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Originally published In Press as doi:10.1074/jbc.M507708200 on October 4, 2005

J. Biol. Chem., Vol. 280, Issue 48, 40152-40160, December 2, 2005
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Nuclear Import of the Retinoid X Receptor, the Vitamin D Receptor, and Their Mutual Heterodimer*

Rubina Yasmin{ddagger}, Rebecca M. Williams§1, Ming Xu{ddagger}, and Noa Noy{ddagger}2

From the {ddagger}Division of Nutritional Sciences and §Departments of Applied and Engineering Physics and Biomedical Sciences, Cornell University, Ithaca, New York 14853

Received for publication, July 15, 2005 , and in revised form, August 29, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The nuclear receptor retinoid X receptor (RXR) can regulate transcription through homotetramers, homodimers, and heterodimers with other nuclear receptors such as the vitamin D receptor (VDR). The mechanisms that underlie the nuclear import of RXR, VDR, and RXR-VDR heterodimers were investigated. We show that RXR and VDR translocate into the nucleus by distinct pathways. RXR strongly bound to importin{beta} and was predominantly nuclear in the absence of ligand. Importin binding and nuclear localization of RXR were modestly enhanced by its ligand, 9-cis-retinoic acid. On the other hand, VDR selectively associated with importin{alpha}. Importin association and correspondingly nuclear import of VDR were markedly augmented by 1,25(OH)2D3. RXR-VDR dimerization inhibited the ability of RXR to bind importin{beta} and to mobilize into the nucleus using its own nuclear localization signal. In contrast, VDR recruited RXR-VDR heterodimers to importin{alpha} and mediated nuclear import of the heterodimers in response to 1,25(OH)2D3. Hence nuclear import of RXR-VDR heterodimers is mediated preferentially by VDR and is controlled by the VDR ligand. The observations reveal a novel mechanism by which an RXR heterodimerization partner dominates the activity of the heterodimers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The retinoid X receptor (RXR)3 is a member of the superfamily of nuclear hormone receptors that is activated by the vitamin A metabolite 9-cis-retinoic acid (9cRA). Like other nuclear receptors, RXR is comprised of several distinct functional domains: an amino-terminal domain, involved in ligand-independent basal transcriptional activity; a DNA-binding domain (DBD) containing two "zinc finger" motifs; a flexible hinge region; and a carboxyl-terminal region, termed the ligand-binding domain (LBD). The LBD contains the ligand-binding pocket as well as regions that mediate multiple protein-protein interactions including association with transcriptional co-regulators, formation of dimers, and, in the case of RXR, formation of tetramers. The LBD of nuclear receptors, including RXR, thus coordinates their ligand-dependent transcriptional activities (1).

In the absence of its cognate ligand, RXR forms high affinity homotetramers. These tetramers are transcriptionally silent, but they rapidly dissociate upon ligand binding (2-4). Hence RXR tetramers appear to serve as an inactive storage pool from which active species can be liberated in response to 9cRA. An additional role for the RXR tetramers was recently suggested by the observations that these oligomers act as DNA architectural factors. It was thus demonstrated that binding of RXR tetramers to promoter regions that contain two RXR response elements in tandem results in a dramatic DNA looping, thereby enabling transcriptional regulation by factors placed far upstream from start sites of target genes (5). Hence by modulating DNA geometry, RXR tetramers can regulate gene expression in a manner that is responsive to 9cRA but is independent of the intrinsic transcriptional activity of the receptor (5). Although RXR can activate transcription as a homodimer (6, 7), this receptor also serves as an obligatory common dimerization partner for numerous other class II nuclear receptors such as the retinoic acid receptor, the thyroid hormone receptor, and the vitamin D receptor (VDR) (1). Through heterodimerization with these receptors RXR functions as a "master regulator" of multiple signaling pathways that are regulated by various hormones and converge at the genome.

Available information suggests that the partitioning of RXR between its different oligomeric complexes is regulated by cognate ligands for this receptor and for its heterodimerization partners. Hence apoRXR is predominantly tetrameric, and ligand binding by this receptor yields RXR homodimers (2, 4, 8). It was also reported that the RXR ligand 9cRA stabilizes RXR homodimers and inhibits the association of RXR with heterodimerization partners such as retinoic acid receptor and VDR (6, 9). Ligand binding by either retinoic acid receptor or VDR was found to overcome this inhibition and to stabilize the respective heterodimers even in the presence of 9cRA. Heterodimerization is thus maximal in the presence of both 9cRA, acting to dissociate RXR tetramers, and the ligand for the partner, which stabilizes the heterodimer (9, 10). Whether enhancement of heterodimerization by the ligand for the partner is a general feature of RXR heterodimers remains to be clarified. As the various RXR-containing oligomers function in the nucleus, an important question for understanding how their activities are regulated relates to the mechanisms that underlie their nuclear import.

Nuclear import of proteins is often mediated by a cluster of basic amino acids in their primary sequence such as the "classical" nuclear localization signals (NLSs) comprised of the sequence K(K/R)X(K/R) (11-14). These sequences are recognized by adapter proteins known as {alpha} importins. Importin{alpha}s contain two distinct NLS binding sites that can each bind a single NLS, or they can associate with a stretch containing a bipartite signal (15, 16). Cargo-loaded importin{alpha} binds to one of the various forms of importin{beta}, which in turn mediates the nuclear import of the complex (12). It was also reported that some cargoes can interact directly with importin{beta}. The sequences recognized by importin{beta} are less well understood and may vary, but in some cases, they are reminiscent of classical NLSs in that they are comprised of regions rich in basic residues (17-19). Several nuclear receptors, including various steroid receptors, RXR, VDR, and thyroid hormone receptor, were found to contain an NLS, termed NL1, between the two zinc fingers of their DBD (20, 21). However, some receptors appear to contain more than one NLS. It was thus reported that the glucocorticoid receptor harbors an additional NLS within its LBD, and that this sequence possesses a different importin selectivity than that displayed by the NL1 (22). VDR appears to harbor a bipartite NLS in the stretch between residues 67 and 108 (21) and another NLS in its hinge region (23).

