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Regulation of Ligand-Induced Heterodimerization and Coactivator Interaction by the Activation Function-2 Domain of the Vitamin D Receptor

Department of Medical Specialties The University of Texas M. D. Anderson Cancer Center Houston Texas, 77030

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Twenty-epi analogs of 1,25-dihydroxyvitamin D₃ (1,25D₃) are 100-1000 times more potent transcriptionally than the natural hormone. To determine whether this enhanced activity is mediated through modulation of the dimerization process or through interaction with coactivators, we performed quantitative protein-protein interaction assays with in vitro translated vitamin D receptor (ivtVDR) and fusion proteins containing glutathione-S-transferase (GST) and either the ligand-binding domain of retinoid X receptor (RXR), or the nuclear receptor-interacting domain of the steroid receptor coactivator 1 (SRC-1), or the glucocorticoid receptor-interacting protein 1 (GRIP-1). We found that heterodimerization of the ligand-binding domains of RXR and VDR was primarily deltanoid dependent as was the interaction of VDR with the SRC-1 or with GRIP-1. The ED₅₀ for induction of heterodimerization was 2 nM for 1,25D₃ and 0.05 nM for 20-epi-1,25D₃. However, the ED₅₀ for induction of VDR interaction with SRC-1 was similar for both 1,25D₃ and the 20-epi analog (ED₅₀ = 0.7–1.0 nM) as was the ED₅₀ for ligand-mediated interaction of VDR with GRIP-1 (ED₅₀ = 0.1–0.3 nM). Mutations in heptad 9 diminished both 1,25D₃ and the 20-epi analog-mediated dimerization, without changing binding of these ligands to VDR. Mutations in VDR’s activation function 2 (AF-2) domain/helix 12 residues diminished the ability of 1,25D₃ to induce heterodimerization and interaction with SRC-1. These mutations did not change the ability of 20-epi-1,25D₃ to induce dimerization but did diminish its ability to induce interaction with SRC-1. We hypothesize that both the hormone and the analog stabilize receptor conformations that expose VDR’s functional interfaces. The mechanisms by which the two ligands expose these functional interfaces differ with respect to participation of the AF-2 domain.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The transcriptional activity of the vitamin D receptor (VDR) depends on binding of VDR/retinoid X receptor (RXR) heterodimers (1, 2, 3, 4) to specific DNA sequences called vitamin D-responsive elements (VDREs) (5) and on interaction of the VDR with transcription coactivators and bridging factors of the basal transcriptional machinery (6, 7, 8, 9). Heterodimerization of VDR with RXR, binding of the dimer to DNA, and interaction of VDR with coactivators of transcription are deltanoidmodulated processes. Therefore, these processes may be subjected to differential regulation by the biologically active metabolite of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25D₃), and natural or synthetic deltanoids. For example, many analogs of 1,25D₃ are significantly more potent transcriptionally than the natural hormone (10, 11, 12, 13, 14) but do not bind the VDR with greater affinity. Several mechanisms have been proposed for this enhanced activity: stability of the analogs, their catabolism to potent by products, poor interaction of the analogs with serum-binding protein (15, 16, 17, 18), or direct potentiation of VDR’s transcriptional activation (10, 19, 20). The steps in VDR activation that have been shown to be differently modulated by 1,25D₃ and its analogs include dimerization with RXR and binding to DNA, mode of interaction of the VDR-RXR complex with DNA, and subtle changes in the affinity of VDR for RXR (10, 13, 19). An additional, recently proposed mechanism for differential activation of the VDR by 1,25D₃ and its analogs is differential recruitment of coactivators/bridging factors of transcription (20).

The 20-epi analogs are a group of deltanoids that attracted significant research interest because they have up to a 1,000 times greater transcriptional potency than the natural hormone. The exceptional transcriptional potency of these superagonists has been proposed to be mediated, in part, through differential modulation of the VDR (10, 14, 18, 19, 20). The 20-epi analogs enhance dimerization and DNA binding of VDR (10, 19) and occupy the ligand-binding pocket of VDR without contacting the C-terminal residues that regulate transcription activation function 2 (AF-2; Refs. 14, 21). The mechanism that leads to the enhanced dimerization by the 20-epi analogs and the consequences of the differential interaction with the AF-2 on their transcriptional potency are not known. However, a well established role of the AF-2 domain is to contribute contact sites for coactivator interaction (6, 7, 21). Therefore, two questions are raised with respect to the mechanism of action of 20-epi analogs: does differential interaction of 1,25D₃ and its 20-epi analogs with the AF-2 residues affect the ability of VDR to recruit coactivators, and, alternately, do AF-2 residues have any role in regulating the dimerization ability of VDR?

