Dimerization with Retinoid X Receptors Promotes Nuclear Localization and Subnuclear Targeting of Vitamin D Receptors

The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor α (RXR) to control transcriptional activity of target genes. To explore the influence of heterodimerization on the subcellular distribution of these receptors in living cells, we developed a series of fluorescent-protein chimeras. The steady-state distribution of the yellow fluorescent protein-RXR was more nuclear than the unliganded green fluorescent protein (GFP)-VDR. Coexpression of RXR-blue fluorescent protein (BFP) promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. FRET also revealed that calcitriol induces formation of multiple nuclear foci of heterodimers. Mutational analysis showed a correlation between hormone-dependent nuclear VDR foci formation and DNA binding. RXR-BFP also promoted hormone-dependent nuclear accumulation and intranuclear foci formation of a nuclear localization signal mutant receptor (nlsGFP-VDR) and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR andnlsGFP-VDR distribution or activity. These experiments suggest that RXR has a profound effect on VDR distribution. This effect of RXR to promote nuclear accumulation and intranuclear targeting contributes to the regulation of VDR activity and probably the activity of other heterodimerization partners. The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor α (RXR) to control transcriptional activity of target genes. To explore the influence of heterodimerization on the subcellular distribution of these receptors in living cells, we developed a series of fluorescent-protein chimeras. The steady-state distribution of the yellow fluorescent protein-RXR was more nuclear than the unliganded green fluorescent protein (GFP)-VDR. Coexpression of RXR-blue fluorescent protein (BFP) promoted nuclear accumulation of GFP-VDR by influencing both nuclear import and retention. Fluorescence resonance energy transfer microscopy (FRET) demonstrated that the unliganded GFP-VDR and RXR-BFP form heterodimers. The increase in nuclear heterodimer content correlated with an increase in basal transcriptional activity. FRET also revealed that calcitriol induces formation of multiple nuclear foci of heterodimers. Mutational analysis showed a correlation between hormone-dependent nuclear VDR foci formation and DNA binding. RXR-BFP also promoted hormone-dependent nuclear accumulation and intranuclear foci formation of a nuclear localization signal mutant receptor (nlsGFP-VDR) and rescued its transcriptional activity. Heterodimerization mutant RXR failed to alter GFP-VDR andnlsGFP-VDR distribution or activity. These experiments suggest that RXR has a profound effect on VDR distribution. This effect of RXR to promote nuclear accumulation and intranuclear targeting contributes to the regulation of VDR activity and probably the activity of other heterodimerization partners. vitamin D receptor human retinoid X receptor α green fluorescent protein yellow fluorescent protein blue fluorescent protein heterodimerization nuclear localization signal 9-cis-retinoic acid 17/2.8 rat osteosarcoma cells CV-1-derived cell line stably expressing YFP-RXR 293-derived cell line stably expressing GFP-VDR luciferase gene that is under control of the 25-hydroxyvitamin D3 24-hydroxylase promoter fluorescence resonance energy transfer glucocorticoid receptor Proteins of the nuclear receptor superfamily mediate response to hormones or intracellular signals into transcriptional responses and regulate an array of important cellular functions. A member of the nuclear receptor superfamily, the vitamin D receptor (VDR)1, mediates effects of calcitriol on bone development and maintenance, calcium homeostasis, immune functions, endocrine functions, vitamin D metabolism, and cellular proliferation and differentiation. Like other class II nuclear receptors, such as the thyroid hormone receptor, the retinoic acid receptor, and many orphan receptors, VDR requires heterodimerization with the retinoid X receptor (RXR) for high affinity binding to target genes (1Sone T. Kerner S. Pike J.W. J. Biol. Chem. 1991; 266: 23296-23305Abstract Full Text PDF PubMed Google Scholar, 2Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (283) Google Scholar). VDR and RXR can heterodimerize in the absence of calcitriol, and these heterodimers regulate basal transcriptional activity of target genes and exert transcriptional silencing functions (3Tagami T. Lutz W.H. Kumar R. Jameson J.L. Biochem. Biophys. Res. Commun. 