A novel approach to investigate the subcellular distribution of nuclear receptors in vivo

Abstract

Subcellular compartmentalisation and the intracellular movement of nuclear receptors are major regulatory steps in executing their transcriptional function.Though significant progress has been made in understanding these regulatory processes in cultured mammalian cells, such results have rarely been confirmed within cells of a living mammal. This article describes a simple, time-efficient approach to study the nuclear versus cytoplasmic accumulation of nuclear receptors and the regions of nuclear receptor proteins that govern subcellular trafficking within hepatocytes of live mice. Pregnane X receptor, a xenobiotic-activated member of the nuclear receptor family, was used to exemplify the approach. Using dual-labeled wild-type and mutant PXR expression constructs, we outline their in vivo delivery, simultaneous cellular expression, visualization and categorical classification within hepatocytes of live mice. Using this approach, we identified three mutants that had an altered subcellular distribution in the presence and absence of a PXR ligand. This novel in vivo method complements the current cell culture-based experimental systems in protein subcellular localisation studies.

Serine 350 of human pregnane X receptor is crucial for its heterodimerization with retinoid X receptor alpha and transactivation of target genes in vitro and in vivo

“…Because several studies suggested that hPXR localization is important for its function [34–36], we next examined whether the S350D mutation can alter the subcellular localization of hPXR. HepG2 cells expressing FLAG-tagged wild-type or mutant hPXR were treated with either DMSO or SR for 12 h; the subcellular localization of hPXR was visualized by fluorescence microscopy and quantified by the proportion of cells with nuclear FLAG staining.…”

Activation of xenobiotic receptors: driving into the nucleus

“…The first evidence of PXR translocation came from studies in mouse liver, indicating that mouse PXR (mPXR) was predominantly localized in the cytoplasm of hepatocytes prior to activation and accumulated in the nucleus following treatment with PCN [9, 105]. Subsequent analysis in the same study, led to the identification of a typical bipartite NLS located in the DBD (66–92) of mPXR, which differs from the leucine-rich XRS of CAR located near the C-terminal within the LBD (Fig.…”

Regulation of aldehyde dehydrogenases by nuclear receptors CAR and PXR in rats genetically predetermined in response to phenobarbital

“…What differentiates PXR from CAR is that PXR is activated only via direct binding to a selective ligand [Sueyoshi and Negishi, 2001]. Although early studies supported the notion that PXR resides in the nucleus, either in absence or in presence of an inducer [reviewed in Sueyoshi and Negishi, 2001], mouse PXR has later been identified to be localized in the cytoplasm, until activated by a ligand and translocated into nucleus [Kawana et al, 2003;Matic et al, 2009].Indeed, mPXR is sequestered in the cytoplasm in the absence of a ligand, bound to a complex consisting of the receptor, the co-chaperone CCRP (for CAR Cytoplasmic Retention Protein, also designated as DNAJC7) and the heat shock 90 (Hsp90) protein.…”