DR4-type RE (B and C). VDR-RXR and CAR-RXR heterodimers were preincubated with solvent, 1 µM ZK168281 (A), 10 µM clotrimazole (B) and 10 µM androstanol (C). Equal amounts of bacterially expressed wild-type GST (–), wild type of mutant GST-NCoR, were then added. Protein-DNA complexes were resolved from free probe through 8% nondenaturing polyacrylamide gels. The relative amounts of supershifted complexes were quantified using a Fuji FLA3000 reader. Representative gels are shown. NS, Nonspecific complexes. Columns represent the mean of at least three experiments, and bars indicate SD. Two-tailed, paired Student’s t test was performed, and P values were calculated in reference to the interaction of wild-type receptor with wild-type NCoR (*, P < 0.05; **, P < 0.01).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Antagonists and Inverse Agonists Have Similar Functional Profiles
In this study, we demonstrate that despite their divergent structure, the VDR antagonist ZK168281, and the CAR inverse agonists clotrimazole and androstanol have comparable effects on the coregulator interaction of their receptors. Therefore, antagonists and inverse agonists may be taken into the same ligand class that can be clearly distinguished from the class of agonists, even if the example of CITCO suggests some overlap between both ligand families exist. Such dual effects are also known for other CAR ligands, such as estrogens (41). According to present knowledge, in very most cases the large ligand-binding pockets of adopted orphan NRs can accommodate only one ligand molecule at the time. This suggests that agonists and antagonists/inverse agonists compete for binding to the pocket. Therefore, effective agonist/antagonist or agonist/inverse agonist ligand couples should have comparable affinities for the NR-LBD. Mutational analyses coupled with molecular modeling of human and mouse CAR (42, 43) as well as the crystal structures of both receptors (34, 35, 36) have indicated that the ligand-binding pocket of the adopted orphan NR has a volume of approximately 600 Å3 and is formed by some 30 amino acid residues of in helices 5–7 and 10. The ligand-binding pocket of VDR is as large as that of CAR (33) but is bound by ZK168281 and 1{alpha},25(OH)2D3 with the same high affinity [Kd = 0.1 nM (23)]. The stricter structural requirement of VDR ligands is related to their contact with H397 in helix 11 (Fig. 4AGo). In addition, the extended side chain of ZK168281 both pushes helix 12 from its agonistic position (26) and directly contacts and stabilizes the CoR-RID. In contrast, clotrimazole and androstanol do not contact the H397 homologs Y326 and Y336 in human and mouse CAR and also do not interact with the CoR-RID (Fig. 4Go, B and C). These properties make ZK168281 a more effective regulator of the coregulator exchange than clotrimazole and androstanol.

The CoA/CoR Ratio May Be the Main Parameter for Changing the Activity State of a NR
Common in the molecular mechanism of antagonists and inverse agonists is their ability to promote the interaction of their receptor with CoR proteins and to inhibit contact with CoA proteins. Classical endocrine NRs, such as VDR, show very low basal activity in the absence of ligand, whereas adopted orphan NRs, such as CAR and PPARs (Molnár, F., and C. Carlberg, unpublished results), display significant amount of constitutive activity. Therefore, antagonists act after a preceding activation of their target receptor by an agonist, whereas inverse agonists antagonize the ligand-independent high basal activity of their receptor. The ligands that were used in this study demonstrate that there are transitions between these two extreme states and that the potential of the ligands to perform each of the two functions is molecule specific. For example, although clotrimazole prevents CoA contact of human CAR, it only weakly recruits CoR binding. In contrast, CITCO supports both CoA and CoR interaction of human CAR. In particular, the latter example raises the question, whether the view that NR ligands actively recruit coregulators of one variety or another is valid for all compounds. Alternatively, it may be that the relatively large ligand-binding pocket of adopted orphan receptors allows the binding of a small ligand, such as CITCO, without inducing significant changes in its conformation. Human and mouse CAR are able to bind in their apo-state both CoA and CoR proteins. This suggests that the ratio between CoA and CoR proteins may be the main parameter for changing the conformation and activity state of a NR, such as CAR, and that the effect of the ligand may be of secondary importance.