1998; 253: 358-363Crossref PubMed Scopus (91) Google Scholar). The addition of calcitriol stabilizes the heterodimers and promotes their binding to the vitamin D response elements (4Carlberg C. Saurat J.H. J. Investig. Dermatol. Symp. Proc. 1996; 1: 82-86PubMed Google Scholar). The importance of heterodimerization in VDR functions led us to investigate the spatial and temporal relationships between these receptors in living cells.Recently we and others have used green fluorescent protein chimeras of VDR to study the receptor distribution in living cells (5Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6Michigami T. Suga A. Yamazaki M. Shimizu C. Cai G. Okada S. Ozono K. J. Biol. Chem. 1999; 274: 33531-33538Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Hsieh J.C. Shimizu Y. Minoshima S. Shimizu N. Haussler C.A. Jurutka P.W. Haussler M.R. J. Cell. Biochem. 1998; 70: 94-109Crossref PubMed Scopus (61) Google Scholar). Unlike the glucocorticoid receptor (GR), which stays in the cytoplasm without the ligand, the unliganded VDR distributes evenly between the cytoplasm and the nucleus. This indicates that the regulation of VDR distribution is more complex than the regulation of GR distribution. The fact that VDR distribution is similar to thyroid hormone receptor distribution (8Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) raised the possibility that heterodimerization with RXR could account for the partial nuclear localization of both VDR and thyroid hormone receptor.Most of what we know about the subcellular distribution of RXR comes from immunohistological and cell fractionation studies. In these studies, RXR was found in the nuclei of normal human skin cells (9Reichrath J. Mittmann M. Kamradt J. Muller S.M. Histochem. J. 1997; 29: 127-133Crossref PubMed Scopus (48) Google Scholar), the rat kidney (10Sugawara A. Sanno N. Takahashi N. Osamura R.Y. Abe K. Endocrinology. 1997; 138: 3175-3180Crossref PubMed Scopus (30) Google Scholar), and the human pituitary (11Sanno N. Sugawara A. Teramoto A. Abe Y. Yen P.M. Chin W.W. Osamura R.Y. Neuroendocrinology. 1997; 65: 299-306Crossref PubMed Scopus (27) Google Scholar). RXR was also detected in the cytoplasm by cell fractionation experiments (12Janssen J.J. Kuhlmann E.D. van Vugt A.H. Winkens H.J. Janssen B.P. Deutman A.F. Driessen C.A. Curr. Eye Res. 1999; 19: 338-347Crossref PubMed Scopus (31) Google Scholar). Although a nuclear localization of RXR is to be expected, looking in live cells sometimes gives surprises, as has been the partial cytoplasmic localization of the thyroid hormone receptor (8Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In a recent paper, focusing on the localization of an orphan nuclear receptor, the transiently expressed mouse GFP-RXRβ was found in the nuclei of phaeochromocytoma cells (36Katagiri Y. Takeda K., Yu, Z.X. Ferrans J. Ozato K. Guroff G. Nat. Cell Biol. 2000; 2: 435-440Crossref PubMed Scopus (161) Google Scholar). Because previous studies indicated that VDR preferentially associates with RXRα to stimulate transcription (13Kephart D.D. Walfish P.G. DeLuca H. Butt T.R. Mol. Endocrinol. 