No abstract

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...