Evidence for Ligand-Dependent Intramolecular Folding of the AF-2 Domain in Vitamin D Receptor-Activated Transcription and Coactivator Interaction

Hisashi Masuyama, Cynthia M. Brownfield, Rene St-Arnaud and Paul N. MacDonald

Department of Pharmacological and Physiological Science (H. M., C. M. B., P. N. M.), Saint Louis University Health Science Center, St. Louis, Missouri 63104, Genetics Unit, Shriners Hospital (R. S.), Montreal, Quebec H3G 1A6, Canada

Address correspondence to: Paul N. MacDonald, Ph.D., Saint Louis University Health Science Center, Department of Pharmacological and Physiological Science, 1402 South Grand Boulevard, St. Louis, Missouri 63104. Reprints are not available.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A ligand-dependent transcriptional activation domain (AF-2) exists in region E of the nuclear receptors. This highly conserved domain may contact several coactivators that are putatively involved in nuclear receptor-mediated transcription. In this study, a panel of vitamin D receptor (VDR) AF-2 mutants was created to examine the importance of several conserved residues in VDR-activated transcription. Two AF-2 mutants (L417S and E420Q) exhibited normal ligand binding, heterodimerization with retinoid X receptor, and vitamin D-responsive element interaction, but they were transcriptionally inactive in a VDR-responsive reporter gene assay. All AF-2 mutations that abolished VDR-mediated transactivation also eliminated interactions between VDR and several putative coactivator proteins including suppressor of gal1 (SUG1), steroid hormone receptor coactivator-1 (SRC-1), or receptor interacting protein (RIP140), suggesting that coactivator interaction is important for AF-2-mediated transcription. In support of this concept, the minimal AF-2 domain [VDR(408–427] fused to the gal4 DNA binding domain was sufficient to mediate transactivation as well as interaction with putative coactivators. Introducing the L417S and E420Q mutations into the minimal AF-2 domain abolished this autonomous transactivation and coactivator interactions. Finally, we demonstrate that the minimal AF-2 domain interacted with an AF-2 deletion mutant of the VDR in a 1,25-(OH)2D3-dependent manner, suggesting a ligand-induced intramolecular folding of the VDR AF-2 domain. The L417S mutant of this domain disrupted the interaction with VDR ligand-binding domain, while the E420Q mutant did not affect this interaction. These studies suggest that the conserved AF-2 motif may mediate transactivation through ligand-dependent intermolecular interaction with coactivators and through ligand-induced intramolecular contacts with the VDR ligand-binding domain itself.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily (1, 2), and it functions as a ligand-induced transcription factor that mediates the genomic effects of 1{alpha},25-dihydroxyvitamin D3 (1,25-(OH)2D3) (3, 4, 5). VDR and other nuclear receptors display a modular structure, with several regions (A/B, C, D, and E) exhibiting different degrees of evolutionary conservation (2, 6). The N-terminal A/B region is the most divergent module in these receptors, and an autonomous activation function, designated AF-1, is present in the A/B region, which activates transcription constitutively in the absence of the ligand binding domain (LBD) (7, 8). The VDR is atypical in this regard since the A/B region of VDR consists of only 20 amino acids, and deletion of these residues does not affect VDR function (9). The highly conserved C domain contains two zinc modules responsible for DNA binding and sequence-specific recognition of vitamin D-responsive elements or VDREs. The D region, or hinge domain, is located between the DNA-binding domain (DBD) and the LBD. The hinge domain is hypothesized to impart flexibility or a high degree of rotational freedom that facilitates receptor binding to a variety of response elements (6). The D region is also implicated in nuclear localization of receptors and in transactivation (2). Finally, region E is responsible for selective binding of the individual ligands with high affinity and selectivity. Moreover, this C-terminal domain contains a dimerization interface and a ligand-dependent transcriptional activation domain designated AF-2 (8, 10, 11, 12, 13).

