CAR2 Displays Unique Ligand Binding and RXR?
Scott S. Auerbach, Joshua G. DeKeyser, Matthew A. Stoner, and Curtis J. Omiecinski
Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington (S.S.A.); and
Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences,
the Pennsylvania State University, University Park, Pennsylvania (J.G.D., M.A.S., C.J.O.)
Received August 30, 2006; accepted December 27, 2006
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 stud-
ies show a marked, ligand-independent, CAR2-mediated transac-
tivation 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
RXR? ligand binding domain was critical for achieving these ef-
fects. CAR2 interaction with SRC-1 was similarly dependent on the
coexpression of RXR?. 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 abil-
ities of these mutagenized forms of CAR2 to transactivate a DR-
4 ? 3 reporter element correlated with their ability to interact with
RxR? and to recruit SRC-1 in a ligand-regulated manner. Together,
these results demonstrate a robust RXR?-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 struc-
turally modifies its ligand binding pocket, suggesting that CAR2 is
regulated by a set of ligands distinct from those governing the
activity of reference CAR.
The constitutive androstane receptor (CAR; NR1I3) is a nuclear
hormone receptor that is predominantly expressed in the liver (Baes et
al., 1994; Wei et al., 2002). It has been implicated in the metabolism
of xenobiotics and drugs (Wei et al., 2000; Zhang et al., 2002; Wang
et al., 2004b), carcinogens (Xie et al., 2003), steroids (Xie et al.,
2003), heme (Huang et al., 2003; Xie et al., 2003; Huang et al.,
2004a), bile acids (Guo et al., 2003; Saini et al., 2004; Zhang et al.,
2004), and thyroid hormone (Maglich et al., 2004). Furthermore, there
is evidence that CAR activity impinges on cholesterol homeostasis
(Kocarek and Mercer-Haines, 2002; Wang et al., 2003b) and signaling
pathways that control food consumption (Qatanani et al., 2004). In
large part, the effects that CAR exerts on these processes are depen-
dent on the receptor’s ability to modulate hepatic gene expression
(Maglich et al., 2002; Ueda et al., 2002). The battery of CAR target
genes include members of all three phases of xeno/endobiotic metab-
olism and clearance, such as certain cytochrome P450, UDP-glucu-
ronosyltransferase, sulfotransferase, glutathione transferase, aldehyde
dehydrogenase, and avidin-biotinylated enzyme complex transporter
families (Maglich et al., 2002; Ueda et al., 2002). Thus far, CAR
response elements have been mapped in a number of the correspond-
ing human genes, including CYP2B6 (Sueyoshi et al., 1999; Wang et
al., 2003a), CYP3A4 (Goodwin et al., 2002), CYP3A5 (Burk et al.,
2004), CYP2C8 (Ferguson et al., 2005) CYP2C9 (Ferguson et al.,
2002; Gerbal-Chaloin et al., 2002), CYP2C19 (Chen et al., 2003),
UGT1A1 (Sugatani et al., 2001), MDR1 (Burk et al., 2005), and
ALAS1 (Podvinec et al., 2004).
The regulation of CAR activity is complex and still poorly under-
stood. Most studies of CAR regulation have focused on mouse CAR.
In mouse, CAR is localized cytosolically in the absence of inducer,
such as the prototypical inducer phenobarbital (Kawamoto et al.,
1999; Zelko et al., 2001). Recent studies have identified a number of
CAR interacting proteins that complex with the cytosolic receptor,
including two heat shock proteins that may function to anchor CAR to
the cytoskeleton (Kobayashi et al., 2003; Yoshinari et al., 2003). Upon
exposure to an inducing agent, CAR is released from this complex by
a mechanism that probably involves protein phosphatase 2A, in turn
accumulating in the nucleus, where the receptor heterodimerizes with
This work was supported by Grant GM66411 from the National Institute of
General Medical Sciences (to C.J.O.) and National Institute of Environmental
Health Sciences Training Grant ES07032 (to S.S.A.).
ABBREVIATIONS: NR, nuclear receptor; CAR, constitutive androstane receptor; RXR, retinoid X receptor; FXR, farnesoid X receptor; DBD,
DNA-binding domain; LBD, ligand-binding domain; DR, direct repeat; PBREM, phenobarbital response enhancer module; XREM, xenobiotic
response enhancer module; CMV, cytomegalovirus; VP16, virus protein 16; EMSA, electrophoretic mobility shift assay; CITCO, 6-(4-chlorophenyl:
imidazo[2,1-b]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime; h, human; m, mouse; SRC-1, steroid receptor coactivator 1; bp, base pair(s);
DMSO, dimethyl sulfoxide; AF-2, activation function 2; RID, receptor interaction domain.
DRUG METABOLISM AND DISPOSITION
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
DMD 35:428–439, 2007
Vol. 35, No. 3
Printed in U.S.A.