The importin selectivity of different receptors and the factors that regulate their nuclear import are incompletely understood. It was reported that some receptors, including RXR and retinoic acid receptor, are constitutively nuclear (24, 25). On the other hand, nuclear translocation of other receptors appears to be a regulated event. For example, nuclear retention of thyroid hormone receptor {alpha}1 was found to be regulated by phosphorylation of one or more residues (26). VDR appears to distribute between the cytoplasm and the nucleus in the absence of its ligand and to undergo nuclear translocation upon ligand binding (21, 27). It was also reported that nuclear import of VDR is promoted in the presence of RXR, suggesting that the process involves RXR-VDR heterodimers (27, 28). However, the mechanisms that underlie the nuclear import of RXR-containing heterodimers are unknown.

The present work was undertaken to better characterize the involvement of cognate ligands in regulating the nuclear localization of VDR, RXR, and their mutual heterodimer and to obtain insight into the mechanisms by which the nuclear import of these receptors is accomplished.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Vectors—Expression constructs for GFP-RXR and BFP-VDR were generated by PCR using mouse RXR{alpha} and human VDR in pSG5 as templates. Resulting PCR products were subcloned into enhanced GFP and enhanced BFP vectors (Clontech), respectively, using the restriction sites 5'-EcoRI and XbaI-3' for GFP-RXR and 5'-XhoI and BamHI-3' for BFP-VDR. In both cases, the fluorescent proteins were fused to the amino termini of the receptors. Constructs were verified by sequencing at the Cornell Biotechnology Center. NLS mutants of the receptors were generated by site-directed mutagenesis using the QuikChangeTM site-directed mutagenesis kit (Stratagene). Mutagenesis was carried out in two rounds. A luciferase reporter construct containing the VDR response element of the vitamin D 24-hydroxylase gene was a gift from Hector DeLuca (University of Wisconsin, Madison, WI) (29). Bacterial expression vectors for importin{alpha}1{Delta}IBB (13) and importin{beta} fused to glutathione S-transferase (30) were gifs from Alec Hodel (Emory University School of Medicine, Atlanta, GA) and from Stephen Adam (Northwestern University, Chicago, IL), respectively.

Cell Culture and Transfection—COS-7 cells (ATCC CRL-1651) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For experimentation, cells were plated in Dulbecco's modified Eagle's medium containing 5% charcoal-treated serum and transfected using FuGENE 6TM (Roche Applied Science). For microscopy, cells were plated in coverglass bottom dishes (Matek Corp.) and transfected with vectors encoding constructs of GFP-RXR (0.25 µg) or BFP-VDR (1.00 µg). Following a 24-h incubation in serum-free Dulbecco's modified Eagle's medium, cells were treated with vehicle or ligand for 30 min prior to imaging; this was carried out in Tyrode's buffer (0.135 M NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 20 mM HEPES, pH 7.4) supplemented with 5 mM glucose.

Transactivation Assays—Cells were cultured in 6-well plates and transfected with GFP-RXR (50 ng), BFP-VDR (100 ng), or both along with a luciferase reporter construct driven by a VDR response element and pCH110 serving as a transfection efficiency control. 24 h following transfection, cells were treated with appropriate ligands for 24 h. Cells were then lysed and assayed for luciferase activity. Luciferase activity was normalized to {beta}-galactosidase activity.

Subcellular Fractionation—Cells were transfected with an expression vector for RXR (3 µg/100-mm plate), maintained in serum-free Dulbecco's modified Eagle's medium for 24 h, and treated with vehicle or 9cRA (1 µM) for 1 h prior to lysis using a buffer containing 20 mM HEPES, pH 7.4, 5 mM KCl, 0.137 mM NaCl, 5.5 mM glucose, 10 µM EDTA, 0.05% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin. Plates were incubated while rocking (at 4 °C for 20 min), and cells were scraped, transferred, and centrifuged at 1000 rpm to separate cytoplasmic fraction (supernatant) from nuclei (pellet). Pellets were washed three times, and nuclei were lysed in 200 µl of buffer containing 1% Triton X-100, 400 mM KCl, 10 mM Tris-HCl, pH 7.5, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1 µg/ml leupeptin. Following a 15-min incubation at 4 °C, mixtures were centrifuged, and supernatant was collected. Protein concentrations were measured by the Bradford assay (Bio-Rad), and an equal amount of proteins was resolved by SDS-PAGE and analyzed by Western blots.

Glutathione S-Transferase (GST) Co-precipitation Assays—Importin{alpha}1{Delta}IBB and importin{beta} were bacterially expressed in fusion with GST. Bacteria were harvested and resuspended in buffer A (20 mM HEPES, pH 7.9, 2 mM dithiothreitol, 1 mM EDTA, 0.5% Nonidet P-40, 0.1% bovine serum albumin, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride). Lysozyme (6 mg/ml) was added, and cells were incubated on ice for 30 min, sonicated, and centrifuged. Supernatants were incubated with GST-Sepharose 4B (Amersham Biosciences) for 2 h. Beads were washed three times and resuspended in buffer A. Concentrations of immobilized proteins were estimated by SDS-PAGE and Coomassie Blue staining using bovine serum albumin as a standard. Receptors used in co-precipitation assays were obtained by ectopic expression in COS-7 cells. Cells were transfected with expression vectors for RXR, for BFP-VDR, or for both. Cells were lysed by a freeze-thaw cycle using buffer A containing aprotinin and leupeptin, and lysates were cleared by centrifugation. Cell lysate was incubated with beads containing 30 µg of the appropriate GST-importin in the presence or absence of ligands. Reaction mixtures were incubated (at 4 °C for 3 h), and beads washed three times in buffer A, resuspended in SDS loading buffer, and boiled. Proteins that co-precipitated with importin were resolved by 10% SDS-PAGE and analyzed by Western blots using GFP antibodies (Clontech) or antibodies for RXR (Santa Cruz Biotechnology).

