Molecular and Cellular Biology, April 2001, p. 2838-2846, Vol. 21, No. 8
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2838-2846.2001
The Peptide Near the C Terminus Regulates Receptor CAR Nuclear
Translocation Induced by Xenochemicals in Mouse Liver
Igor
Zelko,
Tatsuya
Sueyoshi,
Takeshi
Kawamoto,
Rick
Moore, and
Masahiko
Negishi*
Pharmacogenetics Section, Laboratory of Reproductive and
Developmental Toxicology, National Institute of Environmental
Health Sciences, National Institutes of Health, Research Triangle Park,
North Carolina 27709
Received 20 October 2000/Returned for modification 3 January
2001/Accepted 16 January 2001
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ABSTRACT |
In response to phenobarbital (PB) and other PB-type inducers, the
nuclear receptor CAR translocates to the mouse liver nucleus (T. Kawamoto et al., Mol. Cell. Biol. 19:6318-6322, 1999). To define the
translocation mechanism, fluorescent protein-tagged human CAR (hCAR)
was expressed in the mouse livers using the in situ DNA injection and
gene delivery systems. As in the wild-type hCAR, the truncated receptor
lacking the C-terminal 10 residues (i.e., AF2 domain) translocated to
the nucleus, indicating that the PB-inducible translocation is AF2
independent. Deletion of the 30 C-terminal residues abolished the
receptor translocation, and subsequent site-directed mutagenesis
delineated the PB-inducible translocation activity of the receptor to
the peptide L313GLL316AEL319. Ala
mutations of Leu313, Leu316, or Leu319 abrogated the translocation of
CAR in the livers, while those of Leu312 or Leu315 did not affect the
nuclear translocation. The leucine-rich peptide dictates the nuclear
translocation of hCAR in response to various PB-type inducers and
appears to be conserved in the mouse and rat receptors.
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INTRODUCTION |
Organisms are capable of inducing
numerous xenochemical- and steroid-metabolizing enzymes such as
cytochromes P450 (CYPs) in liver microsomes, increasing metabolic
capability against xenochemical toxicity and carcinogenicity.
Phenobarbital (PB), the prototype of structurally diverse
xenochemicals, induces pleiotropically hepatic genes in mammalian
species from mouse to human, with the CYP2B genes being most
dramatically upregulated. A conserved 51-bp PB-responsive enhancer
module (PBREM) has recently been located in the mouse, rat, and human
CYP2B genes (9, 21). PB responsiveness of the
region, including the 51-bp sequence, has been observed independently
by various laboratories (16, 19, 20). Moreover, the
nuclear receptor CAR (constitutive active receptor or constitutive androstane receptor) has been found to regulate the induction of the
CYP2B genes, as well as the transactivation of PBREM
(10, 21). CAR is retained in the cytoplasm of control
hepatocytes and translocates to the nucleus following PB treatment
(13). Nuclear translocation appears to be a general
process through which CAR regulates the CYP2B induction,
since various PB-type inducers (e.g., chloropromazine, chlorinated
biphenyls, and methoxychlor) are also capable of translocating CAR into
the nucleus in livers. The molecular and cellular mechanisms underlying
the xenochemical-responsive nuclear translocation regulated by PB-type
inducers remain a question of major interest.
The fluorescent protein-tagged hCAR, expressed in transformed cell
lines such as HepG2 and HEK293, translocates spontaneously to the
nucleus without exposing the cells to PB-type inducers (13). These in vitro systems provided a practical tool for
the initial identification of signal sequences of CAR that regulate its
nuclear translocation. To understand the molecular basis underlying CAR
for nuclear translocation, we constructed various fluorescent protein-tagged CAR expression vectors and investigated the
nucleocytoplasmic localization of these truncated or mutated CAR
proteins expressed in HEK293 cells. Subsequently, these regulatory
sequences were tested for their functions in livers using in situ
injection of various CAR expression vectors. We herein describe
experimental considerations that lead us to propose that the
leucine-rich sequence near the C terminus regulates the
xenochemical-induced nuclear translocation of CAR in mouse livers in vivo.
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MATERIALS AND METHODS |
Plasmids.