RXR? and subsequently interacts with coregulators such as SRC-1
(Makinen et al., 2002) to regulate target genes. It is currently unknown
whether CAR activity is governed similarly in human hepatocytes
although the available evidence supports such a hypothesis (Pascussi
et al., 2000; Maglich et al., 2003; Wang et al., 2004a). It is odd that
most of the inducing agents that act through the CAR signaling
pathway do not interact directly with the receptor (Moore et al., 2000;
Zhang et al., 2002; Huang et al., 2004a). Only a limited number of
CAR ligands have been identified that regulate the receptor through
interaction with its ligand binding pocket. These include clotrimazole
(Moore et al., 2000; Makinen et al., 2002; Moore et al., 2002),
5?-pregnane-3,20-dione (Moore et al., 2000), CITCO (Maglich et al.,
2003), androstanol (Forman et al., 1998), androstenol (Forman et al.,
1998), 17?-ethynyl-3,17?-estradiol (Makinen et al., 2002), 1,4-bis[2-
(3,5-dichloropyridyloxy)]benzene (Tzameli et al., 2000), and mecliz-
ine (Huang et al., 2004b).
We and others have recently described a number of mRNA splice
variants of the human CAR gene that potentially represent a large
expansion of the CAR proteome (Auerbach et al., 2003; Savkur et al.,
2003; Arnold et al., 2004; Jinno et al., 2004; Lamba et al., 2004). One
of the variant forms results from the use of an alternative splice
acceptor site in intron 6, leading to the insertion of 12 additional
nucleotides. The resultant mRNA has been reported to make up 6 to
10% of the total CAR transcript in human liver (Jinno et al., 2004).
This transcript encodes a protein containing an additional four amino
acids (SPTV) that are predicted to extend helix 6 of the ligand binding
domain and potentially affect the structure of the ligand binding
pocket (Auerbach et al., 2003; Savkur et al., 2003). We term this form
of the receptor CAR2 [CAR1 being the reference form of the receptor
(Baes et al., 1994)]. CAR2 retains a limited ability to transactivate
CAR-responsive reporters (Auerbach et al., 2003; Arnold et al., 2004;
Jinno et al., 2004), a result that correlates with a reduced affinity for
RXR? and in turn a compromised ability to interact with DNA
(Auerbach et al., 2003; Arnold et al., 2004). Ligand studies of CAR2
demonstrated that clotrimazole deactivated the receptor, whereas
CITCO produced a weak, albeit significant activation of CAR2 (Jinno
et al., 2004)—a result that is contrary to mammalian two-hybrid
studies published separately (Arnold et al., 2004). It is noteworthy that
in transfected mouse hepatocytes, nuclear translocation of CAR2 is
not observed after CITCO treatment (Jinno et al., 2004).
Studies presented here now demonstrate that CAR2 constitutively
transactivates DR-3, DR-4, and DR-5 nuclear receptor response ele-
ments in addition to the endogenously encountered PBREM,
CYP2B6-XREM, and CYP3A4-XREM luciferase reporters. The
CAR inverse agonists androstanol and clotrimazole differentially re-
press CAR2 compared with CAR1 at varying doses. CAR2 also seems
to retain the ability to bind CITCO. Furthermore, we report that
RXR? cotransfection greatly enhances the ability of CAR2 to trans-
activate reporter constructs through a mechanism that is highly de-
pendent on the ability of the two receptors to heterodimerize and
requires the DNA binding domains of both receptors together with the
AF2 domain of CAR2. Mammalian 2-hybrid studies revealed that in
contrast to CAR1, the ability of CAR2 to interact with coactivators is
largely dependent on RxR?. Finally, mutagenesis studies conducted
on Ser233 of CAR2 reveal that this site plays an important role in the
ability of the receptor to transactivate reporters, heterodimerize with
RxR?, and recruit SRC-1.
Materials and Methods
Chemicals. Clotrimazole and 5?-androstan-3?-ol were obtained from
Sigma (St Louis, MO). CITCO was purchased from BIOMOL Research
Laboratories (Plymouth Meeting, PA). Dimethyl sulfoxide (DMSO) was pur-
chased from EM Scientific (Gibbstown, NJ). Primers for polymerase chain
reaction and EMSA were purchased from Integrated DNA Technologies
Plasmids. Polymerase chain reaction based cloning was done with Accu-
POL DNA polymerase (GeneChoice, Frederick, MD). The primary structures
of all of the resulting plasmid constructs were verified by DNA sequencing.
The sequences cloned into the respective expression vectors represented only
the protein coding regions and were preceded by a Kozak sequence (Kozak,
1987). Mutagenesis was performed using the indicated primers (Tables 1–4)
and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA)
according to the manufacturer’s protocol. Before transfection, plasmids were
prepped using the Quantum Prep Plasmid Maxiprep Kit (Bio-Rad Laborato-
ries, Hercules, CA). All RXR clones referred to in this manuscript were
derived from human RXR?.
Cell Culture. COS-1 cells (simian virus-40–transformed green monkey
kidney cells) were maintained and transfected in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES,
0.15% sodium bicarbonate, 50 units/ml penicillin G, and 50 ?g/ml streptomy-
cin. Human hepatoma HepG2 cells were maintained and transfected in mini-
mal essential medium; all other components remained the same except for the
This plasmid contains a CMV promoter that yields robust expression of cloned sequences when transfected into mammalian cells. This plasmid is referred to in this text as “CMV2.”