The mechanism through which the nuclear receptor-DNA complex regulates the transcriptional process is largely unknown. Recent data suggest that protein-protein contacts between the receptor and the basal transcriptional machinery are important for ligand-mediated transactivation or repression. Nuclear receptors directly contact several general transcription factors (GTFs) in the preinitiation complex (PIC), including TATA-binding protein (TBP) (14, 15), TBP-associated factors (TAFs) (16, 17), and transcription factor IIB (TFIIB) (18, 19, 20, 21). The interaction of receptors with these GTFs is thought to either recruit these limiting factors to PIC assembly or to stabilize the PIC itself (2). However, other factors in addition to the GTFs are required. This is based on the observation that one nuclear receptor interferes with another receptor’s transcriptional activation pathway without affecting basal transcription or the transcription of other promoters (22, 23, 24). Thus, while the interaction between nuclear receptors and the GTFs may be necessary, it is not sufficient for nuclear receptor-mediated transcription. Another class of factors that contact the nuclear receptors are needed, and these are collectively termed coactivators (review in 25 . Recently described coactivators, including steroid hormone receptor coactivator-1 (SRC-1) (26), estrogen receptor-associated protein (ERAP 160) (27), and receptor interacting protein (RIP140) (28), interact in a ligand-dependent manner with several members of the nuclear receptor superfamily to enhance ligand-induced transactivation. In contrast, several corepressors, such as nuclear receptor corepressor (N-CoR) and silencing mediator for retinoic acid receptors (RARs) and thyroid hormone receptors (SMRT), interact with unliganded receptors to inhibit basal transcription of the associated promoter (6, 25, 29, 30). Interestingly, the LBD (AF-2 domain) of the nuclear receptor is required both for the interaction with the majority of coactivators and for the dissociation of corepressor proteins, suggesting a mechanistic link between transcriptional suppression and activation (6, 30).

The AF-2 activating domain has been characterized in the C-terminal part of region E of the RAR, retinoid X receptor (RXR), thyroid hormone receptor (TR), and estrogen receptor (ER), and this region corresponds to an amphipathic {alpha}-helix motif whose main features are conserved between all known transcriptionally active members of the nuclear receptor superfamily (10, 12, 13). In most instances, the AF-2 motif is transcriptionally silent in the absence of ligand, and ligand binding activates its enhancer potential (10, 12, 13). In this paper, we demonstrate that the conserved AF-2 motif of VDR is required both for 1,25-(OH)2D3-dependent transactivation and for 1,25-(OH)2D3-dependent interactions between VDR and several putative coactivators including SRC-1 and RIP140. Indeed, the minimal AF-2 domain of VDR was sufficient to mediate transactivation as well as to mediate interactions between VDR and SRC-1 or RIP140. Finally, we present evidence for a ligand-dependent intramolecular interaction of the AF-2 helix with the VDR LBD. These data support the hypothesis that the ligand promotes the folding of the AF-2 domain to create a transactivation surface for coactivator interaction and subsequent transactivation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Characterization of the C-Terminal Mutants of VDR
As illustrated in Fig. 1Go, the C-terminal region of VDR examined in this study contained heptad 9, which is an important region of VDR for dimerization with RXR (31), and the AF-2 motif, which is highly conserved throughout the nuclear receptor superfamily (10, 12, 13). A series of point mutations were introduced into this C-terminal region of VDR to test the effects of the mutations on vitamin D-mediated transactivation, ligand binding, RXR heterodimerization, and VDRE interaction. Transient transfection assays in COS-7 cells demonstrated that wild type VDR showed a 60-fold induction of reporter gene expression in the presence of 10-8 M 1,25-(OH)2D3, whereas the C-terminal deletion mutants [VDR(1–386) and VDR(1–403)], which lacked the conserved AF-2 motif, were transcriptionally inactive (Fig. 2AGo). Introducing more subtle mutations into the AF-2 domain itself also abolished VDR-mediated transactivation. The glutamic acid residue at position 420 was changed to glutamine (E420Q) and the leucine residue at position 417 was changed to serine (L417S), and both mutants lost the ability to activate transcription. However, mutation of the glutamic acid located downstream of the AF-2 core motif (E425Q) did not significantly affect VDR-mediated transactivation compared with wild type VDR. Similar results were obtained using another reporter construct (VDRE4-TK-GH) and other cell lines (Hela cells, ROS17/2.8 cells) (data not shown).



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Figure 1. Amino Acid Sequence of C-Terminal Region of VDR

Heptad 9 of the putative dimerization domain and the conserved AF-2 motif are indicated along with several of the VDR point mutations and deletion mutations that were examined in this study. Also illustrated in the box, is an amino acid sequence comparison of the AF-2 region of several nuclear receptors.

