Characterization of the porcine constitutive androstane receptor (CAR) and its splice variants

Abstract

Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5beta-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

Oxazaphosphorine bioactivation and detoxification: the role of xenobiotic receptors

“…Functional analysis of four SNPs, localized in the ligand binding domain of CAR, (His246Arg, Leu308Pro, Asn323Ser, and Val133Gly) revealed that His246Arg is associated with decreased CAR activation by CITCO, while Leu308Pro affect basal but not chemically stimulated CAR activation in cell-based reporter assays 131; 132 . Recently, a number of naturally occurring alternative splicing variants of human CAR have been identified 133; 134; 135 . Functional characterization of these spliced CAR transcripts revealed that some are associated with altered expression, cellular localization, and chemical response of the receptor 136; 137; 138; 139 .…”

The constitutive androstane receptor and pregnane X receptor in the brain

“…One important feature of NR1I2 and NR1I3 is that ligandmediated receptor activation is species dependent, due to differences in the amino acid sequence of the ligand-binding domain (Gray, Peacock, & Squires, 2009;Gray, Pollock, Schook, & Squires, 2010). This issue makes comparisons of NR1I2 and NR1I3 activation between species difficult, although a limited number of selective ligands for rodent and hNR1I2 and hNR1I3 are available (Table 1) (Chan et al, 2011;Lemmen, Tozakidis, Bele, & Galla, 2013;.…”

Small-molecule modulators of PXR and CAR

“…CAR is also absent in most fish species [76]. Compared to the more than 20 functional hCAR splice variants identified, far fewer CAR variants have been detected in other species, and only approximately five in the rat [77] and pig [78], most of which generate truncated protein products. Comparative genomic analyses show that mice, rats, and marmosets do not have a conserved CAR2 site and are thus incapable of generating a protein analogous to hCAR2.…”

“…This means that rodent models of DEHP toxicity will not accurately profile potential human toxicity because of their inability to generate a CAR2-like transcript. Porcine CAR (pgCAR) might be more homologous to human CAR than is rodent CAR, as is evidenced by the responses of pgCAR and hCAR to ligands being more similar than are those of hCAR and mCAR [78]. …”