CAR1NM_005122 Human liver
NACMV2 clone1–352 S233AEcoR1/EcoRV
NACMV2 clone1–352 S233DEcoR1/EcoRV
NA, not available.
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION
This plasmid contains a CMV promoter that yields robust expression of cloned sequences when transfected into mammalian cells. This plasmid is referred to in the text as “3.1.”
CMV2 clone described previouslya
CMV2 clone described previouslya
CMV2 clone described previouslya
CMV2 clone described previouslya;
based on mutagenized mouse cloneb
NA, not available.
aAuerbach et al. (2003).
bVivat-Hannah et al. (2003).
AUERBACH ET AL.
pM and pVP16 (BD Biosciences)
In-frame cloning of sequences into pM and VP16 generate fusion proteins of the GAL4-DNA binding domain and the VP16 activation domain, respectively. These plasmids are used in combination to access interaction of proteins in mammalian
cells. The multiple cloning sites of these two plasmids are identical; therefore, cloned sequences are interchangeable. The luciferase reporter employed in the mammalian two-hybrid assays is pFR-Luc (Stratagene)
CMV2 clone described previouslya
CMV2 clone described previouslya
Human liver cDNA
NA, not available.
aAuerbach et al. (2003).
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION
addition of 1.0 mM sodium pyruvate. All cell culture reagents were purchased
from Invitrogen (Carlsbad, CA).
Reporter and Mammalian Two-Hybrid Assays. All transfections using
COS-1 cells for luciferase reporter assays were performed in a 48-well format.
On the morning of day 1, cells were plated to approximately 50,000 cells per
well. While the cells were attaching, DNA transfection mixtures were assem-
bled using Fugene6 transfection reagent (Roche Applied Science, Indianapolis,
IN). All transfections using HepG2 cells for reporter assays were performed in
a 48-well format using Lipofectamine LTX reagent (Invitrogen). In this case,
DNA transfection mixtures were assembled and placed in the individual wells,
and the cells were then plated directly onto the DNA mixture at approximately
100,000 cells per well. In general, for assays involving standard reporters (not
two-hybrid), 25 ng of CMV2 or CMV2-CAR expression plasmid, 25 ng of
pcDNA3.1 or 3.1-RXR expression plasmid, 100 ng of luciferase reporter, and
10 ng of pRL-CMV (for transfection normalization; Promega, Madison, WI).
All mammalian two-hybrid assays were performed with 40 ng of pVP16
expression plasmid, 10 ng of pM (GAL4) expression plasmid, 100 ng of
pFR-luc reporter, and 10 ng of pRL-CMV. When the pcDNA3.1 expression
plasmid, containing RXR-LBD, was incorporated in to the two-hybrid assay,
10 ng of was used. In all transfections, the transfection reagent was used at a
ratio of 1:3 (micrograms of DNA to microliters of transfection reagent) as
recommended in the manufacturer’s protocol. Within a given experiment, all
transfections contained the same total amount of DNA. At the time of trans-
fection (within 1–6 h after plating), cells were approximately 80% confluent
and had initiated cell division (in the case of COS-1 cells). The following day
(16–18 h after transfection), cells were treated with chemical agents as
indicated in the figures. If chemical treatment was not performed, cells were
lysed and assayed 24 h after transfection. In all treatments, DMSO levels never
exceeded 0.1% (v/v). On day 3 (24 h after chemical treatment), cells were
washed with PBS and luciferase assays were performed using the Dual-
Luciferase Reporter Assay System (Promega, Madison, WI) and a Veritas
Microplate Luminometer (Turner Biosystems, Sunnyvale, CA). Luciferase
assay and stop and glow reagents were diluted with 1? Tris-buffered saline,
pH 8.0, to a 0.5? final concentration. All other aspects of the assay were
performed in accordance to the manufacturer’s protocol. Dilution of luciferase
reagent had no effect on normalized luciferase values.
Statistical Analysis. Quantitative data were examined by analysis of vari-
ance. Unless stated otherwise significance was declared if p ? 0.01. Data are
expressed as means ? S.D. (n ? 4).
CAR2 Activated Various Response Elements in Reporter As-
says, an Effect That Was Greatly Enhanced by Coexpression of
RXR?. Using luciferase reporters designed to preferentially interact
with CAR/RXR heterodimers (Frank et al., 2003), CAR2 produced a
statistically significant transactivation of a DR-4 ? 3 reporter in the
absence of cotransfected RXR? (Fig. 1A). This effect was greatly
enhanced by overexpression of RXR?. Furthermore, the inclusion of
RxR? allowed CAR2 to also transactivate DR-3 and DR-5 reporter
constructs. In parallel assays, CAR1 significantly transactivated the
DR-4 and DR-5 reporters even in the absence of cotransfected RXR?.
The overexpression of RXR? led to a more permissive activation by
CAR1, enabling the receptor to induce all of the direct repeat reporters
and enhancing its overall transactivation potential (Fig. 1A).