1996; 10: 408-419PubMed Google Scholar), the subcellular localization of the RXRα had to be explored in detail. Even less is known about the subcellular location of the VDR/RXR heterodimers. Receptors that heterodimerize with RXR are not only synthesized in the cytoplasm but reside in the cytoplasm to some degree before ligand binding. Thus, heterodimerization may influence subcellular localization of RXR or its partner receptors. Using transcriptionally active and stably expressed yellow, green, and blue fluorescent protein chimeras of RXR and VDR and their mutants, we visualized the differences in the distribution of VDR and RXR in living cells by confocal laser-scanning microscopy.Current developments of multicolor fluorescent protein variants also allow visualization of protein interactions in living cells by fluorescence resonance energy transfer microscopy (FRET) (14Periasamy A. Day R.N. Methods Cell Biol. 1999; 58: 293-314Crossref PubMed Google Scholar). This method was used to demonstrate homodimerization of Pit-1 transcription factor and heterodimerization of Pit-1 with cEts-1 in the cell nucleus (15Day R.N. Mol. Endocrinol. 1998; 12: 1410-1419Crossref PubMed Scopus (116) Google Scholar) as well as the association of retinoic acid receptors and estrogen receptors with coactivators (16Chauchereau A. Georgiakaki M. Perrin-Wolff M. Milgrom E. Loosfelt H. J. Biol. Chem. 2000; 275: 8540-8548Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Here, we adapted FRET to establish that GFP-tagged VDR and RXR heterodimerize in living cells and used this technique to explore the distribution of heterodimers.Recent results reveal that transcriptional activities of nuclear receptors and other signaling proteins could be regulated by the control of translocation across the nuclear membrane (17Johnson C. Van Antwerp D. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar). We generated a cell line stably expressing GFP-VDR that allowed us to correlate nuclear receptor content with basal transcriptional activity. Our experiments revealed that RXR promotes nuclear accumulation of VDR. To study the mechanisms of this RXR effect we explored both nuclear import and export of the unliganded VDR by mutational analysis and by temperature changes.During hormone-induced receptor activation, a redistribution from the cytoplasm into the nucleus has been shown in living cells for GFP chimeras of the mineralocorticoid receptor (18Fejes-Toth G. Pearce D. Naray F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2973-2978Crossref PubMed Scopus (211) Google Scholar), the GR (19Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (326) Google Scholar), the thyroid hormone receptor (8Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), the progesterone receptor (20Lim C.S. Baumann C.T. Htun H. Xian W. Irie M. Smith C.L. Hager G.L. Mol. Endocrinol. 1999; 13: 366-375Crossref PubMed Scopus (123) Google Scholar), and the androgen receptor (21Georget V. Lobaccaro J.M. Terouanne B. Mangeat P. Nicolas J.C. Sultan C. Mol. Cell. Endocrinol. 1997; 129: 17-26Crossref PubMed Scopus (158) Google Scholar). We found earlier that the cytoplasmic portion of VDR-GFP also translocates into the nucleus in a ligand-dependent fashion (5Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6Michigami T. Suga A. Yamazaki M. Shimizu C. Cai G. Okada S. Ozono K. J. Biol. Chem. 1999; 274: 33531-33538Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The mechanisms controlling VDR translocation, however, remained to be elucidated. Nuclear import of proteins involves the binding of their nuclear localization signal (NLS) sequences to import receptors (22Powers M.A. Forbes D.J. Cell. 1994; 79: 931-934Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 23Kohler M. Haller H. Hartmann E. Exp. Nephrol. 1999; 7: 290-294Crossref PubMed Scopus (25) Google Scholar). Recently, NLSs have been identified in the VDR (7Hsieh J.C. Shimizu Y. Minoshima S. Shimizu N. Haussler C.A. Jurutka P.W. Haussler M.R. J. Cell. Biochem. 1998; 70: 94-109Crossref PubMed Scopus (61) Google Scholar, 24Luo Z. Rouvinen J. Maenpaa P.H. Eur. J. Biochem. 1994; 223: 381-387Crossref PubMed Scopus (33) Google Scholar). The effects of mutations in these NLS regions have not yet been evaluated in living cells, and the NLS has not yet been identified in the RXR sequence. We used mutational analysis to clarify the impact of heterodimerization on the rapid calcitriol-induced VDR translocation and correlate it with changes in transcriptional activity. Here we coexpressed a heterodimerization-competent RXR with an NLS mutant of the GFP-VDR to demonstrate that RXR also interacts with the nuclear import machinery.Ligand-induced intranuclear pattern changes, including foci formation, have been reported for the GR (19Htun H. Barsony J. Renyi I. Gould D.L. Hager G.