 


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Figure 2. The Abolishment of VDR-Mediated Transcription by Introducing Mutations into the Conserved AF-2 Motif of VDR

A, The effect of AF-2 motif mutations in a vitamin D-responsive transient gene expression system. COS-7 cells were transfected with 2 µg of the VDRE4-TATA-GH reporter gene construct together with 100 ng of the wild type or mutant VDR expression plasmids. Sixteen hours after addition of the calcium phosphate-DNA precipitate, the cells were washed, media were replaced, and the cells were treated with ethanol vehicle or with 10-8 M 1,25-(OH)2D3. GH secreted into the media was quantitated 24 h following ligand addition using a RIA kit. The results represent the mean ± SD of triplicate determinations. B, High concentrations of 1,25-(OH)2D3 do not rescue transactivation of the L417S and E420Q mutant VDRs. COS-7 cells were transfected as described in panel A. The cells were treated with ethanol vehicle or with 10-10, 10-8, or 10-6 M 1,25-(OH)2D3 for 24 h. GH secretion was quantitated as described in panel A.

 
The loss in transcriptional activation was not due to altered hormone binding since all of the point mutants examined in this study had ligand binding affinities that were comparable to wild type VDR (Table 1Go). In contrast, the VDR(1–386)mutant displayed no detectable ligand binding, and the apparent dissociation constant (Kd) for the VDR(1–403) mutant was approximately 10-fold higher than wild type VDR. However, as demonstrated by Whitfield et al. (32), it is possible that subtle changes in ligand binding may not be apparent in this in vitro binding assay. Therefore, we examined whether high concentrations of ligand could rescue the impairment in transactivation that was observed in the L417S and E420Q mutants. While wild type VDR was maximally activated at 10-10 M 1,25-(OH)2D3, the L417S and the E420Q VDR mutants were completely inactive even when cells were treated with 10,000-fold higher ligand concentrations (10-6 M) (Fig. 2BGo). Thus, the absence of transactivation in the L417S and E420Q mutants was not due to modest alterations in ligand binding in vivo or in vitro.


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Table 1. 1,25(OH)2D3 Binding Affinities of Mutants VDRs

 
We also examined the interaction of these VDR mutants with two factors that are important in VDR-mediated transcription, namely RXR and TFIIB. Disruption of heptad 9 and elimination of the AF-2 motif in the VDR(93–386) mutant resulted in loss of VDR interaction with both RXR and TFIIB. However, removing only the AF-2 motif in the VDR(93–403) construct allowed substantial interaction of VDR with RXR and with TFIIB. Importantly, mutants within the AF-2 domain that are transcriptionally silent (L417S and E420Q) still show considerable interaction with RXR and TFIIB, indicating that the loss of activity is not due to the inability of VDR to interact with these two important factors (Fig. 3Go).



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Figure 3. Interaction of Wild Type and Various Mutant VDRs with RXR or TFIIB in Two-Hybrid System

Yeast expressing AS1-VDR and pGAD.GH-RXR{alpha} or pGAD.GH-TFIIB yeast expression vector were grown for 16 h at 30 C in the absence or presence of 10-8 M 1,25-(OH)2D3. The interactions were analyzed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures.

 
To determine whether these mutations altered the DNA binding properties of the VDR-RXR complex, electrophoretic mobility shift assays were performed using overexpressed pSG5-VDR proteins in COS-7 cells and a [32P]-labeled VDRE. Mutations in the heptad 9 region of VDR were severely compromised in VDRE interactions in this assay. Removal of a portion of heptad 9 [pSG5-VDR(1–386)] eliminated VDR-VDRE interaction, and point mutations of heptad 9 (K386Q, R391Q) resulted in much weaker interactions with the VDRE (Fig. 4AGo). Based on previously published data (31) and on our two-hybrid results (Fig. 3Go), this is likely due to disrupting the ability of VDR to heterodimerize with RXR and subsequent weaker binding of the heterodimer to the VDRE. Importantly, the L417S and E420Q mutants retained the ability to heterodimerize with RXR and bind to the VDRE (lane 6 and 7) compared with wild type VDR (lane 1). Western analysis showed that all VDR proteins were expressed to similar levels in each of these extracts (Fig. 4BGo).



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Figure 4. Gel Mobility Shift Assay of Wild Type and Mutant VDRs

A, Cellular extracts of COS-7 cells transfected with wild type VDR or mutant VDR expression vectors were incubated with a 32P-labeled VDRE, electrophoresed on a 4% nondenaturing polyacrylamide gel, and autoradiographed as described in Materials and Methods. B, These same extracts were analyzed by a Western immunoblot using the 9A7 antibody raised against VDR to determine whether the mutants were expressed in a similar fashion.