The DR reporters used in Fig. 1A contain three copies of an optimal
DR element, a situation that does not exist in the human genome. To
test the question of whether or not CAR2 is capable of transactivation
via the more degenerate elements found in the promoters of CAR
target genes, we created reporters containing previously identified
CAR responsive sequences, the promoters of the CYP2B6 and
CYP3A4 genes, both of these included an upstream enhancer sequence
termed the XREM (Goodwin et al., 1999; Sueyoshi et al., 1999;
Goodwin et al., 2002; Wang et al., 2003a). We also included the
CYP2B6 promoter without the XREM sequence (PBREM) (Fig. 1B).
The PBREM reporter is weakly yet significantly activated by CAR1
(2.6-fold) in the absence of cotransfected RXR?; CAR2 did not
activate the reporter under these conditions. Overexpression of RxR?
allowed for stronger activation by CAR1 and induction by CAR2 on
a level comparable with CAR1 (3.0- and 3.8-fold for CAR2 and
CAR1, respectively). Both CAR1 and CAR2 were able to activate the
The pGL3 basic vector was engineered with the thymidine kinase core promoter as described previously to generate a TK-luc reporter (Auerbach et al., 2003). The DR-1X3 through DR-
5X3 reporters were made with complimentary primers that were annealed and blunt-end–ligated into the Sma1 site upstream of the TK promoter.
aA polymerase chain reaction amplicon was generated from human genomic DNA that contained the 2B6 XREM sequences recently described (Wang et al., 2003a). The amplicon was ligated
upstream of the TK promoter using the KpnI and NheI restriction sites.
bAmplicons encompassing the proximal (p) ER-6 (Barwick et al., 1996) and distal XREM (Goodwin et al., 1999) sequences in the CYP3A4 promoter were amplified separately. Individual
amplicons were digested with EcoRI, purified, and ligated. The ligation was then amplified with the XREMFP and pER6RP. The product from this second amplification was then blunt-end–ligated
into the SmaI site upstream of the thymidine kinase promoter.
cThe PBREM-TK-Luc reporter was described previously (Auerbach et al., 2003).
AUERBACH ET AL.
2B6-XREM reporter in the absence of RXR? (11.1- and 2.5-fold,
respectively), but only CAR1 was able to activate the 3A4-XREM
reporter without cotransfected RxR? (3.1-fold). The addition of
RXR? greatly enhanced the ability of both CAR variants to transac-
tivate these two reporter constructs. In the case of CAR1, the activity
on the 2B6-XREM reporter increased to 40.4-fold over basal an
amount 3.6 times greater than seen without RXR?. Activity on the
3A4-XREM reporter increased to 28.2-fold over basal or 9.1 times
greater than what was seen without the addition of RXR?. CAR2
activities exhibited even greater returns from the addition of RXR?,
activity on the 2B6-XREM reporter increased to 36.4-fold over basal,
14.6 times greater than without RXR?, and activity on the 3A4-
XREM reporter went from undetected to 14.4-fold over basal
Figure 1, A and B, was generated in transformed green monkey
kidney COS-1 cells. These cells are desirable cell models because of
their lack of CAR expression and their ease of transfection. However,
because CAR is mainly expressed in the liver, we decided to confirm
our results for the endogenous promoters in the human hepatoma cell
line HepG2 (Fig. 1C). The results shown in Fig. 1C are in good
agreement with those of Fig. 1B. The most notable difference was that
the overall activity was decreased particularly in the groups with
cotransfected RXR?. The activity of CAR2 on the PBREM was
almost identical to that seen in COS-1 cells, whereas CAR1 activities
were slightly decreased. Activity on the 2B6-XREM reporter by
CAR1, in the absence and presence of RXR?, was 5.1- and 13.6-fold
over basal, respectively; the comparable values for CAR2 were 2.5-
and 20.1-fold over basal. In this case, RXR? increased CAR1 activity
by 2.7-fold and CAR2 activity by 8-fold. For the 3A4-XREM re-
porter, CAR1 activity in the absence of RXR? was 4.2-fold over basal
and 13.2-fold in the presence of RXR? 3.2 times greater than without.
CAR2 activity was undetectable in the absence of RXR?; addition of
RXR? to the system resulted in activities that increased up to 8.5-fold
CAR2 Displayed Altered Activity in Response to Different
Concentrations of the Inverse Agonists Clotrimazole and Andro-
stanol Compared with CAR1 and Retained the Capacity to Bind
CITCO. The four amino acid insertion of CAR2 is within the vicinity
of the ligand-binding pocket. To test whether or not this insertion
would alter ligand binding, we investigated the effects of the inverse
agonists clotrimazole and androstanol and the CAR ligand CITCO on
CAR1 and CAR2. All the transfections performed for Fig. 2 were
FIG. 1. CAR2 transactivation potential is greatly enhanced by RxR?. 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 RXR?. Data are presented as normalized
and adjusted luciferase values in which the activity of the 3.1 (or RXR?)/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 RxR? in COS-1
cells. B, transactivation of multiple endogenous response elements by CAR1 and CAR2 in the presence and absence of RxR? in COS-1 cells. C, transactivation of multiple
endogenous response elements by CAR1 and CAR2 in the presence and absence of RxR? in HepG2 cells.