Helix 12 Takes a Stabilized Position during CoR Interaction
According to the mouse-trap model the helix 12 of a NR has a central role in determining the agonist-triggered interaction of NR-LBDs with CoA proteins (10), and this has been proven extensively in many studies. For apo-NRs, which should be able to interact with CoR proteins, the model suggests free movement of helix 12 as observed in the RXR-LBD crystal structure (44). Amino acids that were identified in this study as being important for CoR interaction, such as the conserved lysines in helices 3 and 4, have already been described to be critical for the CoA contacts of the respective receptors (38). Although we did not directly address the hydrophobic residues on the surface of the NR-LBDs and the CoR-RID, our investigation of charged and polar amino acids suggests that both CoA and CoR proteins contact the same surface region on NR-LBDs. However, the larger RIDs of CoRs compared with CoAs (9) make a move of helix 12 necessary for a coregulator exchange. In fact, in vitro a complete truncation of helix 12 is favorable for CoR interaction (Ref.40 and data not shown).

In living cells, NRs have to deal with a transiently dispensable helix 12, although CoR binding. Therefore, our model of a stabilized position of helix 12 in case of CoR contacts (Fig. 4Go) represents energetically a more favorable state than that of a free-floating helix as suggested in the mouse-trap model (10). Our model (Fig. 4Go) suggests that the contact between the CoR-RID and helix 12 stabilizes the position of the latter helix. This means that, in contrast to CoA interaction, the stabilized position of helix 12 does not support CoR interaction, but that the CoR helps the receptor to get its flexible helix under control. This would explain why the mutagenesis of the contact points between helix 12 and the CoR-RID has less consequences than the mutagenesis of the salt bridge between the conserved lysine in helix 4 and E2278 in the CoR-RID. For the CoR-NR-LBD interaction, the latter salt bridge seems to have a similar impact than the charge clamp between the conserved lysine in helix 3 and the glutamate in helix 12 has for CoA interaction. However, in contrast to the glutamate in helix 12, which changes its distance in relation to its partner lysine with every ligand-triggered move of helix 12, the lysine in helix 4 has a stabilized, ligand-independent position. This explains why in the absence of ligand the LBDs of endocrine NRs favor CoR interaction. Adopted orphan NRs, which display constitutively activity seem to be an exception because their mechanisms of stabilizing helix 12 in the absence of ligand (38) prevent access of CoR-RID to their interface on the surface of the LBD.

Conclusion
The ligand-triggered dynamic exchange of CoA and CoR proteins binding to NRs is the molecular basis of the action of agonists, inverse agonists, and antagonists. The structural determinants of the antagonist- and inverse agonist-triggered interaction VDR and human and mouse CAR with the second RID of NCoR led to the main conclusion of this study that antagonists of endocrine NRs and inverse agonists of adopted NRs have a comparable functional profile. A second, important finding of this study is the stabilization of helix 12 in all three receptors by direct contacts with residues of the CoR. However, in contrast to the CoA interaction, which is dependent of a fixed position of helix 12, the helix is not needed for CoR interaction. In fact, helix 12 has to move from its position in the agonistic LBD conformation to a perpendicular position, where it does not disturb the contact between LBD and CoR. Therefore, fixation of helix 12 in the antagonistic/inverse agonistic conformation seems to be only energetically favorable but of no specific function. The comparable molecular mechanisms that explain the comparable functional profile of antagonist and inverse agonists are likely to be extended from VDR and CAR to other members of the NR superfamily and may lead to the design of even more effective ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Compounds
1{alpha},25(OH)2D3 was kindly provided by Dr. L. Binderup (Leo Pharma, Ballerup, Denmark), ZK168281 was a gift from Dr. A. Steinmeyer (Schering, Berlin, Germany) and TCPOBOP was synthesized and purified according to Honkakoski et al. (45). CITCO was obtained from Biomol (Copenhagen, Denmark), androstanol from Steraloids (Newport, RI) and clotrimazole from Sigma-Aldrich (St. Louis, MO). The two-dimensional structures of the ligands are shown in Fig. 1DGo. The VDR ligands were dissolved in propan-2-ol, whereas the other compounds were dissolved in dimethylsulfoxide. Further dilutions were made in either dimethylsulfoxide (for in vitro experiments) or ethanol (for cell culture experiments).