Multiphoton Microscopy—Multiphoton microscopy was performed using an apparatus similar to that described previously (31). Multiphoton excitation was generated using a Spectra Physics Ti:Sapphire laser (Millenium-Tsunami combination) providing ~100-fs pulses with an 80-MHz repetition rate at 730-nm wavelengths for BFP and fluorescence resonance energy transfer (FRET) imaging and 850 nm for GFP imaging. Beam scanning and image acquisition were performed with a Bio-Rad MRC-1024 scanning system interfaced with an Olympus IX70 inverted microscope. A Conoptics (Danby, CT) 350-80 BKLA Pockel's Cell provided beam intensity modulation and blanking during scanner flyback when data were not being collected. The beam was focused, and the resulting fluorescence was collected with an Olympus 40x/1.3 numerical aperture oil UPlanFl objective. The specimen fluorescence was spectrally selected from the excitation beam (before descanning) using a 670-nm long pass dichroic filter (670DCXXRU, Chroma Technology Corp., Rockingham, VT) and then further separated into either blue or green detection channels using a 490DCXR dichroic and BGG22 and hq575/150 filters (Chroma). Subsequently the fluorescence was detected using two bialkali photomultiplier tube (PMT) assemblies (Hamamatsu, HC125-02), and the resulting signal was transmitted to the standard MRC-1024 integrators by intercepting the photomultiplier inputs on the power cable. Imaging data consisted of at least three imaging sessions with ~30 cells analyzed for each condition.



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  		FIGURE 1.
VDR interacts with importin{alpha}, whereas RXR associates with importin{beta}. A, transcriptional activation by BFP-VDR and GFP-RXR. COS-7 cells were transfected with a luciferase reporter vector driven by a VDR response element and with a pCH110 vector (transfection efficiency control). Cells were also transfected with expression vectors for BFP-VDR and GFP-RXR or with an empty vector. Following an overnight incubation, cells were treated with vehicle or with 9cRA (1 µM), D3 (100 nM), or both for 24 h. Cells were lysed, and luciferase expression was measured and normalized to {beta}-galactosidase activity. Data are mean ± S.D. (n = 3). B-E, interactions of RXR and VDR with importins. Importin{alpha}1{Delta}IBB and importin{beta} were bacterially expressed as GST fusion proteins and immobilized on glutathione-Sepharose beads. Receptors were individually expressed in COS-7 cells, and cell lysates containing each receptor were used in co-precipitation assays. The interactions of RXR (B and D), RXRnlsm (D), or BFP-VDR (C and E) with immobilized importin{alpha}1{Delta}IBB (imp{alpha}, B and C) or importin{beta} (imp{beta}, D and E) were examined by co-precipitation assays carried out in the absence or the presence of the denoted ligands. Proteins that precipitated with the respective importins were resolved by SDS-PAGE and visualized by Western blots. Uniform loading of bait was verified by Ponceau S staining of the membranes. Experiments were carried out three times with similar results. luc, luciferase.

 
Image Analysis—To examine individual cell statistics with a statistically significant sampling size, nuclei were automatically selected and analyzed (IDL, Research Systems Inc.) by adopting an object recognition protocol in which the image to be analyzed was thresholded by an amount given by the average background fluorescence (b) plus two standard deviations ({sigma}) of this background value (b + 2{sigma}). Contiguous regions smaller than 5 um2 were assumed to be noise and ignored. From each region average blue and green values were compiled and corrected for the image background (the median pixel value in each image). A similar protocol could not be used for obtaining cytoplasmic statistics because of the abundance of mitochondria-derived cell autofluorescence (presumably due to NADH). Cytoplasmic-to-nuclear fluorescence ratios were determined manually using the histogram function in Adobe Photoshop (San Jose, CA). Regions of interest that contained no mitochondrial autofluorescence were selected (by eye). Average blue or green channel values were corrected by background measurements in cells that were not transfected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of GFP-RXR and BFP-VDR—To enable imaging of RXR and VDR in live cells, mammalian expression constructs for GFP-RXR and for BFP-VDR were generated. To examine the functionality of these proteins, COS-7 cells were transfected with an empty vector or co-transfected with plasmids encoding GFP-RXR and BFP-VDR. Transcriptional activities were examined by transactivation assays using a luciferase reporter driven by a VDR response element. Minimal responses were observed upon transfection of an empty vector, reflecting the low concentration of VDR in these cells (Fig. 1A). Upon transfection of the labeled receptors, the expression of the reporter was markedly up-regulated following treatment with either D3 or the RXR ligand 9cRA. Concomitant treatment with both ligands resulted in synergistic reporter activation. These observations verify that the tagged receptors are fully functional in their ability to form heterodimers, bind DNA, and activate transcription in response to their cognate ligands.