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4845-4850Crossref PubMed Scopus (326) Google Scholar), the androgen receptor (21Georget V. Lobaccaro J.M. Terouanne B. Mangeat P. Nicolas J.C. Sultan C. Mol. Cell. Endocrinol. 1997; 129: 17-26Crossref PubMed Scopus (158) Google Scholar), the mineralocorticoid receptor (18Fejes-Toth G. Pearce D. Naray F.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2973-2978Crossref PubMed Scopus (211) Google Scholar), the estrogen receptor (26Htun H. Holth L.T. Walker D. Davie J.R. Hager G.L. Mol. Biol. Cell. 1999; 10: 471-486Crossref PubMed Scopus (219) Google Scholar, 27Stenoien D.L. Mancini M.G. Patel K. Allegretto E.A. Smith C.L. Mancini M.A. Mol. Endocrinol. 2000; 14: 518-534PubMed Google Scholar), and the VDR (5Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Here, we report similar hormone-induced intranuclear foci of YFP-RXR and GFP-VDR. In addition, we used FRET microscopy to study ligand-induced changes in the intranuclear distribution of the GFP-VDR/RXR-BFP heterodimers. We used mutational analysis to explore a connection between DNA binding and nuclear foci formation and used FRET microscopy to visualize the effect of RXR to promote DNA binding of VDR in living cells.DISCUSSIONOur experiments with green and blue fluorescent chimeras of the receptors and FRET microscopy provided the initial evidence for the distribution of VDR/RXR heterodimers in living cells. Confocal microscopy also allowed us to recognize that dimerizing RXR facilitates nuclear accumulation of VDR. In addition, we observed that VDR, RXR, and their respective heterodimers all responded to hormone by forming intranuclear bright foci. Mutational analysis showed a correlation between receptor distribution and transcriptional activity and between nuclear foci formation and DNA binding.At first, we generated N-terminal-tagged GFP-VDR and YFP-RXR and a C-terminal-tagged RXR-BFP expression plasmids. The transcriptional activities of the tagged receptors were confirmed by luciferase reporter assays, and the expression of the correct size proteins were confirmed by Western blot analysis. We also cloned cell lines to stably express the GFP-VDR (GL48) and the YFP-RXR (CYR) to establish reproducible systems for the investigation of hormone-independent and hormone-dependent receptor trafficking. Previously, we and others used alternate GFP chimeras of VDR to show hormone-dependent translocation from the cytoplasm into the nucleus (5Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6Michigami T. Suga A. Yamazaki M. Shimizu C. Cai G. Okada S. Ozono K. J. Biol. Chem. 1999; 274: 33531-33538Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Confocal microscopy on GL48 cells confirmed our previous findings on the steady-state subcellular distribution of VDR by showing that unliganded GFP-VDR remained evenly distributed between the cytoplasm and nucleus (Fig. 2).Our finding on the steady-state nuclear localization of YFP-RXRα in living cells was expected from previous immunolocalization studies, and it is similar to the nuclear localization of the transiently expressed GFP-RXRβ in PC12 cells (36Katagiri Y. Takeda K., Yu, Z.X. Ferrans J. Ozato K. Guroff G. Nat. Cell Biol. 2000; 2: 435-440Crossref PubMed Scopus (161) Google Scholar). In contrast, the presence of YFP-RXR in the cytoplasm is new and suggests that heterodimers of VDR and RXR could also form in the cytoplasm (Fig. 1). Furthermore, the difference between the steady-state localization of RXR and VDR raised the possibility that heterodimerization could influence the distribution of either VDR or RXR.FRET microscopy has not been used extensively in the nuclear receptor field. Here, utilizing a heterodimerization mutant of RXR as a control, we located dimers of RXR-BFP and GFP-VDR for the first time in intact living cells. These FRET studies showed that the tagged receptors are competent to form heterodimers; therefore, they were suitable