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

Abstract

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...

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).…”

“…YFP, YFP-LXR a, and YFP-LXR b were stably expressed in HEK293 cells (293YFP, 293LXRa, and 293LXRb cells, respectively) and selected using geneticin as described previously [Prufer et al, 2000]. Expression of YFP-LXR a and YFP-LXR b at the expected molecular weight in each three clones was confirmed using Western blot analysis (not shown).…”

“…Transcriptionally active YFP-RXR (RXR a) was generated as described earlier [Prufer et al, 2000]. We received LXR a and LXR b in the pDNR vector from the Harvard Institute of Proteomics.…”

“…HEK293, and F9 cells were obtained from ATCC (Manassas, VA) and grown as described previously [Racz and Barsony, 1999;Prufer et al, 2000]. Briefly, cell cultures were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM glutamine (Invitrogen), and 0.1 mg/ml gentamicin (Invitrogen …”

“…Unliganded androgen receptor (AR) [Tyagi et al, 2000], glucocorticoid receptor (GR) [Picard and Yamamoto, 1987], and vitamin D receptor (VDR) [Barsony et al, 1997] localize at least partially in the cytoplasm but translocate into the nucleus after ligand binding. 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].…”