The dimerization of nuclear receptors, including VDR and RXR, is modulated by regions in their DNA-binding (5) and ligand-binding domains (5, 22, 23, 24). The residues that regulate dimerization in the ligand-binding domain (LBD) of VDR are in conserved regions called heptad 9 [residues 383–390 (23)] and the E1 domain [residues 244–263 (22)], but so far the AF-2 core residues have not been implicated in contributing to the dimerization interface. However, the in vitro systems used to examine DNA-dependent dimerization of full-length VDR with RXR are often independent of ligand binding (25, 26, 27, 28). Because AF-2 functions are ligand dependent, the contributions of the ligand and of the AF-2 to receptor activation, including the dimerization process, may be compromised in vitro and under conditions of excess receptor protein (28). Therefore, to assess the effect of 1,25D₃ and its analog on both heterodimerization and coactivator interaction, we adopted a highly sensitive pull-down assay system to examine the quantitative and qualitative aspects of ligand regulation of heterodimerization and the coactivator-interacting properties of VDR LBD. We show here that 20-epi-1,25D₃ enhanced the dimerization potency of VDR LBD with RXR relative to 1,25D₃, but did not enhance steroid receptor coactivator 1 (SRC-1) or glucocorticoid receptor-interacting protein 1 (GRIP-1) interaction. We also show that heptad 9 and the E1 domain had a similar role in regulating hormone- and analog-mediated dimerization. In contrast, the AF-2 residues preferentially regulated 1,25D₃-induced dimerization with RXR.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Quantification of Ligand-Dependent Interaction of VDR with RXR, SRC-1, and GRIP-1
To study the ligand-dependent interaction of VDR with RXR and with transcriptional coactivators of the p160 family, we used four constructs expressing the following fusion proteins: glutathione-S-transferase (GST)-RXR, which contained the LBD of RXR fused to GST; GST-VDR, which contained the LBD of VDR fused to GST; GST-SRC-1, which contained the SRC-1 peptide [nuclear receptor-interacting motifs 1 and 2, amino acid residues 635–734 (29, 30)]; and GST-GRIP-1, which contained the three nuclear receptor-interacting boxes of GRIP-1 (amino acid residues 636–766) (30). These fusion proteins were used to examine ligand-dependent interaction of the cellular or the in vitro synthesized (ivt) ³⁵S-labeled VDR by affinity binding to glutathione-Sepharose beads. Figure 1A shows that in the absence of 1,25D₃ there was little dimerization of VDR with RXR (3–4% of input receptor), but in the presence of the hormone, significant heterodimerization of either in vitro translated VDR (ivtVDR) or cellular VDR was induced (20- and 17-fold, respectively). The level of dimerization depended on the ligand concentration and appeared to reach a maximum at 10 nM. Only 1.4% of input ivtVDR dimerized with GST-VDR, and this binding neither increased nor decreased in the presence of the hormone. Because this result is in conflict with data reported by other laboratories (31, 32, 33), we tested whether the bacterially expressed GST-VDR was functional and could dimerize in a 1,25D₃-dependent manner with synthetic RXR. Figure 1A shows that ³⁵S-labeled ivtRXR dimerized with GST-VDR in a ligand-dependent manner. Therefore, the bacterially expressed GST-VDR had both significant 1,25D₃ binding activity and a functional dimerization interface in its LBD. From these experiments, we concluded that both cellular and synthetic VDR dimerize effectively with RXR without the DNA-binding domain. This heterodimerization is ligand dependent and is preferable to homodimerization. These assays exclusively tested VDR-RXR interaction through their LBDs, so they did not exclude the possibility that full-length VDR, containing the dimerization interfaces from both the DNA-binding and the ligand-binding domains, forms homodimers in the presence of DNA.

View larger version (47K): [in this window] [in a new window]   Figure 1. Quantification of Ligand-Dependent VDR-RXR and VDR-Coactivator Interactions A, Ligand-dependent heterodimerization was examined by incubating recombinant VDR expressed in COS-1 cells (cellular VDR) or 35S-labeled ivtVDR with bacterially expressed GST-RXR (5–8 µg), and the indicated concentrations of 1,25D3 (upper two panels). As a control, GST-VDR was examined for its ability to dimerize in a ligand-dependent manner with 35S-labeled ivtRXR and with 35S-labeled ivtVDR (lower two panels). The dimers were captured with glutathione-Sepharose beads, eluted, and analyzed by SDS-PAGE and autoradiography (for ivtVDR) or by Western blot analysis (for cellular VDR). NS, Nonspecific binding to the glutathione-Sepharose beads in the presence of GST. Input, is the amount of cellular or synthetic receptor used per incubation; NL, no ligand. B, Ligand-dependent interaction of p160 coactivators with VDR was examined by incubating 35S-labeled ivtSRC-1 with either GST or GST-VDR LBD fusion protein (left panel) or by incubating 35S-ivtVDR with either GST or GST-SRC-1 (middle panel) or with GST-GRIP-1 (right panel), in the presence or absence of 10 nM ligand. The VDR-coactivator complexes were captured with glutathione-Sepharose beads, eluted, and analyzed as described above.

Effect of Side-Chain Stereochemistry on Ligand-Dependent Heterodimerization and Coactivator Interaction Potencies of VDR
We, and others, have shown that the analog 20epi-1,25D₃ is 100 times more potent transcriptionally than 1,25D₃. This enhanced transcriptional activity is associated with the ability of the analog to induce binding of VDR to DNA even at very low concentrations (0.01 nM) (10, 14), suggesting that the enhanced transcriptional activity and DNA binding activity of VDR-20-epi analog complexes are due to greater dimerization with RXR (14, 19). Other studies from our laboratory have also shown that 20-epi-1,25D₃ and other 20-epi analogs interact with the VDR in a manner that does not require the use of the C-terminal residues of the AF-2 domain, whereas the natural hormone does use these residues (14). That these residues are essential for coactivator interaction raises the possibility that the hormone and the 20-epi analogs may also have different potencies or efficacies to induce coactivator interaction. The assay systems described here allowed us to test these two aspects of VDR activation in vitro and to determine whether they are facilitated by the analog. To that end, we performed protein-protein interaction experiments with VDR-ligand complexes and either GST-RXR or GST-SRC-1, or GST-GRIP-1 using ivtVDR and increasing concentrations of the ligands (Fig. 2). The dose-response plots for 20-epi-1,25D₃-mediated transcription (Fig. 2A), dimerization (Fig. 2B), and coactivator interaction (Fig. 2, C and D) were aligned with the dose-response plots for the corresponding 1,25D₃-mediated activities. These experiments showed that the potency of the 20-epi analog to induce transcription (ED₅₀ = 0.01 nM) was 200-fold greater than that of 1,25D₃ (ED₅₀ = 2 nM). Likewise, the 20-epi analog-dependent heterodimerization potency (ED₅₀ = 0.05 nM) was significantly greater than that of 1,25D₃ to promote this activity (ED₅₀ = 2 nM). In contrast, the potencies of the two compounds to promote interaction with SRC-1 (ED₅₀ = 0.7–1.0 nM) or with GRIP-1 (ED₅₀ = 0.1–0.3 nM) were similar. In experiments not shown here we compared the potencies of 1,25D₃ and its 20-epi analog to induce interaction of full-length SRC-1 with GST-VDR and found them also similar. Therefore, we conclude that the greater transcriptional potency of the analog may be attributed primarily to its ability to enhance the dimerization potency of VDR.