DNA Constructs
Protein Expression Vectors. Full-length cDNAs for human VDR (46), human CAR (47), human RXR{alpha} (48) and human TIF2 (49) were subcloned into the T7/SV40 promoter-driven pSG5 expression vector (Stratagene, La Jolla, CA). The full-length cDNAs for mouse CAR (50) and mouse NCoR (51) were subcloned into the T7/CMV promoter-driven pCMX expression vector. The amino acid substitution mutants of human VDR, human CAR, and mouse CAR were generated using the QuikChange point mutagenesis kit (Stratagene) and confirmed by sequencing. The truncation of helix 12 of VDR by four amino acids (N424*) was created by mutating triplet 424 into a stop codon. The extensions of helix 12 in human and mouse CAR by three amino acids were generated by a double mutant that converted the original stop codon into a coding triplet and the third downstream triplet into a stop codon. The same constructs were used for both T7 RNA polymerase-driven in vitro transcription/translation of the respective cDNAs and for viral promoter-driven overexpression of the respective proteins in mammalian cells.

GST Fusion Protein Constructs. Critical domains of human TIF2 (spanning from amino acids 646–926 including three RIDs) (49) and mouse NCoR (spanning from amino acids 2218–2453 including the second RID) (51) were subcloned into the GST fusion vector pGEX (Amersham Pharmacia, Uppsala, Sweden).

Reporter Gene Constructs. The luciferase reporter gene, fused with the thymidine kinase minimal promoter, was driven by four copies of the DR3-type 1{alpha},25(OH)2D3 response element of the rat ANF gene promoter (30) or one copy of the PBREM of the mouse CYP2B10 gene promoter (containing two DR4-type REs) (19).

In Vitro Translation and Bacterial Overexpression of Proteins
In vitro-translated wild-type or mutated human VDR and human and mouse CAR proteins were generated by coupled in vitro transcription/translation using rabbit reticulocyte lysate as recommended by the supplier (Promega, Madison, WI). Protein batches were quantified by test translations in the presence of [35S]-methionine. The specific concentration of the receptor proteins was adjusted to approximately 4 ng/µl after taking the individual number of methionine residues per protein into account. Bacterial overexpression of GST-TIF2, wild-type, and mutant GST-NCoR or GST alone was obtained from the Escherichia coli BL21(DE3)pLysS strain (Stratagene) containing the respective expression plasmids. GST-TIF2 and GST protein expression were stimulated with 0.25 mM isopropyl-ß-D-thio-galactopyranoside for 3 h at 37 C and GST-NCoR expression was induced with 1.25 mM isopropyl-ß-D-thio-galactopyranoside for 5 h at 25 C. The fusion proteins were purified and immobilized by glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer’s protocol. For gel shift experiments, the fusion proteins were eluted by glutathione.

GST-Pull-Down Assays
GST-pull-down assays were performed with 50 µl of a 50% Sepharose bead slurry of GST, GST-TIF2, or GST-NCoR (preblocked with 1 µg/µl BSA) and 20 ng in vitro-translated, [35S]-labeled NRs in the presence or absence of their respective ligands. Proteins were incubated in immunoprecipitation buffer [20 mM HEPES (pH 7.9), 200 mM KCl, 1 mM EDTA, 4 mM MgCl2, 1 mM dithiothretiol, 0.1% Nonidet P-40 and 10% glycerol] for 20 min at 30 C. In vitro-translated proteins that were not bound to GST-fusion proteins were washed away with immunoprecipitation buffer. GST-fusion protein bound, [35S]-labeled NRs were resolved by electrophoresis through 15% sodium dodecyl sulfate-polyacrylamide gels and quantified on a FLA3000 reader (Fuji, Tokyo, Japan) using Image Gauge software (Fuji).

