Retinoid X Receptor Dominates the Nuclear Import and Export of the Unliganded Vitamin D Receptor

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.

Nuclear localization of liver X receptor α and β is differentially regulated

“…Next, we tested the effect of ligands on intranuclear localization of LXR a and LXR b. We and others showed previously that fluorescent chimeras of nuclear receptors bound to ligand accumulate in nuclear foci [Htun et al, 1996Lim et al, 1999;Prufer et al, 2000;Stenoien et al, 2000;Baumann et al, 2001;Prufer and Barsony, 2002]. We treated 293LXRa and 293LXRb cells with either vehicle (DMSO), agonist (a combination of 10 mM 22-(R) hydroxycholesterol and 100 nM 9-cis retinoic acid), or antagonist (1 mM geranyl geranyl pyrophosphate).…”

“…Nuclear export assays were performed as described previously [Prufer and Barsony, 2002]. 293LXRa and 293LXRb cells were plated onto chamber slides coated with gelatin.…”

“…In contrast, progesterone receptor [GuiochonMantel et al, 1991], thyroid hormone receptor (TR) [Bunn et al, 2001], estrogen receptor (ER) , RXR [Prufer et al, 2000], LXR a [Watanabe et al, 2003], and LXR b [Mo et al, 2002] are primarily located in the nuclei with or without bound ligand. However, nucleocytoplasmic trafficking has been shown for both types of nuclear receptors [Dauvois et al, 1993;Guiochon-Mantel et al, 1994;Hache et al, 1999;Baumann et al, 2001;Bunn et al, 2001;Prufer and Barsony, 2002]. We and others showed recently that nuclear receptors such as the GR [Hache et al, 1999], the progesterone receptor (PR) [Guiochon-Mantel et al, 1994], TR [Baumann et al, 2001;Bunn et al, 2001], ER [Dauvois et al, 1993], VDR [Prufer and Barsony, 2002], and RXR [Prufer and Barsony, 2002] are continuously imported into and exported out of the nucleus.…”

“…Export from the nucleus occurs through receptormediated mechanisms. Studies using heterokaryon and imaging experiments showed that exported nuclear receptors are reimported into the nucleus [Guiochon-Mantel et al, 1991;Hache et al, 1999;Prufer and Barsony, 2002]. It is well established that nuclear receptors are imported into the nucleus by karyopherins.…”

“…An NLS has been identified by site-directed mutagenesis and live cell imaging of GFP-tagged receptors in the DNA binding domain of VDR and RXR. This NLS (NLS1) is in a conserved region between the two Zinc fingers [Hsieh et al, 1998;Prufer and Barsony, 2002]. One amino acid within NLS1 is conserved among all type II nuclear receptors as either arginine or lysine.…”