View larger version (33K): [in this window] [in a new window]   Figure 2. Comparison of Transcription, Dimerization, and Coactivator Interaction Potencies of VDR -1,25D3 and VDR- 20-epi Analog Transcription (A) was measured by transfecting CV1 cells with hVDR expression plasmid and the osteocalcin VDRE-thymidine kinase/GH fusion gene. The transfected cells were incubated with the indicated concentrations of hormone or analog, and reporter gene expression (GH) was measured 24 h later. Dimerization (B) or coactivator interaction (C and D) was measured by incubating 35S-labeled ivtVDR with the indicated concentrations of ligand for 10 min at room temperature and then adding GST-RXR or GST-SRC-1 or GST-GRIP-1 (5–8 µg) and 20 µl of glutathione-Sepharose beads. The complexes were extracted from the affinity beads with SDS buffer, detected by SDS-PAGE and autoradiography, and quantified by densitometric scanning. The results are expressed as percentage of maximal arbitrary densitometric units induced by 35S-labeled ivtVDR and are representative of seven to nine experiments. The ED50 for 1,25D3 to induce transcription in CV-1 cells was 2.0 ± 0.4 nM; The ED50 for 1,25D3 to induce dimerization of the ivtVDR was 1.8 ± 0.6 nM, to induce interaction with GST-SRC-1 was 0.7 ± 0.2, and to induce interaction with GRIP-1 was 0.5 ± 0.1 nM. The ED50s (mean ± SE) for the 20-epi analog to induce transcription, dimerization, and interaction with SRC-1 or GRIP-1 were 0.01 ± 0.003 nM, 0.05 ± 0.02 nM, 1.0 ± 0.15 nM, and 0.25 ± 0.03 nM, respectively.

To examine these possibilities, we used a quantitative protease-sensitivity assay to assess directly the interaction of the analog with ivtVDR monomer and with heterodimerized ivtVDR. This assay has been used by us and others to examine the induction of qualitative and quantitative changes in VDR conformation by various ligands (10, 34, 35). Here, we used the autoradiographic intensity of ³⁵S-labeled trypsin-resistant fragments induced by 1,25D₃ and 20-epi-1,25D₃ to examine three parameters of ligand-receptor interaction: whether VDR-ligand conformation is changed by the dimerization, whether the ligand concentrations required to occupy VDR-binding sites are the same as those required to induce conformational changes in VDR, and whether the same ligand concentration induces conformational changes in VDR monomers and heterodimers.

Figure 3A shows that 1,25D₃ induced trypsin-resistant fragments of 34 and 28 kDa in the VDR monomer. The 34-kDa fragment was first detected at 0.1 nM and reached maximal intensity at 10 nM. The 28-kDa fragment was faint, and its intensity did not reach maximum even at 1 µM (not shown). Small changes occurred in the heterodimerized VDR-1,25D₃ complexes: the trypsin-resistant 34-kDa fragment was still first detected at a concentration of 0.1 nM (Fig. 3B), but the 28-kDa trypsin-resistant fragment was not induced. These experiments suggest that the conformation of VDR-1,25D₃ complexes changes slightly with dimerization. However, the potency of 1,25D₃ to induce conformational changes in VDR monomers and heterodimers remained the same and was well correlated with the affinity of 1,25D₃ for VDR.

View larger version (24K): [in this window] [in a new window]   Figure 3. A Comparison of Conformational Changes in VDR-Ligand Monomers and Heterodimers A and C, 35S-labeled ivtVDR was incubated with the indicated concentrations of 1,25D3 or 20-epi 1,25D3 for 10 min at room temperature and then for 1 h at 4 C and then subjected to trypsin digestion for 10 min; B and D, 35S-labeled ivtVDR was incubated with 1,25D3 or with 20-epi 1,25D3 for 10 min at room temperature and then with GST-RXR and glutathione-Sepharose resins for 1 h at 4 C. The VDR-RXR heterodimers were purified by washing the affinity resins three times and then subjected to trypsin digestion for 10 min. All proteolytic products were separated by SDS-PAGE and visualized by autoradiography of the dried gels. The position of the trypsin-resistant fragments and the undigested ivtVDR are indicated by arrows. NL, No ligand added.

When this analysis was repeated with the heterodimerized VDR-analog complexes (Fig. 3D), we found several interesting differences. First, the conformation of the heterodimerized VDR-analog complexes was significantly different from that of the monomer. In the dimerized VDR-analog complexes, the 32- and the 34-kDa fragments had identical intensities at ligand concentration higher or equal to 0.1 nM, but the intensity of the 28-kDa fragment was diminished. Furthermore, the 34-kDa fragment was now detectable even at a concentration of 0.01 nM, and the 32-kDa fragment was detectable at a concentration of 0.1 nM. These results show that dimerization increased the potency of the analog to stabilize VDR’s conformation by more than 10-fold, suggesting that the affinity of the 20-epi analog for the dimerized VDR had changed. Furthermore, as significant levels of heterodimerized VDR-analog complexes were detected by this assay even at 0.01 nM, it also explains how these low analog concentrations can induce significant binding of VDR to DNA (10) and significant transcriptional activity.

Different Roles for the VDR’s AF-2 Domain in Dimerization Induced by 1,25D₃ and Its 20-epi Analog
To determine which residues contributed to the differential dimerization potencies of 1,25D₃ and its 20-epi analog, we considered the possibility that the two ligands shape differently the functional surface of the VDR’s LBD. These differences may be reflected by changes in the amino acids required to form contact of VDR with RXR. To examine this possibility we substituted residues 383 and 385 in heptad 9 of hVDR, a region that regulates heterodimerization of VDR with RXR (23). Figure 4 shows these mutations diminished 1,25D₃-mediated (Fig. 4A) and 20-epi-1,25D₃-mediated dimerization (Fig. 4B) to the same extent without changing ligand binding affinities (data not shown and Ref. 23). These results confirm previous studies by Nakajima et al. (23), showing that these residues were essential for DNA-dependent heterodimerization and transcriptional activity of VDR. These results also suggest that the 20-epi analog does not change the requirement for heptad 9 residues, although additional mutations in this area are necessary to substantiate these observations.