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION
carried out using the 2B6-XREM reporter, RXR?, and CAR1 or
CAR2. Treatments were applied 18 h after transfection and cells were
harvested 24 h after treatment. All data were normalized to the
expression of Renilla reniformis luciferase and adjusted such that
activity of the CAR1/DMSO group in each panel was equal to 1.
In Fig. 2A, CAR1 and CAR2 activity in COS-1 cells was tested
with increasing concentrations of clotrimazole. In the DMSO control
group, the activity of CAR2 was 73.4% of CAR1 activity. At 0.5 ?M
clotrimazole, CAR1 activity was markedly reduced, and then only
modestly decreased in inhibition across the rest of the concentrations
tested. In contrast, CAR2 was only weakly inhibited at lower concen-
trations; its activity equaled CAR1 at 0.5 and 1.0 ?M. At higher
doses, CAR2 activity decreased much more rapidly than CAR1. At 5
?M CAR2, activity again drops below CAR1 and continues to de-
crease up to 25 ?M, at which point it is strongly repressed, exhibiting
only 15.8% of the activity of CAR1 at that concentration. To further
investigate the effect of clotrimazole as well as another CAR1 inverse
agonist, androstanol, similar experiments were conducted in HepG2
cells (Fig. 2, B and C). A comparable trend was seen under these
conditions, such that the response of CAR1 was almost maximal by 1
?M clotrimazole, whereas CAR2 inhibition at 1 ?M was significantly
less than that observed for CAR1. However, as the concentration of
this agent was increased, CAR2 activities exhibited greater levels of
repression. It is noteworthy that the inverse agonism of CAR2 caused
by high concentrations of clotrimazole was considerably less in
HepG2 cells than in COS-1.
No known ligands of hCAR are able to increase its activity above
constitutive levels. However, there are compounds that activate hCAR
by interacting with its ligand binding domain and inducing nuclear
translocation. CITCO is a well characterized ligand that activates
hCAR in this manner (Maglich et al., 2003). It is difficult to study
ligands that work through this mechanism because the nuclear trans-
location of CAR does not recapitulate in transfected cell systems
where CAR spontaneously accumulates in the nucleus. To see
whether CAR2 could interact with CITCO, we decided to test whether
CITCO could reverse the inverse agonism of androstanol through
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 RXR?
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 5?-androstan-3?-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 (5?-androstan-3?-ol), and CITCO.
AUERBACH ET AL.
competition. The data presented in Fig. 2D demonstrate that 10 ?M
CITCO alone exerts no effect on CAR1 or CAR2 and that 10 ?M
androstanol repressed the activity of both receptors. When adminis-
tered together, the activity of both receptors was significantly greater
than that seen with androstanol alone.
Transactivation of the DR-4 ? 3 Reporter Required the CAR2
AF-2 and DBD, and Enhancement of CAR2 Activity by RXR?
Was Dependent on Heterodimerization and the DBD of RXR?.
Using an EMSA protocol, we previously demonstrated a weak inter-
action of a GST-CAR2/GST-RXR? complex with a CYP2B6 NR1
probe (Auerbach et al., 2003). However, additional EMSA studies
using nuclear extracts from transfected COS-1 cells failed to demon-
strate CAR2-DNA binding under conditions that produced robust
DNA interaction of CAR1 (data not shown). This finding is in
agreement with other published results (Arnold et al., 2004). There-
fore, the question remains as to the mechanism of CAR2 transactiva-
tion. We generated CAR2 DNA constructs that contained deletions of
sequences representing the DBD and AF-2 receptor regions. Cotrans-
fection of these constructs in combination with RXR? led to a
complete loss of CAR2-mediated transactivation of the DR-4 ? 3
reporter (Fig. 3A). These findings suggest that CAR2 is directly
interacting with DNA in a cellular context and mediating transacti-
vation by AF-2-dependent coactivator recruitment.
A similar set of experiments were designed to test the influence of
different forms of RXR? on CAR2 activity and are presented in Fig.
3B. Cotransfection of a heterodimerization-deficient form of RXR?
(Y397A) (Vivat-Hannah et al., 2003) yielded a level of activation that
was statistically significant (p ? 0.01) compared with the CAR2/
3.1? control group, however; the magnitude of this activation was
considerably less than that seen with wild-type RxR?. Deletion of the
DBD (LBD) of RXR? inhibited CAR2 transactivation relative to
transfection of CAR2 alone. Deletion of the RXR? AF-2 sequence did
not greatly affect the ability of RXR? to enhance CAR2 activity.
Together, these data suggest that RXR? must heterodimerize with
CAR2 to facilitate its interaction with DNA and, furthermore, that the
interaction of RXR? with coactivators through its AF-2 domain is not
essential to attain CAR2-dependent transactivation.