 
Autonomous Transactivation Function by the VDR AF-2 Motif
To test whether the transactivation domain encompassing the conserved motif could function independently of the ligand binding/dimerization domain, various deletions and point mutants of the VDR LBD were fused to the gal4 DBD [gal4(1–147)]. Transient reporter gene expression assays were performed in COS-7 cells using Gal45-TATA-GH. Expressing the full-length LBD of the VDR fused to the gal4 DBD [gal4-VDR(93–427)] demonstrated no transcriptional activation in the absence of hormone. However, treating these cells with 10-8 M 1,25-(OH)2D3 activated reporter gene expression by 15-fold (Fig. 5AGo). The ligand-dependent transactivation by this heterologous construct was mediated through the AF-2 domain since the AF-2 deletion mutant [gal4-VDR(93–403)] and the AF-2 point mutants [gal4-VDR(L417S and E420Q)] were inactive in the presence or absence of 1,25-(OH)2D3. N-Terminal deletions [gal4-VDR(281–427, 374–427, and 387–427)] that eliminated ligand binding were transcriptionally inactive in both the presence and absence of 1,25-(OH)2D3 ligand. However, a fusion protein consisting of the last 20 amino acids of VDR linked to the gal4 DBD [gal4-VDR(408–427)] showed autonomous transactivation activity that was unaffected by ligand. The level of reporter gene expression with this minimal AF-2 domain fusion was approximately 40% of the activity of the full-length LBD. To examine whether the ligand-independent transactivation by this minimal AF-2 domain has an amino acid requirement similar to that of ligand-dependent transactivation, three-point mutations were introduced into the gal4-VDR(408–427) construct (Fig. 5BGo). The L417S and the E420Q mutants abolished the autonomous transactivation by the minimal AF-2 domain, and E425Q mutation expressed an activity that was comparable to the wild type AF-2 motif (Fig. 5BGo).



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Figure 5. Autonomous Transactivation Function of the Minimal AF-2 Domain of VDR

A, The minimal AF-2 domain mediates autonomous, ligand-dependent transactivation. COS-7 cells were transfected with 2 µg of a reporter construct (Gal45-TATA-GH) and 0.5 µg of various deletion mutants or point mutants of pSG5-gal4-VDR. The cells were treated in the absence or presence of 10-8 M 1,25-(OH)2D3 for 24 h and GH secretion was determined. Values obtained with gal4(1–147) in the absence of 1,25-(OH)2D3 are set arbitrarily as 1.00. B, The L417S and E420Q mutations ablate autonomous transactivation by the minimal AF-2 domain. COS-7 cells were transfected with 2 µg of a reporter construct (Gal45-TATA-GH) and 0.5 µg of pSG5-gal4, pSG5-gal4-VDR(93–427), or wild type or point mutants of pSG5-gal4-VDR(408–427). The cells were treated and GH secretion was determined.

 
Interactions between the AF-2 Domain of VDR and mSUG1, SRC-1, and RIP140 in the Yeast Two-Hybrid System
Our laboratory has been using two-hybrid strategies to identify proteins that interact with the LBD of VDR in a ligand-dependent manner (21, 33). The bait construct used in this screen was AS1-VDR(93–427), which contained the gal4 DBD fused to the LBD of VDR (amino acids 93–427) (21). A mouse osteoblastic MC3T3 cell cDNA library was constructed in the yeast multicopy expression vector pAD-Gal4. Interaction between AS1-VDR and fusion proteins of pAD-GAL4 expressed in the library were characterized by monitoring ß-galactosidase activity and growth on selective media lacking histidine in the presence of 1,25-(OH)2D3. Several cDNA clones that interact with VDR in the presence of 1,25-(OH)2D3 were obtained. DNA sequence analysis identified three clones as full-length mSUG1 (34), the C-terminal region of mSRC-1 (amino acids 1258–1465) (35), and mRIP140, which was 80% identical to human RIP140 (amino acids 867-1158) (28). As demonstrated previously for the interaction of VDR with SUG1 (34) or with another putative coactivator, glucocorticoid receptor interacting protein 1 (36), the interaction between VDR and these three clones in the two-hybrid system was dependent on the 1,25-(OH)2D3 ligand (data not shown).