View larger version (24K): [in this window] [in a new window]   Figure 4. Regulation of Ligand-Induced Dimerization by Heptad 9 and by the AF-2 Domain A, Dimerization assays were performed with 35S-labeled ivtVDR and GST-RXR as described in Materials and Methods by using either WT VDR or VDR mutated at AF-2 residues 419 (L419S), 420 (E420A), or 421 and 422 (V421M/F422A), or the heptad 9 mutant M383G/Q385A and the indicated concentrations of 1,25D3. Shown are representative plots from three to seven dimerization experiments. The ED50 values (mean ± SE) for induction of dimerization were 1.8 ± 0.6 nM (WT VDR), 110 ± 10 nM (V421M/F422A), 80 ± 7 nM (L419S), and 1.2 ± 0.15 nM (E420A). ED50 was not reached with the mutant M383G/Q385A. B, 20-epi-1,25D3-dependent dimerization assays were performed with 35S-labeled ivtVDR and GST-RXR as described above by using either WT VDR or the four mutant VDR constructs. Shown are representative plots from five to seven dimerization experiments. The ED50 (mean ± SE) for induction of dimerization were 0.05 ± 0.02 nM (WT VDR), 0.10 ± 0.02 nM (V421M/F422A), 0.2 ± 0.015 nM (L419S), and 0.15 ± 0.01 nM (E420A). ED50 was not reached with the mutant M383G/Q385A.

We then considered the possibility that the effect of the 20-epi analog on dimerization potency is indirect. Instead of changing the shape of the dimerization interface the analog may have a greater potency to expose it. X-ray crystallography of LBDs of nuclear receptors show the AF-2 residues provide contact points for interaction with coactivators but are also engaged in forming intramolecular interactions that expose or mask the coactivator binding site (29, 36, 37). Therefore, we hypothesized that the AF-2 domain may also contribute to forming or exposing the dimerization interface. For that, we compared the ligand concentrations required to achieve maximal 1,25D₃-dependent dimerization of the wild-type (WT) VDR and VDR mutated at residues 419 (L419S), 420 (E420A), and 421 and 422 (V421M/F422A). These four residues have been shown to be AF-2 residues because their substitution abolished the transcriptional activity of VDR (14, 35). Figure 4A shows that 1,25D₃-dependent dimerization of WT VDR with GST-RXR became detectable at 0.1 nM and reached saturation at 100 nM. The average ED₅₀ for this dose response was 1.8 nM. A similar dimerization potency was induced by 1,25D₃ by using the AF-2 mutant E420A. On the other hand, the 1,25D₃-dependent dimerization of the AF-2 mutants L419S and V421M/F422A became detectable only at 10 nM, and reached maximal level at 1,000 nM. These results clearly indicate that these mutant receptors had a reduced 1,25D₃-dependent dimerization potency. This decrease in sensitivity to the ligand cannot be due to reduced affinity of 1,25D₃ because Scatchard analysis with cellular VDR (14) and competition assays using the human ivtVDR (data not shown) showed only a 3-fold to 5-fold decrease in the affinity of these mutants for the hormone. Furthermore, the synthetic AF-2 mutants E420A and V421M/F422A had similar affinities for the hormone (data not shown), but the former did not lose 1,25D₃-induced dimerization potency, whereas the latter did (Fig. 4A). We next compared the ability of 20-epi-1,25D₃ to induce dimerization through WT and AF-2-mutated VDRs. Figure 4B shows that the AF-2 mutations had little or no effect on dimerization potency of the 20-epi analog: the ED₅₀ values for dimerization of WT VDR and the AF-2 mutants were 0.05–0.2 nM. None of these mutants had a reduced affinity for the 20-epi analog (Ref. 14 and data not shown). We therefore conclude that differential abilities of the AF-2 mutants to dimerize in response to 1,25D₃ or its 20-epi analog does not correlate with the ligand binding activities of these mutants.

To determine whether the changes in dimerization potencies of AF-2-mutated or heptad 9-mutated VDR were also reflected in ligand-dependent binding of VDR to DNA, we performed electrophoretic mobility shift assays with VDR expressed by recombinant expression vectors in COS-1 cells, and the nuclear extracts from the transfected cells were treated in vitro with the ligands. Figure 5A shows a prominent induction of DNA binding activity by 10 nM of either 1,25D₃ or the 20-epi analog, using WT VDR. However, 1,25D₃-induced DNA binding activity of the AF-2 mutant V421M/F422A was diminished, whereas the 20-epi analog-mediated DNA binding activity of this mutant was similar to that of WT VDR. Both 1,25D₃- and 20-epi analog-induced DNA binding activities were diminished with the double substitution of the heptad 9 residues M383G/Q385A. Differences in DNA binding activities of the three receptor preparations were not due to differences in the amount of immunoreactive VDR (Fig. 5B).

View larger version (45K): [in this window] [in a new window]   Figure 5. Regulation of DNA Binding Activity by AF-2- and Heptad 9 Residues A, Electrophoretic mobility shift assays were performed with the 32P-labeled osteopontin VDRE and nuclear extracts from COS-1 cells containing WT VDR, or the AF-2-mutant V421/M/F422A, or the heptad 9 mutant M383G/Q385A. Nuclear extracts were incubated without or with 10 nM of 1,25D3 or 20-epi-1,25D3 during the DNA binding reaction. B, Immunoblots of input VDR (3 µl of nuclear extract from COS-1 cells expressing recombinant VDR). VDR was detected by Western blot using anti-VDR antibodies 9A7 (Affinity BioReagents, Inc.). Lane 1, WT VDR; lane 2, V421/M/F422A; lane 3, M383G/Q385A.

View larger version (29K): [in this window] [in a new window]   Figure 6. AF2-Mediated Regulation of Transcription and SRC-1 Interaction A–C, Transcriptional activity was assessed by cotransfecting CV1 cells with the indicated expression plasmids bearing hVDR and the osteocalcin VDRE-thymidine kinase/GH fusion gene. The transfected cells were incubated with the hormone or analog, and reporter gene expression (GH) was measured 24 h later. WT VDR was included in each transfection experiment, and the amount of reporter gene expression induced by 100 nM 1,25D3 was considered maximal transcriptional activity. The mean ED50 values (n = 2) for induction of transcription by 1,25D3 were 1.5 nM for WT VDR, 250 nM for L419S, and 60 nM for E420A. The ED50 values for induction of transcription by 20-epi-1,25D3 were 0.01 nM for WT VDR, 0.4 nM for L419S, and 0.1 nM for E420A. Transcriptional activity of V421M/F422A was undetectable even at 1 µM 1,25D3 or the 20-epi analog. The maximal induction of transcription with 1,25D3 or the 20-epi analog by the WT VDR was 15- to 20-fold. Maximal induction of transcription by the mutants E420A and L419S was 2- to 3-fold and 3- to 4-fold, respectively. D, SRC-1 interaction was assessed by incubating WT or AF-2 mutated 35S-labeled ivtVDR with 10 nM 1,25D3 or 20-epi-1,25D3 and 5–8 µg of GST-SRC-1, as described above. The SRC-VDR complexes were captured by glutathione-Sepharose and visualized after SDS-PAGE and autoradiography.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

