Abstract
Vitamin D receptor (VDR) is localized in nuclei and acts as a ligand-dependent transcription factor. To clarify the molecular mechanisms underlying the nuclear translocation of VDR, we utilized an in vitro nuclear transport assay using digitonin-permeabilized semi-intact cells. In this assay, recombinant whole VDR-(4-427) and a truncated mutant VDR-(4-232) lacking the carboxyl terminus of VDR were imported to nuclei even in the absence of ligand. In contrast, VDR-(91-427) lacking the amino-terminal DNA-binding domain was not imported to nuclei in the absence of ligand, and was efficiently imported in its liganded form. These results suggested that there are two distinct mechanisms underlying the nuclear transport of VDR; ligand-dependent and -independent pathways, and that the different regions of VDR are responsible for these processes. Therefore, we performed the yeast two-hybrid screening using VDR-(4-232) as the bait to explore the molecules responsible for ligand-independent nuclear translocation of VDR, and have identified importin 4 as an interacting protein. In the reconstruction experiments where transport factors were applied as recombinant proteins, recombinant importin 4 facilitated nuclear translocation of VDR regardless of its ligand, whereas importin beta failed in transporting VDR even in the presence of ligand. In conclusion, importin 4, not importin beta, is responsible for the ligand-independent nuclear translocation of VDR.
Highlights
The active form of vitamin D, 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2D3)3 regulates various biological events through its direct actions on gene expression in its target organs such as intestine, kidney, and bone [1, 2]
When the recombinant protein for whole vitamin D receptor (VDR) was applied to this system, it was imported to nuclei in the presence of cytosolic extracts prepared from HeLa cells as a source of nuclear transport factors, whereas VDR failed to enter nuclei without cytosolic extracts (Fig. 1A)
glutathione S-transferase (GST)-NLSSV40-Green Fluorescent Protein (GFP) was imported to nuclei in the presence of cytosolic extracts and ATP (Fig. 1B)
Summary
The active form of vitamin D, 1␣,25-dihydroxyvitamin D3 (1␣,25(OH)2D3) regulates various biological events through its direct actions on gene expression in its target organs such as intestine, kidney, and bone [1, 2]. Utilizing its DNA-binding domain, VDR recognizes a vitamin D-responsive element (VDRE) in the target genes, and regulates the expressions via forming complexes with retinoid X receptor (RXR) and transcriptional co-factors on its ligand-binding domain. To fulfill these events on the genome, nuclear receptors must be localized in nuclei. In the case of classical nuclear localization signal (NLS)-containing proteins, importin ␣ bridges the interaction between the NLS motif and importin  [14] Another common element in the process is the small GTPase Ran in its GTP-bound form (Ran-GTP), whose binding to importin  triggers the dissociation of the complex consisting of importin ␣, importin , and the cargoes. The exploration of the molecules involved in the nuclear import of VDR utilizing the yeast two-hybrid screening have revealed the interaction of VDR and importin 4, and the recombinant importin 4 was capable of transporting VDR into nuclei regardless of the presence or absence of the ligand
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