Gel Shift and Supershift Assays
Gel shift assays were performed with equal amounts (~10 ng) of the appropriate in vitro-translated protein. The proteins were incubated for 15 min in a total volume of 20 µl binding buffer [10 mM HEPES (pH 7.9), 150 mM KCl, 1 mM dithiothretiol, 0.2 µg/µl poly(deoxyinosine-deoxycytosine) and 5% glycerol]. For supershift experiments, 0.4–3 µg of bacterially expressed wild-type or mutant GST fusion proteins (or GST alone as negative control) were added to the reaction mixture. Approximately 1 ng of [32P]-labeled double-stranded oligonucleotides (50,000 cpm) corresponding to one copy of the DR3- or DR4-type RE (core sequences are indicated in Figs. 2Go, 5Go, and 6Go) was then added and incubation was continued for 20 min at room temperature. Protein-DNA complexes were resolved by electrophoresis through 8% nondenaturing polyacrylamide gels in 0.5x TBE [45 mM Tris (pH 8.3), 45 mM boric acid, 1 mM EDTA] and quantified on a FLA3000 reader using Image Gauge software.

Transfection and Luciferase Reporter Gene Assays
MCF-7 human breast cancer cells were seeded into six-well plates (105 cells/ml) and grown overnight in phenol red-free DMEM supplemented with 5% charcoal-stripped fetal bovine serum. Plasmid DNA containing liposomes were formed by incubating a reporter plasmid and expression vectors for wild-type or mutated human VDR, human CAR, mouse CAR, human TIF2, or mouse NCoR (each 1 µg as indicated) with 10 µg N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Roth, Karlsruhe, Germany) for 15 min at room temperature in a total volume of 100 µl. After dilution with 900 µl phenol red-free DMEM, the liposomes were added to the cells. Phenol red-free DMEM supplemented with 500 µl 15% charcoal-stripped fetal bovine serum was added 4 h after transfection. At this time, NR ligands or control solvents were also added. The cells were lysed 16 h after onset of stimulation using the reporter gene lysis buffer (Roche Diagnostics, Mannheim, Germany) and the constant light signal luciferase reporter gene assay was performed as recommended by the supplier (Canberra-Packard, Groningen, The Netherlands). The luciferase activities were normalized with respect to protein concentration and induction factors were calculated as the ratio of luciferase activity of ligand-stimulated cells to that of solvent controls.

Structural Modeling and MD Simulations
The initial coordinates of VDR were obtained from the crystal structure of the human VDR-LBD-1{alpha},25(OH)2D3 complex [PDB code 1DB1 (33)]. The missing amino acid residues (no. 118, 119, 375–377, and 424–427) were built using the Quanta98 molecular modeling package (Molecular Simulations Inc., San Diego, CA). The four residues missing from the C terminus (424–427) were built in an {alpha}-helical conformation ({phi} = – 57 °, {psi} = – 47 °). The coordinates of mouse CAR were taken from its recently solved crystal structure bound to the inverse agonist androstenol [PDB code 1XNX (36)]. Residues 344–349 of helix 12, which were missing from the structure, were modeled using the targeted MD method (52). In this method, an additional term is added to the energy function of the system based on the mass-weighted root mean square deviation (RMSD) of a set of atoms in the current structure compared with a reference structure. The additional energy term acts as a positional restraint, which forces the current structure to move toward a reference structure during a targeted MD simulation. Here the crystal structure of mouse CAR in the agonistic conformation [PDB code 1XLS (35)], in which the residues 344–349 are present, was used as a starting structure and the mouse CAR-androstenol structure was the target. In practice, during a 95-psec MD simulation at 340 K, the coordinates of residues 326–343 and 350–357 of the starting structure were forced using a force constant of 5 kcal (mol–1Å–2) to move toward the coordinates of the target. The six residues missing from the mouse CAR-androstenol x-ray structure were allowed to move freely. During the targeted MD simulation, the RMSD was linearly decreased from 7.4 Å, which is the RMSD of residues 326–343 and 350–357 of the starting structures, to 0.0 Å. The slightly increased temperature (340 K) was used to speed up the conformational changes taken place in the targeted MD simulation. The conformation of residues 344–349 obtained from the targeted MD was used to complete the mouse CAR-androstenol x-ray structure. The initial coordinates for residues 103–315 of human CAR were taken from the human CAR/RXR{alpha} heterodimer structure [PDB code 1XVP (34)], whereas the residues 316–348 were built using the modeled mouse CAR.