That the increase in potency to induce conformational change correlated with the greater potency of the VDR-20-epi analog complexes to dimerize suggests that the 20-epi analog may expose VDR’s dimerization interface more effectively than 1,25D₃. However, because the 20-epi analog also induced qualitative changes in VDR’s conformation, we had to consider the possibility that it modified the functional surface of VDR to change the profile of interacting proteins or the nature of their binding sites. Our preliminary results, using site-directed mutagenesis at a region that is most likely to provide contact sites for RXR in the VDR’s LBD (heptad 9) (23, 28), strongly suggest that the contact of RXR with VDR through these residues is not significantly altered by the 20-epi analog. Likewise, results from the site-directed mutagenesis of AF-2 residues that directly provide contact sites for coactivators of the p160 family (29, 37) also suggest that the analog does not significantly change the binding site for these coactivators. However, it still remains to be determined whether the nature of the dimerization interface exposed by each of these ligands is different with respect to recruitment of specific dimerization partners. It also remains to be determined whether the 20-epi analog modifies differently the interaction site for bridging factors or components of the basal transcriptional machinery that interact with the VDR through amino acid residues outside the AF-2 core. For example, a study by Yang and Freedman (20) confirmed our findings that 1,25D₃ and 20-epi-1,25D₃ have similar abilities to induce interaction of VDR with SRC-1, but also suggested that the 20-epi analogs enhanced the interaction of a factor called DRIP 205 with VDR’s LBD. Although the binding of this factor to VDR is also ligand dependent and AF-2 dependent, its nuclear receptor-interacting domain is significantly different from that of the p160 family members (39). A detailed mapping of its binding site to VDR-1,25D₃ complexes and VDR-20-epi analog complexes may reveal whether DRIP 205 binding site in VDR is changed by the 20-epi analog. Another candidate for such investigations could be NCoA-62, a coactivator that interacts with the VDR in a ligand-modulated but AF-2-independent fashion (40).

Our experiments showed for the first time that the AF-2 domain was an important regulator of 1,25D₃-mediated heterodimerization of VDR. Mutating this domain reduced the ability of the hormone to induce protease-resistant conformation (data not shown and Ref. 14) and DNA-independent heterodimerization 100-fold. These mutations also diminished 1,25D₃-mediated binding of VDR to DNA. These results suggest that the ligand-induced conformational changes (which for 1,25D₃ are regulated by the AF-2 domain) determine the VDR’s ability to form contact with the LBD of RXR and eventually to bind DNA. On the other hand, other residues probably determine the 20-epi analog’s ability to stabilize VDR conformation and to induce heterodimerization. That the AF-2 residues are differently used by 1,25D₃ and 20-epi analogs for stabilization of VDR conformation and for regulation of heterodimerization may be explained by a recent publication, describing the fine details of 1,25D₃-occupied VDR’s LBD by x-ray crystallography (41). That study revealed that the position of the AF-2 domain is determined by intramolecular interactions between the AF-2 core residues and other amino acid residues in the LBD. It also showed that the AF-2 residues V418 and F422 and the LBD residues V234, I268, H397, and T401 have a double role in VDR-1,25D₃ complexes: they provide contact points for the hormone and form the intramolecular interactions that determine the position of the AF-2 core. Therefore, it is likely that ligand contact with these residues directly modulates the intramolecular interactions that stabilize VDR’s conformation and the position of the AF-2. We have already shown that the AF-2 core does not provide contact for 20-epi analogs (14). Furthermore, in recent studies (data not shown) we have also confirmed that the residue I268 was essential for 1,25D₃ binding but not necessary for 20-epi analog binding. Taken together, these results explain how the AF-2 domain is used by 1,25D₃ to stabilize the VDR’s conformation, and why it is not used by the 20-epi analog for this function. These results also support the hypothesis that the AF-2 is not positioned in the same way in the two receptor-ligand complexes, and therefore, may regulate differently the availability of the functional interfaces in the VDR-hormone and VDR-analog complexes. That the 20-epi analog does not form contact with residues that are used for intramolecular interactions may contribute to its apparent flexibility in the ligand binding pocket when VDR dimerizes with RXR.

In conclusion, we interpret these findings with respect to the mechanism of ligand-mediated transcriptional activities of VDR to mean that the stability of the heterodimerized and DNA-bound VDR-1,25D₃ complexes may be susceptible to AF-2-mediated intermolecular and intramolecular interactions. For example, because the AF-2 regulates both 1,25D₃ binding to VDR and 1,25D₃-mediated dimerization, any interaction of the AF-2 with additional factors in the transcription apparatus (SRC-1, GRIP-1, etc.) may stabilize or destabilize 1,25D₃-VDR complexes and therefore immediately affect VDR’s DNA binding activity and transcriptional potencies. On the other hand, in the VDR-20-epi-1,25D₃ complex, ligand binding is stabilized by dimerization, is not dependent on AF-2 residues, and therefore is probably not further affected by AF-2 interactions with transcription coactivators. Overall, these experiments confirmed our initial hypothesis that the incredible potency of the 20-epi analogs is due primarily to their ability to form a stable complex with the VDR monomer (14) and an even more stable complex with the heterodimer, thereby producing a long-lasting transcriptionally active VDR complex. That VDR heterodimers are stabilized by 20-epi analogs suggest that some of these compounds may be useful for treatment of conditions whereby VDR action is compromised by defective dimerization, such as hereditary vitamin D-resistant rickets (HVDRR) (42).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Reagents
Anti-VDR antibody 9A7 was obtained from Affinity BioReagents, Inc. (Golden, CO) [-³²P]dATP was obtained from ICN Biochemicals, Inc. (Irvine, CA) and [³⁵S]methionine from Amersham Pharmacia Biotech (Arlington Heights, IL). A coupled transcription/translation kit was obtained from Promega Corp. (Madison, WI). The GST purification system was purchased from Pharmacia Biotech (Piscataway, NJ). 1,25D₃ and its analog 20-epi-1,25-dihydroxyvitamin D₃ (MC 1288) were a generous gift from Dr. L. Binderup (Leo Pharmaceuticals, Bellerup, Denmark).