VDR Associates with Importin{alpha}, whereas RXR Binds to Importin{beta}—Nuclear import of proteins is often accomplished by adapter proteins known as {alpha} importins, which in turn associate with an importin{beta} to mediate nuclear localization (12). It was also reported that some cargoes interact directly with importin{beta} and that, in some cases, NLSs that are recognized by importin{beta} are similar to classical NLSs in that they are comprised of a cluster of basic residues. To examine the mechanisms that underlie the nuclear import of VDR and RXR, their association with importin{alpha}1 and importin{beta} and the effect of cognate ligands on these interactions were investigated. As the molecular weights of the two receptors are similar, we used BFP-VDR and untagged RXR to allow for resolution of the receptors by SDS-PAGE. COS-7 cells were transfected with either expression vectors and lysed 24 h after transfection to obtain lysates that contained the respective receptors. In these experiments, importin{alpha}1 lacking its autoinhibitory importin{beta}-binding domain (importin{alpha}{Delta}IBB) was used. The IBB serves to associate importin{alpha} with importin{beta}; this association in turn mediates the nuclear import of the importin{alpha}-cargo complex (32). In the absence of importin{beta}, the domain internally folds to cover the cargo-binding region of importin{alpha} and thus inhibits the association of the protein with its cargo. Hence in the absence of importin{beta}, cargo binding by importin{alpha} is quite weak. Note as well that the IBB is important for the ability of importin{alpha} to release its cargo once in the nucleus but is not involved in cargo binding in the cytoplasm (33). Hence to bypass the need to include importin{beta} in pull-down assays aimed at examining the interactions VDR and RXR with of importin{alpha}, a truncated protein lacking the autoinhibitory domain was used. Importin{alpha}{Delta}IBB and importin{beta} were bacterially expressed as GST fusion proteins and immobilized on glutathione-Sepharose beads, and their interactions with RXR and BFP-VDR were investigated by co-precipitation assays. Lysates were mixed with immobilized importin, beads were centrifuged and washed, and proteins that precipitated with the importin were resolved by SDS-PAGE and visualized by Western blots. As a control, nonspecific association of receptors with immobilized GST alone was examined. In all experiments, neither RXR nor VDR associated with GST alone (e.g. Fig. 1, B and D, and see also Fig. 5C). RXR did not bind to importin{alpha}{Delta}IBB (Fig. 1B). In contrast, VDR weakly associated with importin{alpha}{Delta}IBB in the absence of ligand, and the interaction was markedly stabilized in the presence of D3 (Fig. 1C). Although RXR did not associate with importin{alpha}, this receptor displayed a robust interaction with importin{beta}. The association was readily apparent in the absence of ligand and was further enhanced in the presence of 9cRA (Fig. 1D). VDR displayed a weak and ligand-independent interaction with importin{beta} (Fig. 1E). These observations reveal that, despite the similarities of their NLSs, RXR and VDR are recognized by different importins.



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  		FIGURE 2.
Nuclear translocation of RXR is enhanced by 9cRA and mediated by an NLS in the DBD of the receptor. A, COS-7 cells were transfected with an expression construct for GFP-RXR and imaged prior to and following a 30-min treatment with 9cRA (1 µM). B, quantitation of the fluorescence intensity of GFP-RXR in nuclei of transfected cells. Fluorescence in nuclei of 30 cells was quantitated to obtain mean ± S.E. C, untransfected COS-7 cells or cells transfected with an expression vector for RXR were treated with vehicle or 9cRA (1 µM) for 1 h. Cells were lysed, and cytoplasmic (cyt) and nuclear (nuc) fractions were separated and analyzed for the presence of RXR by Western blots. The cytosolic and nuclear markers {beta}-tubulin and TBP, respectively, were used to verify enrichment of the relevant fractions. Experiments were carried out three times with similar results. D, cells were transfected with an expression construct for GFP-RXRnlsm, treated with vehicle or 9cRA (1 µM) for 30 min, and imaged. WT, wild type.

 
9cRA Enhances the Nuclear Translocation of RXR—To examine the subcellular distribution of RXR, COS-7 cells were transfected with an expression vector encoding GFP-RXR. Cells were grown for 24 h in serum-free medium, treated with vehicle or 9cRA for 30 min, and imaged using multiphoton fluorescence microscopy (Fig. 2A). To obtain statistically meaningful data, the intensity of GFP-RXR fluorescence in the nuclei of 30 cells treated in the absence or presence of ligand was quantitated (Fig. 2B). In agreement with the observations that RXR displays a robust association with importin{beta} in the absence of ligand (Fig. 1D), apoRXR was found to be predominantly nuclear. Consequently fluorescence intensity in the cytoplasm could not be accurately measured on the background of the intense nuclear fluorescence, and data are provided on fluorescence intensity in the nucleus only. Also in agreement with the stabilization of importin{beta} binding by RXR upon addition of 9cRA, treatment of cells with this ligand resulted in a significant enhancement of nuclear GFP-RXR fluorescence. These observations suggest that, in the absence of ligand, a fraction of RXR is retained in the cytosol, and that this fraction is induced to undergo nuclear translocation upon addition of 9cRA. To further examine the subcellular distribution of RXR, subcellular fractionations were carried out. COS-7 cells expressing endogenous RXR or cells ectopically overexpressing the receptor were treated with vehicle or 9cRA for 1 h. Nuclei were separated from cytosolic fractions, and the RXR content in these fractions was monitored by Western blot analyses. The nuclear and cytosolic markers TBP and {beta}-tubulin, respectively, were used as loading controls and to verify successful separation of the respective fractions (Fig. 2C). The data clearly show that, in the absence of ligand, RXR is present both in the cytoplasm and in the nucleus and that it further partitions into the nucleus upon treatment with 9cRA. Note that in the lane showing nuclear RXR in the presence of 9cRA the loading control TBP is weaker as compared with TBP in the lane in which RXR was probed in the absence of the ligand. Hence ligand treatment increased the nuclear fraction of both endogenous and ectopically expressed RXR.