for studies on the impact of dimerization on subcellular trafficking. FRET results demonstrated that most of the VDR formed dimers with the RXR without the ligand and that these heterodimers were predominantly in the nucleus in a diffuse pattern excluding the nucleoli.Our experiments showed that the dimerization-competent RXR can shift the VDR from the cytoplasm into the nucleus, but the dimerization-deficient RXR is ineffective. This effect of RXR on VDR distribution could involve changes in either VDR export or import across the nuclear envelope. We tested the effect of RXR on VDR export by taking advantage of the differences in their temperature sensitivity. Lowering the temperature of GL48 cells induced export of GFP-VDR into the cytoplasm, but GFP-VDR/RXR-BFP heterodimers were less sensitive to temperature change and remained in the nucleus (Fig. 5). This finding suggests that the export mechanisms for the heterodimer differ from that of VDR. It is possible that export receptors are involved because both VDR and RXR have leucine-rich sequences, which could serve as nuclear export signal (37Hopper A.K. Curr. Biol. 1999; 9: 803-806Abstract Full Text Full Text PDF PubMed Google Scholar). Mutational analysis of these GFP-tagged receptors opens several potential avenues for the investigations of these export mechanisms.We also found that RXR influences nuclear import of VDR. This is most apparent in our experiments with the nlsGFP-VDR, which harbors a mutation in one of the nuclear localization signals of the VDR (7Hsieh J.C. Shimizu Y. Minoshima S. Shimizu N. Haussler C.A. Jurutka P.W. Haussler M.R. J. Cell. Biochem. 1998; 70: 94-109Crossref PubMed Scopus (61) Google Scholar). This mutation severely compromised nuclear import of VDR but did not completely abolish it. The remaining import is likely to be mediated by other domains of the VDR, such as the one suggested within the hinge region (24Luo Z. Rouvinen J. Maenpaa P.H. Eur. J. Biochem. 1994; 223: 381-387Crossref PubMed Scopus (33) Google Scholar). However, we did not find any defect in nuclear import when we deleted this region of GFP-VDR. 2J. Barsony, unpublished data. The ability of RXR to bring the NLS mutant GFP-VDR into the nucleus can be explained by a piggyback mechanism. Such a mechanism was reported to bring the NLS mutant of progesterone receptor into the nucleus by homodimerizing wild-type receptors (38Guiochon-Mantel A. Loosfelt H. Lescop P. Christin-Maitre S. Perrot-Applanat M. Milgrom E. J. Steroid Biochem. Mol. Biol. 1992; 41: 209-215Crossref PubMed Scopus (15) Google Scholar). The effect of RXR could also be indirect; RXR could weaken the VDR interactions with cytoplasmic docking sites, as suggested by a recent report showing that RXR increases the solubility and perhaps assists the folding of the retinoic acid receptor inEscherichia coli (39Li C. Schwabe J.W. Banayo E. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2278-2283Crossref PubMed Scopus (86) Google Scholar). Finally, RXR could also act by complexing with Hsp70 and Hsp90 chaperones (40Holley S.J. Yamamoto K.R. Mol. Biol. Cell. 1995; 6: 1833-1842Crossref PubMed Scopus (72) Google Scholar). Interestingly, RXR not only influenced the steady-state distribution of thenlsGFP-VDR but also restored the effect of calcitriol to induce a rapid nuclear accumulation of the mutant receptors (Figs. 6and 7). This effect is unexpected and implies a role for RXR in the regulation of VDR nuclear import.We found a strong correlation between the ability of RXR to shift VDR into the nucleus and to regulate its transcriptional activity. First, the increase in the nuclear VDR content correlated with an increase in basal transcriptional activity by VDR both in GL48 and CV-1 cells. This hormone-like effect of RXR is consistent with the phantom ligand effect of RXR described earlier (41Schulman I.G. Li C. Schwabe J.W. Evans R.M. Genes Dev. 1997; 11: 299-308Crossref PubMed Scopus (114) Google Scholar). This effect on basal activity may be physiologically important, since previous studies indicate that the unliganded VDR can activate several target genes (42Castillo A.I. Jimenez-Lara A.M. Tolon R.M. Aranda A. Mol. Endocrinol. 