Plasmid Construction
The GST-RXR-expressing plasmid was prepared by ligating an NcoI-EcoRI fragment encoding the LBD of the human RXR into the SmaI-EcoRI site of pGEX2T (Pharmacia Biotech). The GST-VDR-expressing plasmid was prepared by insertion of a BamHI linker into the StuI site of hVDR cDNA cloned into pGEM4, digestion with BamHI, isolation of a 1.1-kb fragment encoding the hVDR LBD, and ligating this fragment in the sense orientation into the BamHI site of pGEX2T. GST-SRC-1 expression plasmid was prepared by PCR amplification of the nucleic acid sequence encoding the NR boxes 1 and 2 (amino acids 635–734) (30) and subcloning of the amplified DNA into BamHI and EcoRI sites of pGEX2T. The GST-GRIP-1 plasmid was prepared by RT-PCR amplification of cDNA encoding amino acid residues 636–766 (30) and subcloning the amplified DNA into pGX2T. The preparation of AF-2 mutant VDR plasmids by site-directed mutagenesis has been described previously (14, 35).

Purification of GST Fusion Proteins
Overnight cultures of the Escherichia coli strain BL21 (DE3)pLysS carrying the expression plasmids for GST, GST-VDR, GST-RXR, GST-SRC, and GST-GRIP-1 were inoculated into 250 ml of LB and grown for 3 h at 37 C. The cells were treated for 3 h with 0.1 mM isopropyl-1-thio-ß-D-galactoside, harvested by centrifugation, and resuspended in 10 ml of PBS containing 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride (PBSDP buffer). The cells were frozen and thawed once and sonicated three times for 15 sec each time, and NP-40 was added at a final concentration of 0.5%. The cell homogenate was then centrifuged for 10 min at 10,000 x g, and the supernatant was incubated with 0.7 ml of a 50% slurry of glutathione-Sepharose beads (Pharmacia Biotech) for 1 h at 4 C. The beads were then washed three times in 5 ml of PBSDP, and the GST fusion protein was eluted by incubating the beads for 15 min with 1 ml of 5 mM reduced glutathione at room temperature. The eluate was dialyzed overnight against 800 ml of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10% glycerol, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol. The purity and yield of the eluted protein were assessed by SDS-PAGE and Coomassie blue staining.

Expression of hVDR in Mammalian Cells and Assessment of Transcriptional Activity
For preparation of cellular VDR, the African green monkey kidney cell line COS-1 was maintained in DMEM supplemented with 10% FBS. Forty-eight hours before transfection, the cells were plated in 150-mm dishes at a density of 6 x 10⁵/dish in DMEM and 10% FBS. Then the cells were transfected by the diethylaminoethyl (DEAE) dextran method (43) with 20 µg/dish of recombinant hVDR plasmid and treated for 1 min with 10% dimethyl sulfoxide. Nuclear extracts (for dimerization and DNA binding assays) were prepared 2 days after transfection, as described below.

For assessment of the transcriptional activity of WT and mutant VDR, the African kidney monkey cell line CV-1 was used. These cells were plated at a density of 10⁵/ml in 35-mm dishes in DMEM and 10% FBS, 48 h before transfection. The transfection was performed by the DEAE dextran method with 2 µg/dish of recombinant VDR plasmid and 2 µg/dish of reporter gene containing the osteocalcin VDRE attached to the thymidine kinase promoter/GH fusion gene (10). Twenty-four hours after transfection, the cells were incubated with 1,25D₃ or 20-epi-1,25D₃ for 24 h, and then the culture medium was collected for measurement of reporter gene expression (GH production) by a RIA.

Dimerization and Coactivator Interaction Assays
Each binding reaction contained 11 µl of PBSDP buffer, 3 µl of ³⁵S-labeled ivtVDR or hVDR-containing nuclear extract (0.2–0.4 ng VDR), 1 µl of ethanol without or with various concentrations of ligand, 3–5 µl of purified GST fusion protein, and 20 µl of glutathione-Sepharose beads (equilibrated in PBSDP buffer), and the volume was brought up to 100 µl with NETND buffer [100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 7.8), 0.2% NP-40, and 1 mM dithiothreitol]. This mixture was incubated at room temperature for 10 min and then for 4 h at 4 C, and then the beads were washed once in 0.2 ml of NETND and twice in 0.2 ml of PBSDP. The bound proteins were eluted from the packed beads by boiling in Laemmli buffer for 3 min and were analyzed by SDS-PAGE. The dimerized VDR was detected by Western blotting (in the case of cellular VDR) or by autoradiography of the dried gels (in the case of ³⁵S-labeled ivtVDR).

Preparation of ivtVDR and Assessment of Ligand-Induced Resistance to Trypsin
ivtVDR, unlabeled or labeled with [³⁵S]methionine (1,000 Ci/mmol), was prepared by in vitro coupled transcription/translation in reticulocyte lysates (Promega Corp.) with the hVDR cDNA in pGEM4. The receptor (0.2–0.4 ng) was incubated with various concentrations of 1,25D₃ or analog for 10 min at room temperature and then on ice for 3–4 h. Then, 100 µg/ml trypsin (Sigma, St. Louis, MO) was added, and the mixture was incubated for 10 min. The digestion products were analyzed by 12% SDS-PAGE and autoradiography of the dried gels.

Preparation of Nuclear Extracts
To test the ligand-induced DNA binding and dimerization activity of VDR, nuclear extracts were prepared from COS-1 cells 48 h after transfection with the hVDR plasmids. The transfected cells were scraped from individual dishes into PBS, washed twice in that buffer, and resuspended in 1 ml of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM dithiothreitol] and incubated on ice for 30 min. After they had swollen, the cells were homogenized with 5–10 strokes of a Dounce homogenizer and centrifuged for 30 sec at 14,000 x g. The supernatants were then discarded, and the nuclear pellets were resuspended in 50 µl of buffer C [20 mM HEPES (pH 7.9), 400 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 25% glycerol, and 0.5 mM dithiothreitol], incubated on ice for 30 min, homogenized again, and centrifuged for 30 sec at 14,000 x g. The nuclear extracts were then collected, frozen in dry ice immediately, and stored at -80 C for further analysis.