The nuclear localization signal of several nuclear receptors, including RXR and VDR, was mapped to their DBD where it is found between the two zinc fingers that comprise the DNA-binding motif of the receptors (21). In RXR, this NLS consists of the sequence KRTVRK. To generate an RXR defective in its ability to undergo nuclear localization, the four basic residues of this sequence within the GFP-RXR expression vector were replaced with alanines. The resulting construct (GFP-RXRnlsm) was transfected into COS-7 cells, and the subcellular distribution of the mutant protein was examined. The images (Fig. 2D) show that RXRnlsm was excluded from the nucleus both in the presence and absence of 9cRA. Taken together with the observations that the mutant protein was unable to bind to importin{beta} (Fig. 1D), these findings confirm the identification of the sequence as the RXR NLS and show that it mediates the interactions of the receptor with this importin.

Ligand Binding Enhances the Nuclear Translocation of VDR—To examine the subcellular distribution of VDR and the effect of the receptor's ligand on this distribution, COS-7 cells were transfected with an expression vector for BFP-VDR, grown for 24 h in serum-free medium, treated with vehicle or D3 for 30 min, and imaged (Fig. 3A), and fluorescence intensity in the nuclei and cytoplasm of 30 cells was quantitated (Fig. 3B). In the absence of its ligand, VDR partitioned between the cytoplasm and the nucleus. Treatment with D3 resulted in a marked redistribution, mobilizing a large fraction of the receptor into the nucleus. These observations correlate well with the ability of the VDR ligand to enhance the association of the receptor with importin{alpha} (Fig. 1C).



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  		FIGURE 3.
Nuclear translocation of VDR is induced by D3 and partially mediated by an NLS in the DBD of the receptor. A, COS-7 cells were transfected with an expression construct for BFP-VDR and imaged prior to and following a 30-min treatment with D3 (100 nM). B, the ratio of fluorescence intensity of BFP-VDR in nuclei (nuc) and cytosol (cyt) of 30 transfected cells was quantitated to obtain mean ± S.E. C, COS-7 cells were transfected with an expression construct for BFP-VDRnlsm and imaged prior to and following a 30-min treatment with D3 (100 nM). D, the ratio of fluorescence intensity of BFP-VDRnlsm in nuclei and cytosol of 30 transfected cells was quantitated to obtain mean ± S.E. WT, wild type.

 
Like RXR, the DNA-binding domain of VDR contains a stretch rich in basic amino acid residues, and it was suggested that this stretch, comprised of the sequence RRSMKRK, functions as the NLS of the receptor (21). A BFP-VDR mutant containing the replacements (R/A)(R/A)SM(K/A)(R/A)K (BFP-VDRnlsm) was generated and transfected into COS-7 cells, and the mutant was imaged in the absence or presence of D3. The mutant protein was found to be largely excluded from the nucleus in the absence of ligand (Fig. 3C,left panel). However, it underwent nuclear translocation upon addition of D3 (Fig. 3, C,right panel, and D). Although the ligand-induced nuclear translocation of the mutant did not result in its complete accumulation in the nucleus (Fig. 3, compare D with B), the distinct ligand response of VDRnlsm suggests that, unlike RXR whose nuclear translocation relies solely on the DBD-NLS, VDR contains an additional, ligand-responsive NLS. These observations are thus in agreement with previous reports indicating that VDR harbors more than one functional NLS (21, 23).

RXR-VDR Heterodimerization Is Stabilized in the Presence of both D3 and 9cRA—The observations that cytoplasmic fractions of both RXR and VDR mobilize to the nucleus upon binding of their respective ligands raise the question of how the nuclear localization of the RXR-VDR heterodimer is controlled. It should be noted in regard to this that, in addition to regulating the subcellular distribution of their cognate receptors, ligands affect the stability of the VDR-RXR heterodimer. Specifically previous studies indicated that 9cRA inhibits, whereas D3 strengthens, the interactions between the receptors and that the heterodimer is maximally stabilized in the presence of both ligands (9, 10). To examine whether 9cRA and D3 similarly modulate the interactions between RXR and VDR in cells, we used the BFP-VDRnlsm and GFP-RXRnlsm constructs, which harbor mutations in the NLS of the receptors. Cells were co-transfected with vectors encoding the mutants and imaged. The effect of ligands on BFP-VDRnlsm-GFP-RXRnlsm heterodimerization was imaged by exciting only the BFP, monitoring both green and blue emission channels simultaneously, and extracting the green/blue ratio, so-called FRET microscopy. When the two labels are in close proximity to each other (Förster distances of 1-5 nm for commonly used fluorophore pairs), an excited BFP molecule can transfer energy to a GFP molecule via a radiationless process, resulting ultimately in a green photon. Thus, associated BFP-GFP pairs will appear more green than unassociated BFP and GFP molecules at the same average concentration.

Similarly to their localization when transfected individually, BFP-VDRnlsm and GFP-RXRnlsm displayed nuclear exclusion when expressed in tandem (Fig. 4A). In contrast with the ability of D3 to induce partial nuclear mobilization of BFP-VDRnlsm when transfected alone (Fig. 3C), this receptor remained predominantly extranuclear even in the presence of its ligand when co-expressed with RXRnlsm. The cytosolic retention of VDRnlsm by RXRnlsm indicates the presence of VDR-RXR heterodimers in the cytosol. The inability of D3 to induce nuclear localization of VDR under these conditions thus allows for monitoring the effects of ligands on heterodimer formation without interference from large effects on subcellular redistribution.