To test whether the conserved AF-2 motif mediated the ligand-dependent interaction between VDR and these putative coactivators, the VDR AF-2 mutants were examined in the two-hybrid interaction assay. Deletion of the AF-2 motif [VDR(93–403)] resulted in no or weak interaction of this mutant with the cofactors tested here (Fig. 6Go). Point mutations within the AF-2 core (L417S and E420Q) also disrupted the interaction of VDR with mSUG1, mSRC-1, and mRIP140 whereas mutations flanking the core had little effect (K386Q, R391Q, and E425Q). It is important to note that most of the AF-2 mutants still retained strong interactions with both RXR{alpha} and TFIIB (Fig. 3Go). Interestingly, the minimal AF-2 domain (residues 408–427), which demonstrated ligand-independent, autonomous transactivation (Fig. 5AGo), also showed significant interactions with all of the cofactors tested here. As illustrated in Fig. 7Go, AS1-VDR(408–427) demonstrated significant interactions with mSRC-1 and mRIP140 while pAS1-VDR(281–427), which had no autonomous transactivation activity (Fig. 5Go), also did not interact with these putative coactivators in this system. The introduction of two AF-2 point mutations into pAS1-VDR (408–427) abolished the interaction with all cofactors, while the mutation located downstream of the core AF-2 motif (E425Q) retained the interactions. Interestingly, none of these minimal AF-2 fusions interacted with RXR{alpha} (Fig. 7Go) or with the pAD-Gal4 parent expression vector (data not shown). Thus, ß-galactosidase activity determined in these studies is a reflection of protein-protein interaction and is not due to reporter gene expression driven by autonomous activation by the AS-1-VDR(408–427) construct alone. In fact, these data demonstrate that while the gal4-VDR(408–427) is an autonomous activator in mammalian cells, it does not express detectable constitutive transactivation in this yeast system.



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Figure 6. The Effect of Deletion or Point Mutations in the AF-2 Activation Domain on the Interaction between VDR and Coactivators

Yeast expressing the wild type, deletion, or point mutations of AF-2 activation domain of AS1-VDR or pAS1 yeast expression vector alone and pAD-SUG1, pAD-SRC-1, or pAD-RIP140 were grown for 16 h at 30 C in the presence of 10-8 M 1,25-(OH)2D3. The interactions were assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures.

 


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Figure 7. The AF-2 Domain Is Sufficient to Mediate Interaction with Putative Coactivator Proteins

Yeast expressing the AS1-VDR(93–427, 281–427, 408–427), mutations of AS1-VDR(408–427) or pAS1 yeast expression vector alone, and pAD-SUG1, pAD-SRC-1, pAD-RIP140, or pAD-RXR{alpha} were grown for 16 h at 30 C in the absence or presence of 10-8 M 1,25-(OH)2D3. The interactions were assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures.

 
Intramolecular Interaction between the Minimal AF-2 Domain and an AF-2 Deletion Mutant of VDR
These data suggest that the AF-2 domain functions in transactivation perhaps by contacting coactivator proteins and that ligand promotes these interactions by altering the conformation of the AF-2 region. A comparison of the crystal structures of unliganded RXR and liganded RAR indicates that, upon binding of ligand, the AF-2 domain folds down on the receptor to close off the ligand-binding pocket (11, 37). This suggests that the AF-2 domain forms ligand-dependent intramolecular contacts with residues in the VDR LBD. To test this hypothesis, we examined the interaction of minimal AF-2 domain [VDR(408–427)] with the AF-2 deletion mutant of VDR [VDR (93–403)]. Since VDR(93–403) retains ligand binding, albeit with somewhat reduced affinity, the effect of ligand on the interaction of AF-2 domain with VDR(93–403) was also examined. As illustrated in Fig. 8AGo, the minimal AF-2 domain showed weak, but significant, interactions with VDR(93–403) in the presence of 1,25-(OH)2D3. This interaction was markedly reduced in the absence of ligand and was also not apparent using the AS1-VDR(93–386) bait or the pAS1 parent expression vector. Interestingly, introducing the E420Q or E425Q mutations into the minimal AF-2 domain did not affect its interaction with VDR(93–403), while the L417S mutant disrupted this interaction (Fig. 8BGo).



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Figure 8. Intramolecular Interaction between Minimal AF-2 Domain and AF-2 Deletion Mutant of VDR

