Structural Determinants of Constitutive Androstane Receptor Required for Its Glucocorticoid Receptor Interacting Protein-1-mediated Nuclear Accumulation

Abstract

Nuclear translocation of constitutive androstane receptor (CAR) is a primary mechanism for the induction of cytochrome P450 genes by phenobarbital (PB). We have shown that exogenous expression of the p160 coactivator glucocorticoid receptor interacting protein-1 (GRIP1) in hepatocytes in vivo can mediate PB-independent nuclear accumulation of murine CAR (mCAR). To understand the mechanism of this PB-independent nuclear accumulation, we have examined the mCAR structural determinants of its GRIP1-mediated nuclear localization. Mutations of the xenobiotic response sequence (XRS), which had been shown to block PB-dependent nuclear translocation of human CAR in mouse hepatocytes in vivo, also blocked GRIP1-mediated nuclear accumulation of mCAR in mouse hepatocytes in vivo and further blocked nuclear localization in cultured HepG2 cells. A leucine 326 XRS mutant retained partial transcriptional activity, but mutations of three leucines in the XRS eliminated transcriptional activity in HepG2 cells, suggesting that the translocation function of the XRS overlaps with transcriptional functions. Mutation of the activation function 2 motif, by deletion of the C-terminal 8 amino acids, also reduced nuclear localization by both PB treatment and GRIP1 expression in hepatocytes in vivo, suggesting that either interaction with GRIP1 through this motif or active CAR was required for the nuclear localization. The localization of a DNA-binding domain mutant was essentially unchanged by coexpression of GRIP1, although without GRIP1 coexpression, this mutant expressed exhibited a more nuclear localization compared with wild type. The results are most consistent with a model in which GRIP1 interaction and activation of mCAR in the nucleus result in retention and accumulation of mCAR in the nucleus in untreated animals. The model requires that mCAR is constantly shuttling between the nucleus and cytoplasm even in untreated animals in which mCAR is predominantly cytoplasmic.

Redundant enhancement of mouse constitutive androstane receptor transactivation by p160 coactivator family members

“…1), consistent with previous studies [14]. When SRC-1 or SRC-2 was transfected alone, no enhancement of luciferase activity was detected.…”

“…Unexpectedly, exogenous expression of a p160 coactivator (SRC2/GRIP1) also resulted in accumulation of CAR in the nuclei of hepatocytes in vivo [7]. Mutations in either the DNA binding domain (DBD), XRS, or the transactivation motif (AF-2) motif reduced the GRIP1-mediated nuclear accumulation of CAR [14]. The results were most consistent with an interaction of GRIP1 with CAR in the nucleus mediating the nuclear retention.…”

“…Cells were transfected with pPBRU-2C1-luc (firefly luciferase reporter) and pRL-SV40 (luciferase reporter) and expression plasmids for mCAR and p160 coactivators as indicated in the figure legends. Transfection of cells using Lipofectamine 2000 and analysis of firefly and Renilla luciferase activities 24 h after transfection were as described [14]. For in vivo transfections, plasmid DNA was isolated and injected into tail veins of six to eight week-old (20-25 g) BALB/c male mice (Harlan Labs) using the TransIT In Vivo Gene Delivery System (Mirus Bio Corp.) as described [14].…”

“…Transfection of cells using Lipofectamine 2000 and analysis of firefly and Renilla luciferase activities 24 h after transfection were as described [14]. For in vivo transfections, plasmid DNA was isolated and injected into tail veins of six to eight week-old (20-25 g) BALB/c male mice (Harlan Labs) using the TransIT In Vivo Gene Delivery System (Mirus Bio Corp.) as described [14]. For the luciferase reporter assay, 10 μg of pPBRU2C1-luc DNA, 0.3 μg of pRL-SV40 DNA, and, as indicated, 1 or 10 μg of expression vector DNA for mCAR or p160 coactivators, respectively, were injected.…”

“…For the luciferase reporter assay, 10 μg of pPBRU2C1-luc DNA, 0.3 μg of pRL-SV40 DNA, and, as indicated, 1 or 10 μg of expression vector DNA for mCAR or p160 coactivators, respectively, were injected. Twenty-four h after the injection, the mice were sacrificed, the liver tissue was homogenized, and luciferase activity was determined by the dual luciferase assay as described [14].…”