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Liganded and unliganded vitamin D receptors (VDRs) carry out distinct functions; both types of functions require heterodimerization with retinoid X receptors (RXRs). Our recent studies with fluorescent protein chimeras of VDR and RXR, termed GFP-VDR, YFP-RXR, and RXR-BFP, indicated that RXR regulates VDR functions in part by regulating subcellular localization. Here we explored the mechanisms of this regulation. Photobleaching experiments demonstrated that YFP-RXR and both unliganded and liganded GFP-VDR shuttle constantly between nucleus and cytoplasm. To characterize RXR import, we identified a nuclear localization sequence (NLS) in the DNA-binding domain. Mutations in this NLS caused predominant cytoplasmic localization of nlsYFP-RXR and prevented transcriptional activity. The nlsRXR-BFP retained unliganded GFP-VDR in the cytoplasm and reduced baseline transcriptional activity. After calcitriol exposure, however, both GFP-VDR and nlsRXR-BFP entered the nucleus. We characterized receptor export rates and mechanisms using permeabilization experiments. Mutations in the calreticulin binding region slowed both GFP-VDR and YFP-RXR export. Coexpression of RXR-BFP slowed the export of unliganded GFP-VDR, whereas calcitriol treatment tripled the rate of GFP-VDR export. Treatment with leptomycin B, an inhibitor of CRM-1 receptor-mediated export, inhibited export of unliganded GFP-VDR but did not influence export of liganded GFP-VDR or YFP-RXR. Leptomycin B added before calcitriol similarly decreased hormone-induced luciferase activity but was ineffective when added subsequent to calcitriol. These results indicate that the unliganded and liganded VDR interact differently with the import and export receptors and with RXR. Most likely, the regulation of VDR nuclear import by RXR is essential for ligand-independent functions.

The vitamin D receptor (VDR) acts as heterodimer with the retinoid X receptor ␣ (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 and nlsGFP-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.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 (1, 2). 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 (3). The addition of calcitriol stabilizes the heterodimers and promotes their binding to the vitamin D response elements (4). 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 (5-7). Unlike the glucocorticoid receptor (GR), which stays in the cytoplasm without the ligand, the unliganded VDR distributes evenly between the cytoplasm and the nucle...

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We used immunocytochemistry to obtain a complete cellular and subcellular mapping of the 1,25-dihydroxyvitamin D3 receptor protein (VDR) in the rat limbic system. We observed specific VDR immunostaining in the nucleus as well as in the perinuclear cytoplasm of neuronal cells. The limbic system consists of a variety of neuronal structures, and is known to have influence on memory, behavior, emotions and reproduction. In the hippocampal formation, we found strong nuclear staining as well as less distinguished cytoplasmic VDR staining in CA1, CA3 and CA4. The CA2 area showed a unique cytoplasmic predominance of VDR. The amygdala was found to exhibit specific patterns of VDR distribution in the various regions of the nucleus. We observed distinct differences of VDR localization within the limbic preoptic areas of the hypothalamus. Further parts of the brain we analyzed included the mammillary bodies, the indusium griseum and the cingulate cortex. The subcellular distribution of VDR in regions of the limbic system suggests a specific functional role of the receptor protein and indicates a role for calcitriol as a neuroactive steroid.

Activity of nuclear receptors is regulated by their nuclear localization. Liver X receptors (LXR) a and b are nuclear receptors that regulate transcription of genes for cholesterol metabolism, cholesterol transport, and lipogenesis. While LXR a and b are very similar in structure and exhibit similar ligand binding properties, their physiological roles are quite different. Since the LXRs fall into a class of receptors that move between the nucleus and cytoplasm, experiments were conducted to determine whether LXR a and LXR b show differences in their nuclear localization pattern. To determine the location of each receptor, cell lines stably expressing yellow fluorescent protein (YFP) chimeras with either LXR a or LXR b were examined. Retention in the nucleus of the chimeric proteins in the presence or absence of ligands was assessed using fluorescence microscopy coupled with digitonin permeabilization assays. Surprisingly, differences were found between LXR a and LXR b. Whereas unliganded LXR a was retained in the nucleus, unliganded LXR b was partially exported. Mutations were then introduced into putative nuclear localization sequences (NLS) to determine which sequences are important for nuclear localization and function. Mutation in one such sequence abolished nuclear localization of LXR a, whereas the analogous change in LXR b had a much less dramatic effect. Mutations in analogous putative NLS also differentially affected transcriptional activation by LXR a and LXR b. These data demonstrate for the first time that nuclear retention and localization as well as function of LXR a and LXR b are differentially regulated.

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