1999; 13: 1141-1154Crossref PubMed Scopus (48) Google Scholar, 43Dabrowski M. Robinson E. Hughes S.V. Bland R. Hewison M. Mol. Cell. Endocrinol. 1998; 142: 131-139Crossref PubMed Scopus (6) Google Scholar). Second, RXR facilitated hormone-independent nuclear localization of thenlsGFP-VDR and increased basal transcriptional activity by the nlsGFP-VDR. Third, RXR restored the ability of thenlsGFP-VDR to accumulate in the nucleus after calcitriol addition and to activate the reporter gene after calcitriol addition. This indicates that the regulation of VDR nuclear import by RXR may play a role in hormone-dependent transcriptional regulation. Such regulation could explain how, in some target tissues, changes in the ratio between RXR and VDR can influence calcitriol-induced transcription (44Whitfield G.K. Hsieh J.C. Nakajima S. MacDonald P.N. Thompson P.D. Jurutka P.W. Haussler C.A. Haussler M.R. Mol. Endocrinol. 1995; 9: 1166-1179Crossref PubMed Google Scholar, 45Kahlen J.P. Carlberg C. Nucleic Acids Res. 1997; 25: 4307-4313Crossref PubMed Scopus (14) Google Scholar). Potentially, the regulation of protein trafficking across the nuclear envelope by RXR is a general mechanism that controls the activity of many transcriptional regulators.Regulation of subnuclear trafficking can be one of the ways RXR influences the speed and specificity of transcriptional activities. Here we found that hormone induces YFP-RXR and GFP-VDR to accumulate in foci (Figs. 1 and 2). Our earlier finding that a DNA binding mutant of GFP-GR failed to form foci (46Barsony J. Carroll J. McKoy W. Renyi I. Gould D.L. Htun H. Hager G.L. Microsc. Microanal. 1997; 3 Suppl. 2: 131-132Crossref Google Scholar) and our present finding that the DNA binding mutant of GFP-VDR also fails to form nuclear foci suggest that these foci could signify receptor binding to DNA target sites. This notion is further supported by the FRET experiments, which show that calcitriol treatment causes focal accumulation of heterodimers in the nucleus (Fig. 3). The use of the heterodimerization mutant of RXR-BFP for control makes this argument even more powerful. Moreover, coexpression of RXR-BFP restored the ability of thenlsGFP-VDR to form foci and activate transcription after calcitriol treatment (Figs. 7 and 9). The dimerization-deficient form of RXR-BFP had no effect. This observation is in agreement with earlier findings on the effect of calcitriol to promote binding of VDR/RXR heterodimers to vitamin D response elements (47Thompson P.D. Jurutka P.W. Haussler C.A. Whitfield G.K. Haussler M.R. J. Biol. Chem. 1998; 273: 8483-8491Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 48Lemon B.D. Freedman L.P. Mol. Cell. Biol. 1996; 16: 1006-1016Crossref PubMed Scopus (81) Google Scholar) and suggests that RXR could promote DNA binding by influencing intranuclear targeting of VDR. A local change in receptor dynamics could account for the calcitriol-induced accumulation of heterodimers in discrete subnuclear regions and warrant further investigations by photobleaching techniques.Multicolor fluorescent protein tagging and advanced microscopy methods allowed us to visualize subcellular distribution of VDR, RXR, and their respective heterodimers in living cells. These studies revealed that RXR heterodimerization plays a key role in the translocation and nuclear targeting of VDR, which could explain tissue-specific differences in VDR distribution observed previously by immunocytology (25Prüfer K. Veenstra T.D. Jirikowski G.F. Kumar R. J. Chem. Neuroanat. 