Electrophoretic Mobility Shift Assays
A HindIII fragment containing the mouse osteopontin VDRE (7) was labeled with ³²P to a specific activity of 1–5 x 10⁸ cpm/µg. Each binding reaction contained 50 mM KCl, 12 mM HEPES-NaOH (pH 7.9), 1 mM EDTA, 1 mM dithiothreitol, 12% (vol/vol) glycerol, 0.5 ng of the DNA probe, 10 µg of total nuclear proteins containing 0.2–0.4 ng VDR, and 1 µg of poly(dI-dC) as nonspecific competitor DNA. The binding reactions were performed at room temperature for 30 min in the absence or presence of 10 nM of ligand. The complexes were resolved by electrophoresis through 4% polyacrylamide gels at 4 C, and VDR-DNA complexes were detected by autoradiography.

	ACKNOWLEDGMENTS

 
We thank Dr. L. Binderup (Leo Pharmaceuticals) for the generous gift 1,25D₃ and the 20-epi analog MC 1288, Dr. J. W. Pike for the human vitamin D receptor expression plasmid, Dr. R. M. Evans, Dr. D. Mangelsdorf, and the Salk Institute for the RXR- expression plasmid, Dr. M.-J. Tsai for the generous gift of the SRC-1 expression plasmid, and Dr. M. Goode for her helpful comments on this manuscript.

	FOOTNOTES

 
Address requests for reprints to: Sara Peleg, Section of Endocrinology, Box 15, Department of Medical Specialties, 1515 Holcombe Boulevard, Houston, Texas 77030. E-mail: speleg{at}mail.mdanderson.org

This study was supported by NIH Grant DK-50583 to S.P.