Cells co-transfected with vectors encoding BFP-VDRnlsm and GFP-RXRnlsm were treated with vehicle, 9cRA, D3, or both ligands, and the interactions between the two mutant receptors were monitored by FRET imaging. Treatment with either D3 or 9cRA individually had little effect, but addition of both ligands resulted in a 22% increase in FRET (Fig. 4B). The magnitude of the increase was relatively small, but this ratio change corresponds to that measured in dialyzed cell extracts under conditions in which these probes undergo heterodimerization. Hence although some of the details of the effects of ligands on the RXR-VDR heterodimer, such as destabilization by 9cRA, could not be visualized in this setting, the observations do suggest that, similarly to their behavior in vitro, ligand binding by both RXR and VDR significantly strengthens their interactions in live cells.

VDR Recruits RXR-VDR Heterodimers to Importin{alpha}—The observations that VDR associates with importin{alpha} whereas RXR binds to importin{beta} (Fig. 1) raise the question of which of these pathways mediate the nuclear localization of RXR-VDR heterodimers. To address this question, BFP-VDR and RXR were co-expressed in COS-7 cells, and whole cell lysates containing both proteins were obtained. The interactions of these proteins with importin{alpha} and importin{beta} were then examined by co-precipitation assays. Similarly to its behavior when present alone, VDR co-expressed with RXR associated with importin{alpha}{Delta}IBB in a D3-dependent manner (Fig. 5A,top). Reprobing the same membrane using RXR antibodies showed that RXR co-precipitated with VDR and importin{alpha}{Delta}IBB and that the interaction was strengthened by D3 but not by 9cRA (Fig. 5A,bottom). Similar results were obtained upon co-precipitation of BFP-VDR and RXRnlsm (Fig. 5B). Hence VDR recruits the RXR-VDR heterodimer to importin{alpha} in a process controlled by its ligand.



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  		FIGURE 4.
D3 and 9cRA stabilize VDR-RXR association in cells. A, COS-7 cells were co-transfected with expression vectors for GFP-RXRnlsm and BFP-VDRnlsm. Cells were treated with the denoted ligands for 30 min, and heterodimerization between the labeled receptors was visualized by FRET imaging. Imaging was carried out by excitation of BFP-VDR and monitoring the ratio of green/blue fluorescence emission (see "Experimental Procedures" for details). B, green/blue ratio in cytoplasm (cyto) of cells co-transfected with BFP-VDRnlsm and GFP-RXRnlsm. Data are mean ± S.E. (n = 30).

 



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  		FIGURE 5.
VDR recruits the RXR-VDR heterodimer to importin{alpha}, whereas RXR does not mediate heterodimer association with importin{beta}. Importin{alpha}1{Delta}IBB and importin{beta} were bacterially expressed as GST fusion proteins and immobilized on glutathione-Sepharose beads. BFP-VDR and RXR or BFP-VDR and RXRnlsm were co-expressed in COS-7 cells. Co-precipitation assays were carried out to examine the interactions of BFP-VDR and RXR (A) or the interactions of BFP-VDR and RXRnlsm (B) with importin{alpha}1{Delta}IBB (imp{alpha}) in the absence or presence of ligands. The interactions of RXR and BFP-VDR with importin{beta} (imp{beta}, C) were similarly examined. Proteins that precipitated with the respective importins were resolved by SDS-PAGE and visualized by Western blots. Membranes were probed using VDR antibodies and then reprobed for RXR. In all experiments, uniform loading of bait was verified by Ponceau S staining of the membranes. Experiments were carried out three times with similar results.

 
RXR Does Not Mediate Binding of RXR-VDR Heterodimers to Importin{beta}—The ability of RXR to recruit RXR-VDR heterodimers to importin{beta} was then examined. Co-precipitation assays were carried out using immobilized importin{beta} and cell lysates containing RXR and BFP-VDR (Fig. 5C). Similarly to its behavior when present alone, RXR associated with importin{beta}, and the interaction was slightly enhanced in the presence of 9cRA. Interestingly the interactions of RXR with importin{beta} were inhibited upon addition of D3, and the inhibition was more pronounced in the presence of both D3 and 9cRA. Considering that the RXR-VDR association is stabilized by D3 and is further strengthened in the presence of ligands for both receptors, these observations are likely to indicate that RXR associates with importin{beta} as a homodimer and that this receptor dissociates from this importin upon heterodimer formation. Like its behavior when present alone, the interactions of VDR with importin{beta} in the presence of RXR were weak and ligand-independent (Fig. 5C,bottom), indicating that heterodimeric RXR does not recruit VDR to importin{beta}.

Nuclear Translocation of the RXR-VDR Heterodimer Is Mediated by VDR—Multiphoton microscopy was then used to examine the subcellular distributions of VDR and RXR in cells that ectopically express both receptors. In these experiments, concomitant imaging of GFP-RXR and BFP-VDR to determine relative subcellular concentrations was complicated by a small amount of FRET when receptors were heterodimerized. To achieve the cleanest possible signal, each partner within the RXR-VDR heterodimer was separately visualized. Cells were co-transfected with either GFP-RXR and a construct encoding untagged VDR or with a construct for untagged RXR in conjunction with BFP-VDR. In the absence of ligands, co-expression of the receptors did not significantly alter their subcellular localization; i.e. GFP-RXR was present predominantly in the nucleus, and BFP-VDR partitioned between the cytoplasm and the nucleus.