Polymerase chain amplification was employed using
pT7HisMyc-hCAR plasmid, Pfu DNA polymerase, and specific
sets of primers to generate deletion mutants of CAR with newly created
XhoI and EcoRI sites at the 5' and 3' ends,
respectively. PCR amplification was done with primers that amplified
the nucleotides encoding amino acids 1 to 348 (hCARwt), 1 to 338 (hCAR1-338), 1 to 328 (hCAR1-328), 1 to 318 (hCAR1-318), and 1 to 308 (hCAR1-308).
Amplified fragments were digested with these two enzymes and inserted
into pEGFP-C1, pECFP-C1, or pEYFP-C1 vectors (Clontech, Palo Alto,
Calif.) to give an in-frame N-terminal fusion with one of the green
(GFP), cyan (CFP), or yellowish (YFP) fluorescent proteins. A human
glucocorticoid receptor (hGR)cDNA (provided by John Cidlowski) was also
cloned into pEYFP-C1 or pECFP-C1 vector. Site-directed mutagenesis of
hCAR and mouse CAR (mCAR) in pEYFP-C1 was conducted according to the
instruction manual for the QuickChange site-directed mutagenesis system
(Stratagene, La Jolla, Calif.). Nucleotides encoding leucines 312, 313, 315, 316, and 319 in hCAR and leucines 326 and 329 in mCAR, as well as
Gly314 and Glu318 in hCAR, were mutated to encode alanines. All
mutations and deletions were confirmed by sequencing, and plasmids
bearing the CAR cDNAs with mutations or deletions were prepared using
Qiagen Plasmid Maxi Kit (Qiagen, Valencia, Calif.). PBREM-tk-luciferase
reporter gene was constructed in the pGL3 plasmid (21). A
mouse SRC-1 cDNA, corresponding to amino acid residues from 633 to
1405, was amplified by using Pfu DNA polymerase and a
specific set of primers having EcoRI and
HindIII restriction sites at the 5' and 3' ends,
respectively. Amplified cDNA was cloned into pcDNA3.1 Myc/His plasmid
(Invitrogen) with the Kozak sequence at the 5' end. The SRC-1 cDNA was
verified by sequencing.
DNA transfections and visualization of fluorescent protein-tagged
CAR in HEK293 cells.
Cells were cultured in Dulbecco modified
Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 100 U
of penicillin per ml and 100 µg of streptomycin per ml at 37°C
under a 5% CO2 atmosphere. For analysis of nuclear
translocation, cells in an 8-well Lab-Tek Chamber Slide (Nalge Nunc,
Naperville, Ill.) were switched to DMEM without phenol red, transfected
with various plasmids using the calcium phosphate method (CellPhect
Kit; Pharmacia), and cultured for an additional 24 h.
Fluorescence-positive cells were identified with Axiovert 35 inverted
fluorescence microscope (Zeiss) equipped with filters from Omega optics
(Brattleboro, Vt.). YFP and GFP fluorescences were detected by using an
XF104 filter, while CFP fluorescence was visualized by using an XF113 filter. Fluorescent images of liver cells were captured with Spot II
cooled charge-coupled device camera (Diagnostic Instruments, Sterling
Heights, Mich.) and processed using the accompanying software package.
Nucleocytoplasmic localization of fluorescent protein-tagged CAR or GR
was determined by counting cells. GFP fluorescence-positive cells were
classified into three different categories: N<C for predominantly
cytoplasmic fluorescence, N=C for equal fluorescence distribution in
both the cytoplasmic and the nuclear regions, and N>C for
preferentially nuclear fluorescence.
For analysis of SRC-1 coactivation HEK293 cells were transfected with
pGL3-tk-mPBREM and pRL-CMV plasmids using calcium phosphate coprecipitation. The pcDNA3.1 Myc/His (Invitrogen) plasmid containing cDNA for wild-type hCAR and its mutant forms was cotransfected alone or
with pcDNA3.1-mSRC-1 vector, and the luciferase activity was assayed
24 h after transfection. The promoter activities were determined
from three independent transfections and normalized against Renila
luciferase activities.
