Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR

Abstract

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells

“…CAR translocates to the nucleus in response to agonists or activators and transactivates target gene expression. In contrast, exogenously expressed CAR accumulates in the nucleus without any stimuli in cultured cell lines Kanno et al, 2005Kanno et al, , 2007, exhibiting high levels of constitutive transactivation of target genes (Baes et al, 1994). The chemicals that inhibit the constitutive transactivation potential of CAR such as androstane metabolites, androstanol and androstenol, are called "inverse agonists" (Forman et al, 1998).…”

Subcellular Trafficking Signals of Constitutive Androstane Receptor: Evidence for a Nuclear Export Signal in the DNA-Binding Domain

“…In cultured HepG2 cells, mouse CAR (mCAR) is predominantly nuclear, but mutation of the XRS results in cytoplasmic localization, and mutation of the XRS also blocks GRIP1-mediated nuclear translocation in untreated mice (Xia and Kemper, 2005). These results suggest that the XRS may be a PB-responsive NLS, but rat CAR fragments containing the XRS did not exhibit NLS activity (Kanno et al, 2005). Furthermore, the XRS motif does not resemble a typical NLS signal but is similar to leucine-rich protein interaction domains or, paradoxically, shares some sequence similarity with an NES found in the Ah receptor (Fischer et al, 1995;Wen et al, 1995;Ikuta et al, 1998).…”

“…This xenochemical response signal (XRS) is conserved in mouse and rat CAR and plays similar roles in their localization (Zelko et al, 2001;Kanno et al, 2005;Xia and Kemper, 2005). In cultured HepG2 cells, mouse CAR (mCAR) is predominantly nuclear, but mutation of the XRS results in cytoplasmic localization, and mutation of the XRS also blocks GRIP1-mediated nuclear translocation in untreated mice (Xia and Kemper, 2005).…”

“…Nucleocytoplasmic shuttling of nuclear receptors requires specific nuclear localization signals (NLSs) (Kaffman and O'Shea, 1999) and nuclear export signals (NESs) (Fischer et al, 1995;Wen et al, 1995). Two NLSs and a cytoplasmic retention region have been identified in rat CAR (Kanno et al, 2005). A specific NLS, 99 LRRARQAR-RRA 109 (critical residues underlined) in the hinge region was identified by mutational analysis.…”

“…Deletion of the 110 amino acids in the N-terminal, including the hinge NLS, however, did not eliminate nuclear localization, and a second diffuse NLS was proposed within the sequence from amino acid residues 111 to 320. In addition to the NLSs, a cytoplasmic retention region (CRR), which is a potential binding site for CCRP, was defined in the LBD by deletional analysis (Kanno et al, 2005).…”