To study the effects of cognate ligands on the subcellular distribution of VDR when co-expressed with RXR, cells were transfected with BFP-VDR and untagged RXR, cultured in a serum-free medium for 24 h, and imaged prior to and following a 30-min treatment with vehicle, 9cRA, D3, or both ligands. The partitioning of BFP-VDR between the nucleus and the cytoplasm in 30 cells was quantitated (Fig. 6A). BFP-VDR co-expressed with RXR mobilized to the nucleus upon treatment with D3 or both 9cRA and D3. Treatment with 9cRA had a modest effect. As RXR does not recruit heterodimers to its "cognate" importin, the response of VDR to 9cRA suggests that, even when co-expressed with RXR, this receptor can move to the nucleus on its own. Specifically this effect can be understood to reflect that inhibition of RXR-VDR heterodimerization by 9cRA (9, 10) results in release of VDR monomers from the heterodimer, enabling their nuclear localization. As D3 enhances the recruitment of VDR as well as of RXR-VDR heterodimers to importin{alpha}, nuclear mobilization by D3 may reflect import of either VDR, the heterodimers, or both. The conclusion that the modest induction of VDR translocation by 9cRA is not mediated by RXR was further supported by examining the behavior of VDR in the presence of RXRnlsm (Fig. 6B). These data showed that 9cRA also somewhat induced nuclear translocation of VDR under conditions where RXR cannot move to the nucleus using its own NLS. We then examined the ability of the VDR mutant lacking its DBD-NLS to mobilize to the nucleus in the presence of RXR (Fig. 6C). The behavior of VDRnlsm in the presence of RXR mimicked its behavior when transfected alone (Fig. 3D); i.e. the mutant displayed partial nuclear translocation in response to D3. Hence RXR does not mediate the nuclear translocation of VDR; i.e. it is not actively involved in nuclear import of the RXR-VDR heterodimer.



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  		FIGURE 6.
VDR mediates the nuclear localization of RXR-VDR heterodimers. A-C, COS-7 cells were transfected with RXR and BFP-VDR (A), with RXRnlsm and BFP-VDR (B), or with RXR and BFP-VDRnlsm (C). Cells were treated with the denoted ligands, and the ratio of fluorescence intensity of BFP-VDR in nuclei (nu) and cytosol (cyt) of transfected cells was quantitated. D and E, COS-7 cells were transfected with expression vectors for GFP-RXR and untagged VDR (D) or for GFP-RXRnlsm and VDR (E). Cells were treated with the denoted ligands, and the fluorescence intensity of GFP-RXR in nuclei of 30 transfected cells (D) or the ratio of fluorescence intensity of GFP-RXRnlsm in nuclei and cytosol of 30 transfected cells (E) was quantitated. In all cases, data are mean ± S.E. (n = 30).

 
To visualize RXR in the presence of VDR, cells were co-transfected with GFP-RXR and untagged VDR. As wild type RXR is predominantly nuclear, the fluorescence intensity of GFP-RXR in the nucleus over-shadows the cytoplasmic signal, rendering it difficult to accurately obtain the nuclear/cytosolic ratio. Hence the effect of ligands on nuclear localization in this setting was obtained by monitoring the fluorescence of GFP-RXR in the nucleus (Fig. 6D). A small fraction of GFP-RXR co-expressed with VDR translocated to the nucleus in response to either 9cRA or D3. However, the movement was most pronounced upon treatment of cells with both ligands. Considering that the stability of the RXR-VDR heterodimer is maximal when both receptors are ligated and that VDR recruits heterodimers to its cognate importin, the observations that efficient translocation required the presence of both 9cRA and D3 suggest that, when co-expressed with VDR, a significant fraction of cytosolic RXR translocates to the nucleus as a heterodimer. This conclusion was strongly supported by the findings that, in the presence of VDR, the nuclear translocation of GFP-RXRnlsm was also induced by D3 and, more prominently, by D3 in conjunction with 9cRA (Fig. 6E). Hence the NLS of RXR was dispensable for its D3-induced nuclear translocation, demonstrating that, in the presence of VDR, RXR moves to the nucleus as a heterodimer via a process mediated by VDR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented above demonstrate that RXR and VDR are imported into the nucleus by distinct pathways; RXR selectively associates with importin{beta}, whereas VDR binds to importin{alpha}. The observations further indicate that association of both receptors with their cognate importins and hence their nuclear import are both enhanced by the respective ligands but that the magnitude of the ligand response is markedly different. In the absence of its ligand, VDR weakly interacts with importin{alpha}, and the association is considerably enhanced in the presence of D3. Correspondingly apoVDR partitions between the cytoplasm and the nucleus, and ligand binding triggers massive nuclear import of this receptor. In contrast, RXR robustly binds to importin{beta} and is predominantly nuclear even in the absence of ligand. Importin binding and nuclear import of RXR are modestly enhanced upon addition of 9cRA.

In agreement with previous reports, the nuclear localization of both receptors was found to be mediated by an NLS located between the zinc fingers of their DBD, the so-called NL1. Although NL1 appears to comprise the sole such signal in RXR, VDR is likely to contain other ligand-responsive NLSs (Fig. 3 and Refs. 21, 23, and 27). We note, however, that mutation of NL1 of VDR abolished its ability to associate with importin{alpha}1 both in the absence and in the presence of D3 and that VDRnlsm also did not bind to importin{beta} (data not shown). The mechanism by which the additional NLS(s) of VDR mediate(s) nuclear import thus remain to be clarified. The data demonstrate that, despite the similarities of the positions and the sequences of the NL1 of the two receptors, this NLS is highly accessible for importin binding in apoRXR but is sequestered in VDR in the absence of ligand. The ligand responsiveness of the NL1 indicates that, within each receptor, there must exist intramolecular communication between the LBD and the DBD and that such a communication allows the ligand to regulate the functionality of the NLS.