A, Interaction between the minimal AF-2 domain and the AF-2 deletion mutant of VDR. Yeast expressing the AS1-VDR(93–386, 93–403) or pAS1 yeast expression vector alone and pGAD.GH-VDR(408–427) or pGAD.GH expression vector alone were grown for 16 h at 30 C in the absence or presence of 10-8 M 1,25-(OH)2D3. The interactions were assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures. B, Interaction between point mutants of minimal AF-2 domain and AF-2 deletion mutant of VDR. Yeast expressing the AS1-VDR(93–403), and wild type and point mutations of pGAD.GH-VDR(408–427) were grown for 16 h at 30 C in the absence or presence of 10-8 M 1,25-(OH)2D3. The interactions were assessed in a ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The C-terminal, 50-amino acid residues of the VDR encompass two key structural motifs that play a central role in 1,25-(OH)2D3-mediated transcription. One region corresponds to heptad 9 (residues 383–390), which apparently functions as a protein-protein interaction interface that is essential for VDR heterodimerization with RXR. This is based on studies of VDR (31) and related receptors (38, 39), which demonstrate that mutations in this region disrupt the ability of these receptors to heterodimerize with RXR and to bind their cognate response elements. Crystallography of the RXR homodimer clearly established the role of an {alpha}-helical region encompassing heptad 9 (helix 11) as the central dimerization interface in RXR and strongly implicated similar roles in structurally related receptors (11). A second important domain in the C terminus of VDR is the AF-2 motif, which functions as a ligand-dependent transactivation domain. Mutation of conserved residues in RARs and TRs ablate hormone-induced transactivation, implicating a predominant role of the AF-2 domain in nuclear receptor-signaling pathways (10, 13). Although the precise mechanisms involved in the AF-2 transactivation function are not well understood, recent data indicate that this {alpha}-helical domain functions as an interaction surface for transcriptional coactivator proteins that are putatively involved in nuclear receptor-mediated transcription (6, 25). A detailed functional analysis of the VDR AF-2 domain and the role of the 1,25-(OH)2D3 ligand in the activity of this domain have been lacking. In this paper, we demonstrate the requirement of an intact AF-2 core domain for 1,25-(OH)2D3-activated transcription. Furthermore, we show that the AF-2 domain alone is sufficient to mediate transactivation in a heterologous system and that it is also sufficient to mediate interaction with several putative transcriptional coactivator or adapter proteins.

Nakajima et al. (31) demonstrated that deletion of the VDR AF-2 domain abolished VDR-mediated transactivation without compromising heterodimerization of VDR with RXR and subsequent binding of the VDR-RXR heterodimer to the VDRE. These data indicated a selective role of the VDR AF-2 domain in 1,25-(OH)2D3-activated transcription. However, 1,25-(OH)2D3 binding to the VDR(1–403) mutant was reduced by an order of magnitude compared with wild type VDR, suggesting an additional role for the AF-2 domain in high- affinity ligand binding (31). Similar results were obtained with VDR(1–403) in the transactivation, two-hybrid interaction, and ligand-binding assays in the present study ( Figs. 2–4GoGoGo and Table 1Go). Indeed, the crystal structures of liganded RAR and TRs show that the AF-2 motif is packed onto the body of the receptor with a portion of it forming part of the ligand-binding pocket (37, 40). This dual role of the AF-2 domain in both transactivation and in high-affinity ligand binding complicates the functional analysis of this domain in VDR-mediated transactivation. However, in the present study, we defined point mutations within the VDR AF-2 core motif (Fig. 1Go) that effectively discriminated between transactivation and ligand-binding effects. While the VDR(E420Q) and the VDR(L417S) mutants bound 1,25-(OH)2D3 with equilibrium binding constants similar to wild type VDR (Table 1Go), both point mutants were transcriptionally silent. Furthermore, transactivation was not evident when the mutant receptors were incubated with ligand concentrations that were 10,000-fold greater that the apparent Kds (Fig. 2BGo). Altering a glutamic acid residue that is adjacent to the conserved region (E425) did not significantly affect transactivation. Consequently, these data highlight the central importance of the charged and hydrophobic residues within the AF-2 core motif in VDR-activated transcription.

Fusing the minimal AF-2 domain of VDR (residues 408–427) to a heterologous DBD [Gal4(1–147)] produced a hybrid protein that expressed significant transactivation potential in mammalian cells (Fig. 5Go). These data are consistent with previous findings that similar regions in TR, RAR, and RXR also function as autonomous transactivation domains (10, 12, 13). Introducing the E420Q and L417S mutations into this hybrid construct abolished transactivation by the minimal VDR AF-2 motif. Thus, E420 and L417 are essential for ligand-activated AF-2 activity in the intact receptor and for autonomous transactivation mediated by the minimal AF-2 domain. Interestingly, Gal4-VDR hybrids that expressed additional N-terminal sequences along with the minimal AF-2 domain [i.e. VDR(383–427), VDR(373–427), and VDR(281–427)] were transcriptionally silent in this assay. Perhaps additional N-terminal sequence adjacent to the AF-2 core motif may mask the AF-2 domain and inhibit its transactivation potential. Alternatively, additional factors such as corepressor proteins may interact with the VDR constructs containing this additional N-terminal sequence that may inhibit or suppress AF-2 activity (29, 30). Regardless, the chimera containing the full-length, intact LBD [VDR(93–427)] was inactive in the absence of ligand, but it expressed substantial transactivating activity in the presence of 10-8 M 1,25-(OH)2D3. These data indicate that the AF-2 domain, while active on its own, is not in the appropriate orientation or conformation for efficient participation in the transactivation process in the intact receptor in the absence of ligand. However, in the context of the intact receptor, binding of ligand may alter the conformation of the AF-2 to promote transactivation.