The LBD of RXR? Facilitated the Interaction of CAR2 with the
RID (Receptor Interaction Domain) of SRC-1. Considering the
noted influence of RXR? on CAR2 activity and previously docu-
mented effects of RXR? on CAR1 interaction with coactivators
(Dussault et al., 2002), two hybrid experiments were performed to
assess the effect of RXR? on CAR2/coactivator interaction. The
results presented in Fig. 4A demonstrate a strong, RXR?-dependent
interaction of CAR2 with GAL4-SRC-1 (RID). In the case of CAR1,
inclusion of RXR? yielded only a modest increase in the interaction
between it and SRC-1. In other experiments, it was determined that
VP16-CAR1 or -2 did not interact with unfused (empty) GAL4 (data
To further verify the results in Fig. 4A, an experiment was per-
formed in which CAR1 or 2-LBD and SRC-1 (RID) were inserted into
the GAL4 and VP16 vectors, respectively (Fig. 4B). Transactivation
by the LBD of CAR2 was observed only in the presence of overex-
pressed RXR?-LBD. Cotransfection of VP16-SRC-1 (RID) further
enhanced the RXR?-dependent transactivation by CAR2. It is note-
worthy that the inclusion of RxR? had no effect on the ability of the
GAL4-CAR1 to transactivate the reporter or its ability to recruit VP16
via the SRC-1 (RID) construct in these experiments. This result is a
stark contrast to those observed with CAR2.
Mutation of the Ser233 Site in CAR2 Modified Receptor Ac-
tivity. In our previous study (Auerbach et al., 2003), we identified a
serine residue, Ser233, in the inserted SPTV sequence of CAR2 as a
putative target of phosphorylation. To determine the potential impact
of Ser233 phosphorylation, we mutated the residue either to an alanine
(CAR2-A) to prevent phosphorylation or to an aspartate (CAR2-D) to
mimic constitutive phosphorylation. CAR2-A produced a signifi-
cantly greater transactivation response than CAR2, on all reporters
and in all experimental contexts tested, with the exception of the
2B6-XREM reporter in the presence of RXR? cotransfection (Fig. 5).
CAR2-D exhibited a compromised ability to transactivate relative to
CAR2. The latter effect was most robust when using the CYP3A4
reporter in the presence of RXR? over expression. The data in Fig. 5
also show that CAR2-A acts more like the reference form of the
receptor (CAR1) in its ability to transactivate the three reporter
To further characterize the effects of these mutations, we employed
them in our mammalian two-hybrid system to assess their effects on
RXR? heterodimerization and SRC-1 recruitment (Fig. 6). In Fig. 6A,
the ligand-binding domain of RXR? and the CAR2, CAR2A, and
CAR2D ligand binding domains were fused into the GAL4 and VP16
vectors, respectively. CAR2 had a limited ability to interact with
FIG. 3. Enhancement of CAR2 activity by RXR? is dependent on heterodimeriza-
tion with RXR? and the AF-2 domain of CAR2. Transfection assays were per-
formed 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 RxR? and 10 ?M
clotrimazole. B, transactivation of the DR-4 ? 3 response element by CAR2 in the
presence of different forms of RxR? and 10 ?M 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
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION
RXR?, whereas CAR2A displayed a greatly enhanced interaction and
CAR2D had no ability to interact with RXR?. In Fig. 6B, we tested
the ability of these mutants to recruit the RID of SRC-1 in the
presence and absence of RXR? and 10 ?M clotrimazole. As expected,
none of the CAR2 constructs were able to transactivate the reporter
very well in the absence of RXR?. When RXR? was included in the
assay, both CAR2 and CAR2A were able to recruit SRC-1 in an equal
capacity; however, CAR2D displayed little ability to interact with
SRC-1 under these conditions. Furthermore, 10 ?M clotrimazole was
much more disruptive of this process on CAR2 than it was for CAR1.
This result is in strong agreement with the result presented in Fig. 2A.
CAR2 contains a four-amino-acid insertion (SPTV) in its LBD.
Initial studies of the variant receptor demonstrated a severely com-
promised capacity for transactivation (Auerbach et al., 2003; Arnold
et al., 2004; Jinno et al., 2004). Here, we show that cotransfection of
RXR? markedly enhances the constitutive activity of CAR2 in re-
porter assays. The enhancement of CAR2 transactivation seems inde-
pendent of an RXR?-mediated interaction of the receptor with coac-
tivators. We also assessed the ability of the known CAR ligands
clotrimazole and androstanol to function as inverse agonists of CAR2
at various concentrations and found marked differences in the re-
sponse of CAR2 to these compounds compared with CAR1. Further
studies also demonstrated that CITCO reverses the inverse agonism of
androstanol on both CAR1 and CAR2, providing strong evidence that
CITCO is a CAR2 ligand. Mammalian two-hybrid studies showed that
the ability of CAR2 to recruit coactivators has a stronger dependence
on RXR? than CAR1 and that CAR2 displays a compromised ability
to heterodimerize with RXR?. This later effect can be circumvented
in part by mutating the Ser233 residue of CAR2 to an alanine and is
completely ablated by changing the same residue to an aspartate. The
S233D mutation also lost all ability to recruit SRC-1. Finally, and in
agreement with the COS-1 dose-response data, the recruitment of
SRC-1 by CAR2 was abrogated more effectively by 10 ?M clotrim-
azole than for CAR1.