The helices 12 of VDR and mouse and human CAR were repositioned on the basis of the crystal structure of the human PPAR{alpha}-CoR-RID peptide-GW6471 complex [PDB code 1KKQ (37)]. The CoR-RID peptide was build and docked to the LBDs of human VDR, human CAR, and mouse CAR using the coordinates of the CoR-RID peptide of the PPAR{alpha} structure. Finally, ligands were placed to the ligand-binding sites of LBD-CoR-RID peptide complexes. ZK168281 was docked to VDR on the basis of earlier MD simulation results (26) and androstanol on the basis of the mouse CAR-androstenol x-ray structure (36). Clotrimazole was docked to the ligand-binding site of human CAR, respectively, using the GOLD protein-ligand docking program (53). For the energy minimizations and MD simulations, human VDR, human CAR, and mouse CAR complexes were solvated by 11379, 12256, and 11178 TIP3P water molecules in a periodic box of 63 x 69 x 91 Å, 62 x 75 x 89 Å and 62 x 76 x 83 Å, respectively. The water molecules of the complexes were first energy-minimized for 1000 steps, heated to 300 K in 5 psec and equilibrated by 10 psec at a constant temperature of 300 K and pressure of 101,300 Pa. After that, the simulation systems were minimized for 1000 steps, the temperature of the systems was increased to 300 K in 5 psec and equilibrated for 100 psec while keeping the protein backbone atoms (N, C{alpha}, C) restrained by an atom-based harmonic potential of 1 kcal mol–1Å–2. The purpose of these simulation steps was to remove atom-atom clashes and let the protein side chains pack efficiently. After that, the restraints were removed and 150 psec MD simulations were carried out. In the simulations, the electrostatics were treated using the particle-mesh Ewald method. A time step of 1.5 fsec was used, and bonds involving hydrogen atoms were constrained to their equilibrium lengths using the SHAKE algorithm. The simulations were done using the AMBER8.0 simulation package (University of California, San Francisco, CA) and the parm99 parameter set of AMBER. The parameters of the ligands were generated with the Antechamber suite of AMBER8.0 in conjunction with the general amber force field. The atomic point charges of the ligands were calculated with the two-stage RESP (54) fit at the HF/6–31G* level using ligand geometries optimized with the semiempirical PM3 method using the Gaussian03 program (Gaussian Inc., Pittsburgh, PA).


   ACKNOWLEDGMENTS
 
We would like to thank Drs. S. Kliewer (Southwestern Medical School, Dallas, TX) for CAR expression vectors, L. Binderup (LEO Pharma, Ballerup, Denmark) for 1{alpha},25(OH)2D3, A. Steinmeyer (Schering AG, Berlin, Germany) for ZK168281, P. Honkakoski (University of Kuopio) for TCPOBOP and discussions, and T. W. Dunlop for critical reading of the manuscript.


   FOOTNOTES
 
This work was supported by the Academy of Finland (Grants 50319, 50331, and 203926).

First Published Online May 19, 2005

Abbreviations: ANF Atrial natriuretic factor; CAR constitutive androstane receptor; CITCO 6-(4-chlorophenyl)imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime; CoA, coactivator; CoR corepressor; CYP P450 cytochrome mono-oxygenase; DR direct repeat; GST glutathione-S-transferase; LBD ligand binding domain; MD molecular dynamics; NCoR, nuclear receptor corepressor; NR nuclear receptor; 1{alpha},25(OH)2D3; 1{alpha},25-dihydroxyvitamin D3; PBREM phenobarbital-responsive enhancer module; PDB, Protein Data Bank; PPAR peroxisome proliferator-activated receptor; RA retinoic acid; RE response element; RID receptor interaction domain; RMSD root mean square deviation; RXR retinoid X receptor; TCPOBOP 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene; TIF2, transcription intermediary factor 2; VDR 1{alpha},25(OH)2D3 receptor.

Received for publication December 24, 2004. Accepted for publication May 3, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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NURSA Molecule Pages Link:

Nuclear Receptors:   PPARα  |  VDR  |  CAR  |  RXRα
Coregulators:   GRIP1  |  NCOR
Ligands:   Calcitriol  |  CITCO  |  TCPOBOP  |  Androstanol



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J. Y. Kim, Y. L. Son, and Y. C. Lee
Involvement of SMRT Corepressor in Transcriptional Repression by the Vitamin D Receptor
Mol. Endocrinol., February 1, 2009; 23(2): 251 - 264.
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