In the context of RXR-VDR heterodimers, VDR was competent for importin{alpha} binding, recruited the heterodimers to this importin (Fig. 5, A and B), and efficiently mediated the nuclear import of the complex (Fig. 6). In contrast, the interactions of RXR with importin{beta} were inhibited upon heterodimerization (Fig. 5C), providing a rationale for understanding the observations that the RXR NLS was not involved in the nuclear import of the heterodimer (Fig. 6, B and E). Association of RXR with its heterodimerization partners is mediated primarily by an interaction interface located in the LBD with an additional binding energy contributed by association through the DBD (7, 34, 35). However, the dimerization of the DBD is weak, occurs only when the heterodimers are bound to DNA, and, in some cases, is not observed at all (36). As importin binding takes place in solution, the findings that the accessibility of the NLS of RXR is regulated by heterodimerization thus further indicate that the LBD exerts a control over the conformation of the DBD. The three-dimensional structures of DNA-bound DBDs and of LBDs of various receptors, including RXR and VDR, have been solved (37-40). However, insights into the basis of functional communication between the domains has long been hampered by the inability to obtain precise structural information on full-length receptors. Delineation of the intramolecular networks through which ligand binding and heterodimer formation regulate the accessibility of the NLS thus awaits further studies.



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  		FIGURE 7.
Model for nuclear import of RXR, VDR, and RXR-VDR heterodimers. Nuclear import of RXR and VDR is mediated by importin{beta} and importin{alpha}, respectively. RXR strongly associates with importin{beta} and localizes to the nucleus in the absence of ligand. Importin{beta} binding and nuclear import of RXR are modestly enhanced by 9cRA. On the other hand, association of VDR with importin{alpha} and its nuclear translocation are considerably stabilized by D3. RXR-VDR heterodimerization is inhibited by 9cRA and maximally stabilized in the presence of both D3 and 9cRA. Nuclear import of RXR-VDR heterodimers is mediated by VDR through its D3-induced association with importin{alpha}.

 
It is well established that ligands control the transcriptional activities of nuclear receptors by inducing structural rearrangements in the AF-2 region at the carboxyl terminus of their LBDs (41). In turn, these changes triggers the exchange of receptor-bound transcriptional repressors with activators, resulting in up-regulation of target gene transcription (for a review, see Ref. 42). Previous studies (e.g. (9, 10, 21, 27)) and the present findings demonstrate that, in addition to regulating the interactions of receptors with transcriptional co-regulators, ligands also control the behavior of their receptors in solution. Hence ligands direct the partitioning of RXR between its various oligomers, and they control the nuclear import of receptors. As depicted in the model shown in Fig. 7, the data presented above indicate that, although RXR and VDR can independently move to the nucleus, the nuclear import of their mutual heterodimer is controlled predominantly by VDR, mediated by importin{alpha}, and regulated by D3. It is worth noting that, in addition to its role as a common binding partner for multiple other receptors, RXR displays important nuclear activities on its own; holo-RXR can activate transcription as a homodimer (6, 7), and the unliganded RXR homotetramer functions as a DNA architectural factor (5). These activities require the presence of RXR in the nucleus and indeed RXR is predominantly nuclear in the absence of either ligand or a heterodimerization partner. However, a fraction of RXR remains cytoplasmic under these conditions. This fraction can be mobilized into the nucleus upon addition of an RXR ligand presumably to enable transcriptional activation by RXR homodimers. The cytosolic fraction of RXR is also imported into the nucleus as a heterodimer in a process that requires the presence of both the heterodimerization partner and the ligand for this partner. It is tempting to speculate that such a regulatory mechanism may operate to ensure that mobilization only occurs under conditions where the heterodimer is fully functional, i.e. when the partner is activated.

Whether the regulatory features reported here to underlie the nuclear import of the RXR-VDR heterodimer are shared by other RXR heterodimers remains to be clarified. However, the transcriptional activity of heterodimeric RXR appears to depend on the nature of the partner (43, 44). In the context of what is termed "permissive heterodimers," e.g. RXR-PPAR (peroxisome proliferator-activated receptor), both the partner and RXR can mediate ligand-induced transcriptional activation. In contrast, in the context of "non-permissive" heterodimers, e.g. RXR-VDR, the complex can be activated by the ligand for the partner but only poorly responds to RXR-specific ligands. This inhibition is relieved upon ligation of the partner, resulting in synergistic activation in the presence of both ligands. The data reported here suggest that the subjugation of RXR to a non-permissive partner and its ligand may be explained, at least in part, by the dominance of the partner over the nuclear import of the complex.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant RO1-CA68150 (to N. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by NIBIB/National Center for Research Resources, National Institutes of Health Grant 9 P41 EB001976-16. Back

2 To whom correspondence should be addressed: Division of Nutritional Sciences, 222 Savage Hall, Cornell University, Ithaca, NY 14853. Tel.: 607-255-2490; Fax: 607-255-1033; E-mail: nn14{at}cornell.edu.

3 The abbreviations used are: RXR, retinoid X receptor; BFP, blue fluorescent protein; DBD, DNA-binding domain; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GST, glutathione S-transferase; IBB, importin{beta}-binding domain; LBD, ligand-binding domain; NLS, nuclear localization signal; VDR, vitamin D receptor; 9cRA, 9-cis-retinoic acid; D3, 1,25(OH)2D3; TBP, TATA-binding protein. Back


   ACKNOWLEDGMENTS
 
We are grateful to Alec Hodel (Emory University School of Medicine, Atlanta, GA), Stephen Adam (Northwestern University, Chicago, IL), and Hector DeLuca (University of Wisconsin, Madison, WI) for sharing constructs.



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 RESULTS
 DISCUSSION
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