CAR2 displays unique ligand binding and RXRalpha heterodimerization characteristics

Abstract

The constitutive androstane receptor (CAR; NR1I3) regulates the expression of genes involved in xenobiotic metabolism. Alternative splicing of the human CAR gene yields an array of mRNAs that encode structurally diverse proteins. One form of CAR, termed CAR2, contains an additional four amino acids (SPTV) that are predicted to reshape the ligand-binding pocket. The current studies show a marked, ligand-independent, CAR2-mediated transactivation of reporters containing optimal DR-3, DR-4, and DR-5 response elements, and reporters derived from the natural CYP2B6 and CYP3A4 gene promoters. Overexpression of the RXRalpha ligand binding domain was critical for achieving these effects. CAR2 interaction with SRC-1 was similarly dependent on the coexpression of RXRalpha. Mutagenesis of Ser233 (SPTV) to an alanine residue yielded a receptor possessing higher constitutive activity. Alternatively, mutating Ser233 to an aspartate residue drastically reduced the transactivation capacity of CAR2. The respective abilities of these mutagenized forms of CAR2 to transactivate a DR-4 x 3 reporter element correlated with their ability to interact with RxRalpha and to recruit SRC-1 in a ligand-regulated manner. Together, these results demonstrate a robust RXRalpha-dependent recruitment of coactivators and transactivation by CAR2. In addition, CAR2 displays novel dose responses to clotrimazole and androstanol compared with the reference form of the receptor while at the same time retaining the ability to bind CITCO. This result supports a hypothesis whereby the four-amino-acid insertion in CAR2 structurally modifies its ligand binding pocket, suggesting that CAR2 is regulated by a set of ligands distinct from those governing the activity of reference CAR.

Fig. 1

CAR2 transactivation potential is greatly enhanced by RxRα. Transfection assays were performed in COS-1 or HepG2 cells with plasmids indicated/illustrated in the figure and as described under Materials and Methods. Experiments were performed in the presence or absence of cotransfected RxRα. Data are presented as normalized and adjusted luciferase values in which the activity of the 3.1 (or RxRα)/CMV2 group is adjusted to 1 for each respective response element. Each data point represents the mean (± S.D.) of four separate transfections. A, transactivation of multiple DR response elements by CAR1 and CAR2 in the presence and absence of RxRα in COS-1 cells. B, transactivation of multiple endogenous response elements by CAR1 and CAR2 in the presence and absence of RxRα in COS-1 cells. C, transactivation of multiple endogenous response elements by CAR1 and CAR2 in the presence and absence of RxRα in HepG2 cells.

Fig. 2

CAR2 displays altered activity in response to different concentrations of the inverse agonists clotrimazole and androstanol compared with CAR1 and retains the capacity to bind CITCO. Transfection assays were performed in COS-1 or HepG2 cells. All transfections were conducted on the 2B6-XREM reporter and included RxRα and either CAR1 or CAR2. Treatments were administered 18 h after transfection and the cells were harvested 24 h after treatment. Data are presented as normalized luciferase values adjusted so that the CAR1/DMSO group in each panel is equal to 1. Each data point represents the mean (± S.D.) of four separate transfections. A, inverse agonism of CAR1 and CAR2 in response to clotrimazole in COS-1 cells. B, inverse agonism of CAR1 and CAR2 in response to clotrimazole in HepG2 cells. C, inverse agonism of CAR1 and CAR2 in response to 5α-androstan-3α-ol in HepG2 cells. Asterisks denote data points at which CAR2 is significantly different from CAR1 at a specific concentration of chemical. (mean ± S.D., n = 4, *, p < 0.01;**, p < 0.05) D, reversal of androstanol’s inverse agonism by CITCO. The asterisks indicate that the activity of CAR1 and CAR2 is significantly greater in the groups treated with both androstanol and CITCO compared with the respective androstanol-only groups (mean ± S.D., n = 4;*, p < 0.01), clot (clotrimazole), Andro (5α-androstan-3α-ol), and CITCO.

Fig. 3

Enhancement of CAR2 activity by RxRα is dependent on heterodimerization with RxRα and the AF-2 domain of CAR2. Transfection assays were performed in COS-1 cells with plasmids indicated/illustrated in the figure and as described under Materials and Methods. Chemical treatments indicated in the figure were done 18 h after transfection, and the cells were harvested 24 h after treatment. All experiments were conducted using the DR-4 × 3 reporter construct. Data are presented as normalized luciferase values. Each data point represents the mean (± S.D.) of four separate transfections. A, transactivation of the DR-4 × 3 response element by various forms of CAR2 in the presence and absence of RxRα and 10 µM clotrimazole. B, transactivation of the DR-4 × 3 response element by CAR2 in the presence of different forms of RxRα and 10 µM clotrimazole. In A, the full-length CAR2 expression plasmid encodes a N-terminal hemagglutinin-tagged form of the receptor. The epitope tag does not affect the activity of the receptor (data not shown).

Fig. 4

CAR2 recruitment of SRC-1 is RxRα-dependent. Mammalian two-hybrid experiments were performed in COS-1 cells with plasmids indicated in the figures/illustrations and as described under Materials and Methods. Data are presented as normalized and adjusted luciferase values in which the activity of the VP16 (empty)/3.1 (empty)/DMSO (A) or GAL4/VP16/DMSO (B) data points are adjusted to 1. Each data point represents the mean (± S.D.) of four separate transfections. A, SRC-1 recruitment of CAR1 or CAR2 in the presence and absence of RxRα. B, CAR1 and CAR2 recruitment of SRC-1 in the presence and absence of RxRα.

Fig. 5

Mutation of the Ser233 site in CAR2 modifies its transactivation potential. Transfection assays were performed in COS-1 cells with plasmids indicated/illustrated in the figure and as described under Materials and Methods. Transfections were performed in the presence or absence of cotransfected RxRα. Data are presented as normalized and adjusted luciferase values in which the activity of the 3.1/CMV2 group is adjusted to 1 for each respective response element. Each data point represents the mean (± S.D.) of four separate transfections. Figure 5 shows transactivation of the DR-4 × 3, 2B6-XREM and the 3A4-XREM reporters by CAR1, CAR2, and the CAR2 mutants S233A and S233D in the presence and absence of RxRα. Within group comparisons, data points are denoted with an asterisk to indicate that they deviate significantly from the relevant control. For example, on the DR4 × 3 reporter, CAR2A/3.1+ is compared with CAR2/3.1+ and found to have a level of activation significantly greater, the same is found comparing the CAR2A/RxRα group with the CAR2/RxRα group. This process of comparing the CAR2 mutants back with the wild-type CAR2 construct, with or without RxRα, is repeated for each reporter (mean ± S.D., n = 4; p < 0.01).

Fig. 6

Effects of Ser233 mutagenesis on heterodimerization and SRC-1 recruitment. Mammalian two-hybrid experiments were performed in COS-1 cells with plasmids indicated in the figures/illustrations and as described under Materials and Methods. Chemical treatments indicated in the figure were done 18 h after transfection and the cells were harvested 24 h after treatment. Data are presented as normalized luciferase values. Each data point represents the mean (± S.D.) of four separate transfections. A, interaction between CAR2 and its S233A and S233D mutants with RxRα. B, interaction between CAR2 and its S233A and S233D mutants with SRC-1 in the presence and absence of RxRα and clot (clotrimazole).