Received for publication October 22, 1999. Revision received August 10, 2000. Accepted for publication August 14, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Liao J, Ozono K, Sone T, McDonnell DP, Pike JW 1990 Vitamin D receptor interaction with specific DNA requires a nuclear protein and 1,25-dihydroxyvitamin D₃. Proc Natl Acad Sci USA 87:9751–9755[Abstract/Free Full Text]
2. MacDonald PN, Haussler CA, Terpening CM, Galligan MA, Reeder MC, Whitfield GK, Haussler MR 1991 Baculovirus-mediated expression of the human vitamin D receptor: functional characterization, vitamin D response element interactions and evidence for a receptor auxiliary factor. J Biol Chem 266:18808–18813[Abstract/Free Full Text]
3. Yu V, Delsert C, Anderson B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin JM, Glass CK, Rosenfeld MG 1991 RXRß: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251–1266[CrossRef][Medline]
4. Kliewer SA, Umensono K, Mangelsdorf DJ, Evans RM 1992 Retinoid x receptor interacts with nuclear receptors in retinoic acid, thyroid, and vitamin D signaling. Nature 355:446–449[CrossRef][Medline]
5. Freedman LP, Lemon BD 1997 Structural and functional determinants of DNA binding and dimerization by the vitamin D receptor. In: Feldman D, Glorieux FH, Pike JW (eds) Vitamin D. Academic Press, San Diego, CA, pp 127–148
6. Gill RK, Atkins LM, Hollis BW, Bell NH 1998 Mapping the domains of the interaction of the vitamin D receptor and steroid receptor coactivator-1. Mol Endocrinol 12:57–65[Abstract/Free Full Text]
7. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
8. Baudino TA, Kraichely DM, Jefcoat Jr SC, Winchester SK, Partridge NC, MacDonald PN 1998 Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription. J Biol Chem 273:16434–16441[Abstract/Free Full Text]
9. Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM, Erdjument-Bromage H, Tempst, P, Freedman LP 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828[CrossRef][Medline]
10. Peleg S, Sastry M, Collins ED, Bishop JE, Norman AW 1995 Distinct conformational changes induced by 20-epi analogues of 1,25-dihydroxyvitamin D₃ are associated with enhanced activation of the vitamin D receptor. J Biol Chem 270:10551–10558[Abstract/Free Full Text]
11. Ferrara J, McCuaing K, Hendy GN, Uskokovic M, White JH 1994 Highly potent transcriptional activation by 16-ene derivatives of 1,25-dihydroxyvitamin D₃. J Biol Chem 269:2971–2981[Abstract/Free Full Text]
12. Sasaki H, Harada H, Hand Y, Morino H, Suzawa M, Shimpo E, Katsumata T, Masuhiso Y, Matsuda K, Ebihara K, Ono T, Masushige S, Kato S 1995 Transcriptional activity of a fluorinated vitamin D analog on VDR-RXR-mediated gene expression. Biochemistry 34:370–377[CrossRef][Medline]
13. Cheskis B, Lemon BD, Uskokovic M, Lemedico PT, Freedman LP 1995 Vitamin D₃-retinoid x receptor dimerization, DNA binding and transactivation are differently affected by analogs of 1,25-dihydroxyvitamin D₃. Mol Endocinol 9:1814–1824[Abstract]
14. Liu Y-Y, Collins ED, Norman AW, Peleg S 1997 Differential interaction of 1,25-dihydroxyvitamin D₃ analogues and their 20-epi homologues with the vitamin D receptor. J Biol Chem 272:3336–3345[Abstract/Free Full Text]
15. Dilworth FJ, Calverley MJ, Makin HLJ, Jones G 1994 Increased biological activity of 20-epi-1,25-dihydroxyvitamin D₃ is due to reduced catabolism and altered protein binding. Biochem Pharmacol 47:987–993[CrossRef][Medline]
16. Kissmeyer AM, Mathiasen IS, Latini S, Binderup L 1995 Pharmacokinetics studies of vitamin D analogues: relationship to vitamin D binding protein (DBP). Endocrine 3:263–266
17. Siu-Caldera ML, Clark JW, Santos-Moore A, Peleg S, Liu Y-Y, Uskokovic MR, Sharma S Reddy GS 1996 1,25-dihydroxy-24-oxo-16-ene vitamin D₃, a metabolite of a synthetic vitamin D₃ analog, 1,25-dihydroxy-16-ene vitamin D₃, is equipotent to its parent in modulating growth and differentiation of human leukemic cells. J Steroid Biochem Mol Biol 59:405–412[CrossRef][Medline]
18. Honda A, Nakashima N, Mori Y, Katsumata T, Ishizuka S 1992 Effects of vitamin D binding proteins on HL-60 cell differentiation induced by 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D₃. J Steroid Biochem Mol Biol 41:109–112[CrossRef][Medline]
19. Zhao XY, Eccleshall TR, Krishnana AV, Gross C, Feldman D 1997 Analysis of vitamin D analog-induced heterodimerization of vitamin D receptor with retinoid X receptor using the yeast two-hybrid system. Mol Endocrinol 11:366–378[Abstract/Free Full Text]
20. Yang W, Freedman LP 1999 20-epi analogues of 1,25-dihydroxyvitamin D₃ are highly potent inducers of DRIP coactivator complex binding to the vitamin D receptor. J Biol Chem 274:16838–16845[Abstract/Free Full Text]
21. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 11:1025–1033[Medline]
22. Rosen ED, Beninghof EG, Koenig RJ 1993 Dimerization interfaces of thyroid hormone, retinoic acid, vitamin D and retinoid X receptors. J Biol Chem 268:11534–11541[Abstract/Free Full Text]
23. Nakajima S, Hsieh J-C, MacDonald PN, Galligan MA, Haussler CA, Whitfield GK, Haussler MR 1994 The C-terminal region of the vitamin D receptor is essential to form a complex with a receptor auxiliary factor required for high affinity binding to the vitamin D responsive element. Mol Endocrinol 8:159–172[Abstract]
24. Nishikawa J, Kitaura M, Imagawa M, Nishikara T 1995 Vitamin D receptor contains multiple dimerization interfaces that are functionally different. Nucleic Acids Res 23:606–611[Abstract/Free Full Text]
25. Ross TK, Darwish HM, Moss VE, DeLuca HF 1993 Vitamin D-influenced gene expression via a ligand-independent receptor-DNA complex intermediate. Proc Natl Acad Sci USA 90:9257–9260[Abstract/Free Full Text]
26. Nishikawa J, Kitaura M, Matsumoto M, Imagawa M, Nishikara T 1994 Difference and similarity of DNA sequence recognized by VDR homodimer and VDR/RXR heterodimer. Nucleic Acids Res 22:2902–2907[Abstract/Free Full Text]
27. Kephart DD, Walfish PG, DeLuca H, Butt TR 1996 Retinoid x receptor isotype identity directs human vitamin D receptor heterodimer transactivation from the 24-hydroxylase vitamin D response elements in yeast. Mol Endocrinol 10:408–419[Abstract]
28. Thompson PD, Jurutka PW, Haussler CA Whitfield CK, Haussler MR 1998 Heterodimeric DNA binding by the vitamin D receptor and retinoid X receptors is enhanced by 1,25-dihydroxyvitamin D₃ and inhibited by 9-cis-retinoic acid: evidence for allosteric receptor interactions. J Biol Chem 273:8483–8491[Abstract/Free Full Text]
29. Nolte RT, Wisley GB, Stefan W, Cobb JE, Lambert MH, Kurokawa R, Rosenfeld MG, Willson TM, Glass CK, Milburn MV 1998 Ligand binding and co-activator assembly of the peroxisome proliferator-activated receptor . Nature 395:137–143[CrossRef][Medline]
30. Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ, Stallcup MR 1998 Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities. Mol Endocrinol 12:302–313[Abstract/Free Full Text]
31. Towers TL, Luisi BL, Asianov A, Freedman LP 1993 DNA target selectivity by the vitamin D receptor: mechanism for dimer binding to an asymmetric repeat element. Proc Natl Acad Sci USA 90:6310–6314[Abstract/Free Full Text]
32. Freedman LP, Arce V, Perez Fernandez R 1994 DNA sequences that act as high affinity targets for the vitamin D₃ receptor in the absence of the retinoid X receptor. Mol Endocrinol 8:265–273[Abstract]
33. Koszewski NJ, Reinhardt TA, Horst RL 1996 Vitamin D receptor homodimer interactions with the murine osteopontin response element. J Steroid Biochem Mol Biol 59:377–388[CrossRef][Medline]
34. Nayeri S, Danielson C, Kahlen J-P, Schrader M, Mathiasen IS, Binderup L, Carlberg C 1995 The anti-proliferative effect of vitamin D₃ analogues is not related to the AP-1 pathway, but related to promoter selectivity. Oncogene 11:1853–1858[Medline]
35. Peleg S, Nguyen C, Woodard BT, Lee J-K, Posner GH 1998 Differential use of transcription activation function 2 domain of the vitamin D receptor by 1,25-dihydroxyvitamin D₃ and its A ring-modified analogs. Mol Endocrinol 12:525–535[Abstract/Free Full Text]
36. Brozozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom O, Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389:753–758[CrossRef][Medline]
37. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL 1998 The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95:927–937[CrossRef][Medline]
38. Bourguet W, Ruff M, Chambon P, Gronemeyer H, Moras D 1995 Crystal structure of the ligand-binding domain of the human nuclear receptor RXR-. Nature 375:377–382[CrossRef][Medline]
39. Rachez C, Gamble M, Chang CP, Atkins GB, Lazar MA, Freedman LP 2000 The DRIP complex, SRC1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. Mol Cell Biol 20:2718–2726[Abstract/Free Full Text]
40. Baudino TA, Kraichely DM, Jefcoat SC, Winchester SK, Partridge NC, MacDonald PN 1998 Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription. J Biol Chem 273:16434–16441
41. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173–179[CrossRef][Medline]
42. Malloy PJ, Pike JW, Feldman D 1999 The vitamin D receptor and the syndrome of hereditary 1,25-dihydroxyvitamin D-resistant rickets. Endocr Rev 20:156–188[Abstract/Free Full Text]
43. Peleg S, Abruzzese RV, Cote GJ, Gagel RF 1990 Transcription of the human calcitonin gene is mediated by a C-cell specific enhancer containing E-box-like elements. Mol Endocrinol 4:1750–1758[Abstract]

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