Importantly, we demonstrate that the minimal AF-2 domain of VDR interacts with an AF-2 deletion mutant of VDR [VDR(93–403)] in a ligand-dependent manner. We interpret this trans interaction between the isolated AF-2 domain and the remainder of the VDR LBD as a ligand-induced intramolecular folding of the AF-2 domain. Whereas the AF-2 mutation (E420Q) did not affect this ligand-dependent intramolecular contact, the L417S mutation severely impaired AF-2 interaction with the VDR LBD. These data imply that these two residues may, in fact, play distinct roles in the transactivation process. The hydrophobic residues (e.g. L417) may be involved in forming the hydrophobic core of the ligand-binding pocket and may be required for 1,25-(OH)2D3-dependent intramolecular folding of the AF-2 domain. In contrast, the charged residues (e.g. E420) may not be involved in intramolecular folding of the AF-2 domain, but instead this may be surface exposed and required for coactivator interaction. These observations are consistent with the putative model of ligand-induced conformational changes in the AF-2 domain based on the crystal structures of liganded RAR compared with unliganded RXR (11, 37). Interestingly, the minimal AF-2 domain [gal4-VDR(408–427)] expresses only 40% of the transcriptional activity and coactivator interaction observed with the full-length LBD in the presence of 1,25-(OH)2D3. This observation suggests that additional residues outside the AF-2 domain contribute to the transactivation surface. Additional studies are required to refine various aspects of this model and identify other residues that comprise the transactivation surface. Strong candidates may reside within the two activation domains identified in the VDR LBD in a yeast-based system (41).

Our ongoing studies to identify putative transcriptional adaptor proteins that interact with the VDR led to the isolation of several VDR-interactive clones from a murine osteoblast cDNA library in the yeast two-hybrid system. Sequence analysis revealed that three of the cDNA clones encoded putative coactivators implicated in nuclear receptor-mediated transcription including SRC-1 (26), RIP140 (28), and SUG1 (34). Importantly, these putative coactivators interacted with the VDR in a 1,25-(OH)2D3-dependent manner with the AF-2 domain of the VDR playing an essential role in this interaction. This is best exemplified in the observation that mutations in the AF-2 core motif disrupt interactions between VDR and SRC-1 or RIP140. These same mutations that abolish interaction with the coactivators also abolish transactivation. Moreover, the VDR AF-2 domain alone was sufficient to mediate interaction with these coactivators and to mediate autonomous transactivation. Although a direct examination of the effects of these coactivators on VDR-mediated transcription needs to be addressed, the strong correlation between coactivator interaction and transactivation observed in these studies supports a role for these putative coactivators in the mechanism of 1,25-(OH)2D3-activated transcription mediated through the VDR AF-2 domain.

In summary, we have analyzed the functional role of the extreme C-terminal region of VDR. Our data illustrate the distinct roles of heptad 9 (helix 11) in VDR dimerization with RXR as well as the central role that the conserved AF-2 motif plays in ligand-dependent coactivator interaction and transactivation. Importantly, these data support structural studies in which ligand binding induces a dramatic conformational change in the AF-2 domain mediated through an intramolecular folding of the AF-2 domain that creates a transcriptional surface for coactivator binding.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transient Transfection Studies
The VDRE4-TATA-GH plasmid contained four copies of the rat osteocalcin VDRE adjacent to the rat osteocalcin promoter (-40 to +32) driving to a human GH reporter sequence (33). The Gal45-TATA-GH plasmid contained five copies of the gal4-responsive element, an Elb promoter fragment, and the human GH reporter sequence. The pSG5-VDR expression plasmid was described previously (42, 43). The pSG5-gal4 was constructed by inserting the gal4 cDNA encoding amino acids 1–147 into the pSG5 expression plasmid (Stratagene, La Jolla, CA). Point mutations were introduced into the human VDR cDNA using oligonucleotide-directed mutagenesis (44, 45), and C-terminal deletions were generated by introducing stop codons at the indicated positions. All of the mutants were confirmed by DNA sequencing. VDR mutants were subcloned into pSG5 or pSG5-gal4. To generate pSG5-gal4-VDR(408–427), oligonucleotides corresponding to the last 20 amino acids of human VDR with EcoRI and BamHI overhangs at the 5'- and the 3'-end, respectively, were subcloned in frame into pSG5-gal4.