It was reported previously that mouse CAR interaction with coac-
tivators was enhanced by the overexpression of RXR? (Dussault et
al., 2002). This phenomenon has also been observed with FXR
(Pineda, 2004). In the case of CAR, it was suggested that RXR?
produces an allosteric effect on the receptor and was not directly
involved in coactivator recruitment (Dussault et al., 2002). Data
presented in this study for human CAR are in agreement with the
mouse report (Dussault et al., 2002) and suggest that RXR?, inde-
pendent of its AF-2 domain, allosterically modifies the activity of
CAR2, without directly interacting with coactivator when in complex
with CAR2. Therefore, these data imply that the AF-2 function of
CAR2, and not that of RXR?, mediates coactivator recruitment, a
conclusion that is further supported by the results demonstrating the
complete ablation of CAR2 activity after a deletion of its AF-2 motif.
During the course of these studies, we noticed that CAR2 was being
repressed more strongly by higher concentrations of clotrimazole than
those observed for CAR1. To further investigate this, CAR1 and
CAR2 were both assayed for activity on the 2B6-XREM promoter
FIG. 4. CAR2 recruitment of SRC-1 is RXR?-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 RxR?. B,
CAR1 and CAR2 recruitment of SRC-1 in the presence and absence of RxR?.
FIG. 5. Mutation of the Ser233 site in CAR2 modifies its transactivation potential.
Transfection assays were performed in COS-1 cells with plasmids indicated/illus-
trated in the figure and as described under Materials and Methods. Transfections
were performed in the presence or absence of cotransfected RXR?. 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 RxR?. 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/RxR? group with the CAR2/RxR? group. This process of comparing
the CAR2 mutants back with the wild-type CAR2 construct, with or without RxR?,
is repeated for each reporter (mean ? S.D., n ? 4; p ? 0.01).
AUERBACH ET AL.
with multiple concentrations of clotrimazole and androstanol. The
results of Fig. 2 demonstrate that CAR2 exhibits an altered response
to inverse agonists compared with CAR1. Further studies also re-
vealed that CAR2 retains the ability to interact with CITCO, a well
characterized CAR1 ligand. Therefore, it is conceivable that CAR2
may be regulated by a distinct yet overlapping set of ligands in
comparison with CAR1, a concept that is supported by molecular
modeling approaches indicating that the differential splicing of CAR2
results in an insertion of four amino acids (SPTV) in close proximity
to the receptor ligand binding pocket (Auerbach et al., 2003). Large
chemical screens should reveal whether unique pharmacophores exist
that specifically modulate the CAR2 receptor and its respective biol-
ogy. This process is complicated by CAR’s unique regulation, allow-
ing certain ligands to act as agonists, such as has been shown with
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene on mCAR (this effect has
not yet been seen on hCAR) (Forman et al., 1998), inverse agonists,
as well as general activators of receptor translocation, such as phe-
Why is transfection of RXR? necessary in COS-1 cells that already
express levels of the receptor detectable by Western immunoblotting?
The RXR antibody we have used (?N197; Santa Cruz Biotechnolo-
gies, Santa Cruz, CA) is not RXR isotype-selective. Isotype-selective
heterodimerization has already been documented for nurr1 (Sacchetti
et al., 2002) and vitamin D receptor (Kephart et al., 1996). In our
studies, certain receptors, such as PXR and FXR, are unaffected by
overexpression of RXR? in COS-1 cells (S. S. Auerbach, J. G. De
Keyser, and C. J. Omiecinski, unpublished data). Perhaps PXR and
FXR preferentially interact with an abundant isotype of RXR ex-
pressed in COS-1 cells, whereas CAR more selectively heterodimer-
izes with RXR? that is not expressed in high abundance in these cells.
Furthermore, data from HepG2 cells (Fig. 1C) showed that CAR1 and
CAR2 had similar transactivation potentials in the absence of cotrans-
fected RXR? compared with COS-1 cells (Fig. 1B). The addition of
exogenous RXR? yielded a greater increase in activity in COS-1 cells
than in HepG2 cells. In our experience, COS-1 cells have transfection
efficiencies 5- to 10-fold greater (based on the activity of R. renifor-
mis luciferase) than that seen in HepG2 cells. The effect of cotrans-
fected RXR? is compared within the same cell line in contrast to
transfection efficiency that is compared across lines. Therefore, it
would be expected that if everything else were equal, the maximal
activity would be lower in HepG2 cells because of the lower trans-
fection efficiency but that the percentage of increase imparted by
cotransfected RXR? should be equal between the two cell lines. It is
possible that HepG2 cells express more RXR? compared with the
other RXR isotypes, creating an environment more favorable for CAR
activity compared with COS-1 cells. This idea is further supported by
the data from Fig. 2A. The strong repression of CAR2 by clotrimazole
in COS-1 cells was surprising and may indicate that a combination of
an unfavorable RXR isotype background acts synergistically with
CAR2’s strong dependence on RXR? for coactivator recruitment,
essentially silencing the receptor. Finally, it seems reasonable to
speculate that heterodimeric complexes composed of different RXR
isotypes may preferentially interact with distinct subsets of DNA
response elements. If this hypothesis is correct, expression ratios of
RXR isotypes in hepatocytes may significantly influence expression
of drug metabolizing enzymes. Consistent with this idea, ablation of
RXR? in mouse hepatocytes substantially reduces CAR-mediated
gene expression, although other RXR isotypes are expressed in the
liver (Mangelsdorf et al., 1992; Cai et al., 2003).
