Molecular Mechanism of Nuclear Translocation of an Orphan Nuclear Receptor, SXR

Abstract

The steroid and xenobiotic receptor (SXR) is an orphan nuclear receptor that plays a key role in the regulation of xenobiotic response by controlling the expression of drug metabolizing and clearance enzymes. We observed that pregnane X receptor (PXR), the mouse ortholog of SXR, was retained in the cytoplasm of hepatic cells of untreated mice, whereas PXR was translocated to the nucleus after administration of a ligand, pregnenolone 16␣-carbonitrile. To understand the molecular mechanisms underlying the xenochemical-dependent nuclear translocation of SXR, we identified the signal sequence of SXR that regulates its nuclear translocation; using an in vitro expression system, we allocated the nuclear localization signal (NLS) to amino acid residues 66 to 92 within the DNA binding domain of SXR. The NLS of SXR is characterized as the bipartite type, and is recognized by the three molecular species of importin ␣: Rch1 (PTAC58), NPI1, and Qip1, in the presence of PTAC97 of importin ␤ to target the nuclear pore. The nuclear translocation of SXR was observed as an essential regulatory event for transcription of its target genes such as CYP3A4. These results strongly suggest that the molecular mechanism of the nuclear import of SXR was different from that of another xenosensor, the constitutively active receptor, whose translocation into the nucleus is mediated by a leucine-rich xenochemical response signal in its ligand binding domain.

Purification of full-length human Pregnane and Xenobiotic Receptor: polyclonal antibody preparation for immunological characterization

“…Hence, using PXR antiserum we also investigated the expression of PXR variants in a liver cell line (HepG2) and intestinal adenocarcinoma cell line (COLO) by Western blot analysis. Based on this study, as well as others [20], transfected PXR is observed to be a predominantly nuclear protein. Therefore, we prepared PXR-enriched nuclear extract from cultured cells for our Western blot analyses.…”

“…Furthermore, endogenously expressed PXR protein has been shown to be localized in cytoplasm of cultured HepG2 cells [21] while it was undetectable in same cell line by other group [17]. When expressed by transfection in cultured cells, ligand-independent nuclear localization was observed [20,22]. On the other hand, 'nuclear extracts' that were prepared from untreated adult male Wistar rats offered nuclease protection in DNase I foot print analysis of rifampicin-responsive region.…”

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

“…We found that a basic amino acid rich sequence in the hinge region of LXR a (NLS3) is important for nuclear localization. An analogous sequence in MR [Tanaka et al, 2005], GR [Freedman and Yamamoto, 2004], and SXR [Kawana et al, 2003] binds to importin a. In addition, we identified a basic amino acid rich sequence (NLS4) À12 to À9 amino acids N-terminal of the DBD in both LXR a and LXR b that is important for nuclear localization.…”

“…Another nuclear localization sequence (NLS3) has been identified in the hinge region of nuclear receptors including the GR [Picard and Yamamoto, 1987], the AR [Zhou et al, 1994], the SXR [Kawana et al, 2003], and the VDR [Luo et al, 1994]. Analogous regions exist in both LXR a and LXR b (Fig.…”

“…Analogous amino acids in the second Zinc finger exist in other nuclear receptors such as RXR, LXR a, and LXR b. Another NLS (NLS3) has been identified in the hinge region of many nuclear receptors including PR [Guiochon-Mantel et al, 1991], ER [Ylikomi et al, 1992], GR [Picard and Yamamoto, 1987], AR [Zhou et al, 1994], the steroid and xenobiotic receptor (SXR) [Kawana et al, 2003], the mineralocorticoid receptor (MR) [Pearce et al, 2002], and the VDR [Luo et al, 1994]. Analogous regions exist in other nuclear receptors such as LXR a and LXR b, but not in RXR.…”