COS-7 cells were cotransfected with reporter gene constructs (VDRE4-TATA-GH or Gal45-TATA-GH) and with receptor expression vectors (pSG5-VDR or pSG5-gal4-VDR). In all transfections, the amount of total DNA was kept constant at 10 µg by adding pTZ18U (U.S. Biochemical, Cleveland, OH) as a carrier plasmid. The cells were transfected by standard calcium phosphate coprecipitation procedures as described previously (43). Transfected cells were treated with the indicated concentrations of 1,25-(OH)2D3 or ethanol vehicle for 24 h, and the amount of secreted GH was determined with a RIA kit (Nichols Institute, San Juan Capistrano, CA).

Ligand-Binding Assay
COS-7 cell lysates expressing wild type or mutant human VDRs were prepared as described previously (32). The lysates were incubated with five different concentrations of 1,25-(OH)2-[3H]D3 (18 Ci/mmol) overnight at 4 C in the presence or absence of a 400-fold molar excess of unlabeled 1,25-(OH)2D3. Bound and free ligand were separated with dextran-coated charcoal and analyzed by Scatchard plots to determine the dissociation constant.

Gel Mobility Shift Analysis
The VDRE oligomer corresponding to the rat osteocalcin VDRE was described previously (46). The VDRE oligomer was labeled to high specific activity by a fill-in reaction with Klenow fragment of DNA polymerase I and [{alpha}-32P]dCTP (3000 Ci/nmol). Ten micrograms of the lysates containing wild type or mutant human VDRs were incubated with 32P-labeled VDRE probe for 30 min at 22 C in 10 mM Tris-HCl, pH 7.6, 100 mM KCl, 1.0 mM dithiothreitol, 15% glycerol, 0.1 µg/ml BSA, and 50 µg/ml poly(deoxyinosinic-deoxycytidylic)acid. Unbound probe and protein-DNA complexes were separated by nondenaturing electrophoresis on a 4% polyacrylamide gel in 0.25xTris-borate-EDTA. Gels were dried and exposed for autoradiography.

Preparation of Two-Hybrid Expression Vectors and cDNA Library Screening
All two-hybrid plasmids constructs used the pAS1 (47) and the pGAD.GH (48) or pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression vectors. AS1-VDR constructs, containing the full-length and deletion mutants of VDR [(93–427), (93–386), (281–427)], were described previously (21). Other deletions [(93–403), (387–427), (408–427)] and point mutations (K386Q, R391Q, L417S, E420Q, E425Q) of VDR were also subcloned into the pAS1 or the pGAD.GH vector for examination in the two-hybrid assay. The MC3T3-E1 cell cDNA library was prepared in the pAD-GAL4 vector. For cDNA library screening, the library was cotransformed with pAS1-VDR(93–427) into the yeast strain Hf7c, which was made competent with lithium acetate (49). Transformants were plated on media lacking leucine, tryptophan, and histidine (SC-leu-trp-his) and containing 10-8 M 1,25-(OH)2D3 and 10 mM 3-amino-1,2,4-triazole. Histidine-positive colonies were assayed for ß-galactosidase expression using a colony lift filter assay (47).

ß-Galactosidase Assays
The pAD-GAL4-mSUG1, -SRC-1, -RIP140, -RXR{alpha}, and the pGAD.GH-TFIIB, -RXR{alpha}, -VDR were cotransformed with wild type and mutant pAS1-VDR into the yeast strain Hf7c as described above. Transformants were plated on media lacking leucine and tryptophan (SC-leu-trp) and were grown for 4 days at 30 C to select for yeast that had acquired both plasmids. Triplicate independent colonies from each plate were grown overnight in 2 ml of SC-leu-trp with or without 10-8 M of 1,25-(OH)2D3. Cells were harvested and assayed for ß-galactosidase activity as described (50).


    ACKNOWLEDGMENTS
 
We would like to thank The Endocrine Society and its Student Affairs Committee for the generous support of C. M. B.


    FOOTNOTES
 
This work was supported in part by NIH Grants R29DK-47293 and R01DK-50348 (to P.N.M.) and an Endocrine Society Student Research Fellowship (to C.M.B.). R. St-A. is a chercheur-boursier from the Fonds de la Recherche en Santé du Québec.

Received for publication April 21, 1997. Revision received June 5, 1997. Accepted for publication June 9, 1997.


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