1999; 16: 135-145Crossref PubMed Scopus (210) Google Scholar). We also visualized the ligand-dependent interaction of heterodimers with possible DNA binding sites by FRET imaging and found a correlation between intranuclear foci formation and transcriptional activity by mutational analysis. Mutational analysis also indicated that nuclear targeting is independent of DNA binding. As the RXR forms heterodimers with other nuclear receptors and associates with coactivators and corepressors, RXR could also regulate the activity of other partners by regulating subcellular trafficking. The techniques described here open the possibility of exploring the location and kinetics for a multitude of regulatory protein interactions and exploring the mechanisms of transcriptional regulation in living cells. Proteins of the nuclear receptor superfamily mediate response to hormones or intracellular signals into transcriptional responses and regulate an array of important cellular functions. A member of the nuclear receptor superfamily, the vitamin D receptor (VDR)1, mediates effects of calcitriol on bone development and maintenance, calcium homeostasis, immune functions, endocrine functions, vitamin D metabolism, and cellular proliferation and differentiation. Like other class II nuclear receptors, such as the thyroid hormone receptor, the retinoic acid receptor, and many orphan receptors, VDR requires heterodimerization with the retinoid X receptor (RXR) for high affinity binding to target genes (1Sone T. Kerner S. Pike J.W. J. Biol. Chem. 1991; 266: 23296-23305Abstract Full Text PDF PubMed Google Scholar, 2Blumberg B. Evans R.M. Genes Dev. 1998; 12: 3149-3155Crossref PubMed Scopus (283) Google Scholar). VDR and RXR can heterodimerize in the absence of calcitriol, and these heterodimers regulate basal transcriptional activity of target genes and exert transcriptional silencing functions (3Tagami T. Lutz W.H. Kumar R. Jameson J.L. Biochem. Biophys. Res. Commun. 1998; 253: 358-363Crossref PubMed Scopus (91) Google Scholar). The addition of calcitriol stabilizes the heterodimers and promotes their binding to the vitamin D response elements (4Carlberg C. Saurat J.H. J. Investig. Dermatol. Symp. Proc. 1996; 1: 82-86PubMed Google Scholar). The importance of heterodimerization in VDR functions led us to investigate the spatial and temporal relationships between these receptors in living cells. Recently we and others have used green fluorescent protein chimeras of VDR to study the receptor distribution in living cells (5Racz A. Barsony J. J. Biol. Chem. 1999; 274: 19352-19360Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 6Michigami T. Suga A. Yamazaki M. Shimizu C. Cai G. Okada S. Ozono K. J. Biol. Chem. 1999; 274: 33531-33538Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Hsieh J.C. Shimizu Y. Minoshima S. Shimizu N. Haussler C.A. Jurutka P.W. Haussler M.R. J. Cell. Biochem. 1998; 70: 94-109Crossref PubMed Scopus (61) Google Scholar). Unlike the glucocorticoid receptor (GR), which stays in the cytoplasm without the ligand, the unliganded VDR distributes evenly between the cytoplasm and the nucleus. This indicates that the regulation of VDR distribution is more complex than the regulation of GR distribution. The fact that VDR distribution is similar to thyroid hormone receptor distribution (8Zhu X.G. Hanover J.A. Hager G.L. Cheng S.Y. J. Biol. Chem. 1998; 273: 27058-27063Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) raised the possibility that heterodimerization with RXR could account for the partial nuclear localization of both VDR and thyroid hormone receptor. Most of what we know about the subcellular distribution of RXR comes from immunohistological and cell fractionation studies. In these studies, RXR was found in the nuclei of normal human skin cells (9Reichrath J. Mittmann M. Kamradt J. Muller S.M. Histochem. J. 1997; 29: 127-133Crossref PubMed Scopus (48) Google Scholar), the rat kidney (10Sugawara A. Sanno N. Takahashi N. Osamura R.Y. Abe K. Endocrinology. 1997; 138: 3175-3180Crossref PubMed Scopus (30) Google Scholar), and the human pituitary (11Sanno N. Sugawara A. Teramoto A. Abe Y. Yen P.M. Chin W.W. Osamura R.Y. Neuroendocrinology. 1997; 65: 299-306Crossref PubMed Scopus (27) Google Scholar). RXR was also detected in the cytoplasm by cell fractionation experiments (12Janssen J.J. Kuhlmann E.D. van Vugt A.H. Winkens H.J. Janssen B.P. Deutman A.F. Driessen C.A. Curr. Eye Res. 1999; 19: 338-347Crossref PubMed Scopus (31) Google Scholar). Although a nuclear localization of RXR is to be expected, looking in live cells sometimes gives surprises, as has been the partial cyt