CAR1 and CAR2 probably coexist within the human liver and may
have negative or synergistic effects on each other. Over the course of
these studies, we also performed cotransfection experiments to inves-
tigate the activity of the 2B6-XREM reporter in response to varying
ratios of CAR1 to CAR2, with concomitant treatment by DMSO,
androstanol, clotrimazole, and CITCO (data not shown). The results
of these studies showed no differences between groups that contained
CAR1 and CAR2 compared with each receptor alone. These results do
not rule out the possibility that CAR1 and CAR2 interact in vivo, in
that our studies were conducted on an idealized reporter, containing
endogenous promoter sequences known to be highly responsive to
CAR and removed from their native chromatin environment. In the
context of a full-length endogenous promoter, it is conceivable that
there are many situations in which gene regulation could be modified
through the combined effect of the two isoforms. Furthermore, en-
dogenous expression levels of CAR are likely much less than those
achieved in the transfection experiments conducted here. Therefore, it
is possible that the high concentrations achieved in the in vitro assays
mask certain potential effects. Further experiments will be required to
more thoroughly determine potential interactions between the CAR
At this point, it is unclear whether the activities of CAR, or the
respective CAR variants, may be subject to regulation through differ-
ential phosphorylation. In our studies, we used site-specific mutagen-
esis to evaluate the potential contributions of differential phosphory-
lation of the Ser233 residue in CAR2. We observed significant
FIG. 6. Effects of Ser233 mutagenesis on heterodimerization and SRC-1 recruit-
ment. 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 trans-
fection 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 RxR?. B, interaction between CAR2 and its S233A and S233D
mutants with SRC-1 in the presence and absence of RxR? and clot (clotrimazole).
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION
differences in transactivation potential among the modified CAR2
proteins. One potential explanation for these differences among
CAR-2, CAR2-A, and CAR2-D is that Ser233 serves as a site of
phosphorylation functioning to negatively regulate the activity of
CAR2 by modifying its ability to heterodimerize with RXR? and
engage in subsequent coactivator recruitment. This hypothesis is
consistent with the transactivation capacities of the different CAR2
forms: with CAR2-A ? CAR2 ? CAR2-D. This pattern of activity
matches the ability of the receptors to interact with RXR? and to bind
coactivator in an RXR?-dependent fashion. Despite the intriguing
nature of these results, it is still premature to suggest that CAR2 is
regulated by phosphorylation in vivo. However, it is clear that the
amino acid constituency of the inserted SPTV sequence in CAR2
imparts unique functional attributes to the receptor in both its ability
to heterodimerize with RXR? and its response to inverse agonists.
The biological implications of these findings, in vivo, will require
Current estimates place the number of human genes at 20,000 to
25,000, approximately equal to the number of genes in Arabidopsis
thaliana and only approximately 4 times more than the number of
genes found in Saccharomyces cerevisiae (Blencowe, 2006). A pos-
sible explanation for increased complexity in higher organisms could
be their more extensive use of alternative splicing. An estimated 40 to
60% of all human genes undergo alternative splicing (Modrek and
Lee, 2002). On the other hand, S. cerevisiae has been shown to use
very few alternative-splicing events (Sapra et al., 2004). The CAR
gene has been shown to undergo extensive alternative splicing. Along
with the pregnane X receptor, CAR mediates a defense against po-
tentially toxic exogenous and endogenous chemicals, along with an
ever-growing list of other roles. Regulation of CAR activity is com-
plex and poorly understood. The studies presented here indicate a
number of biological differences between the reference form of CAR
and an alternatively spliced isoform, CAR2. Alternative splicing of
the CAR gene may be an evolutionarily derived mechanism that
allows it to produce an array of proteins that are able to carry out
separate tasks by using subtle differences in their regulation and
interactions with chemical activators.
Acknowledgments. We are grateful to Denise Weyant and
Shengzhong Su for their skilled technical assistance and are thankful
to Jack Vanden Heuvel and Eric Tien for gifts of plasmids and advice
with respect to the mammalian two-hybrid assay.
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Address correspondence to: Dr. Curtis J. Omiecinski, Center for Molecular
Toxicology and Carcinogenesis, Department of Veterinary & Biomedical Sci-
ences, The Pennsylvania State University, University Park, PA 16802. E-mail:
CAR2 LIGAND BINDING AND RXR? HETERODIMERIZATION