Expression of fluorescent protein-tagged CAR in livers.
A
portion of the liver was exposed through a ventral midline incision,
and 80 µg of a given plasmid DNA in 300 µl of minimal essential
medium was injected into three different spots, as previously described
(21), or the expression plasmids (10 µg) were injected through the tail vein using TransIT In Vivo Gene Delivery
System (Mirus, Madison, Wis.) according to the manufacturer's
protocol. Then, two doses of xenochemicals were administered
intraperitoneally at 2 and 5 h after the injection: chloropromazine
(CPZ; 50 and 25 mg/kg, respectively), PB (100 mg/kg), or
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP; 0.33 mg/kg). The
mice were sacrificed 3 h after the last treatment with
xenochemical, and a block of the liver was frozen for preparing the
sections (8-µm thickness) using Tissue-Tek OTC compound (Sakyra
Finetek, Torrance, Calif.). One of every other four consecutive
sections was placed on a cover glass (three sections per glass). Liver
sections on cover glasses were fixed with 4% paraformaldehyde, washed
once with phosphate-buffered saline, dehydrated in methanol, and
stained for nuclei using 0.5 µg of Hoechst S-33258 per ml in 80% glycerol.
Gel shift assays.
The TNT-Coupled Reticulocyte Lysate System
(Promega) was used to prepare in vitro-translated CAR and RXR
as
described in our previous study (21). These in
vitro-translated receptors were incubated with 10 µl of HEPES buffer
(pH 7.6) containing 0.5 mM dithiothreitol, 15% glycerol, 2 µg of
poly(dI-dC), 0.05% NP-40, 50 mM NaCl, and approximately 30,000 cpm of
32P-end-labeled oligonucleotide.
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RESULTS AND DISCUSSION |
Nuclear localization of hCAR in HEK293 cells.
The
PBREM-tk-CAT reporter gene is activated by hCAR in cotransfected
cells such as HepG2 and HEK293 in the absence of inducers such as PB
and TCPOBOP (10). The activation appears to occur because
hCAR is inherently active and always in the nucleus of the transfected
cells (13). YFP-tagged hCAR was expressed in the nucleus
of the transfected HEK293 cells, whereas CFP-tagged hGR was expressed
in the cytoplasm and dexamethasone treatment translocated the GR into
the nucleus (Fig. 1A).
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FIG. 1.
Nucleocytoplasmic distribution of the wild-type and
truncated CARs in HEK293 cells. (A) Both YFP-CAR and CFP-GR expression
plasmids were cotransfected into HEK293 cells. After being cultured for
24 h, the transfected cells were treated with dexamethasone (+DEX,
0.1 µM) or dimethyl sulfoxide ( DEX) for 3 h and visualized
under a fluorescence microscope. (B) Various truncated hCARs were
constructed, expressed in HEK293 cells, and visualized under a
fluorescent microscope as described in Materials and Methods. (C)
Deletion mutants are shown underneath the wild-type
(hCARwt) with numbers indicating deletions from the
C-terminal end. The cells expressing hCAR were visualized and evaluated
for the nucleocytoplasmic distribution of the receptor: N<C for
cytoplasmic dominant fluorescence; N=C for equal fluorescence
distribution in both cytoplasmic and nuclear regions; and N>C for
nuclear dominant fluorescence.
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Residues were successively deleted from the C terminus of hCAR, and the
C-terminal truncated receptors were constructed into pEGFP-C1 vector
and transfected into HEK293 cells (Fig. 1B and C). Even when the first
10 residues were removed, the receptor (hCAR1-338) still
accumulated in the nucleus, suggesting that hCAR1-338
retained the translocation capability. However, deletion of the
additional 10 residues decreased dramatically the nuclear translocation
capability of the receptor. In fact, none of the cells displayed
nuclear dominant localization of the hCAR1-328. The lack
of the nuclear accumulation was not due to a stability of the deleted
CAR protein since Western blot analysis of the cell extracts showed the
expression of both hCARwt and hCAR1-328 with
the expected sizes at similar levels in the transfected HEK293 cells
(data not shown). In further deletion, hCAR1-318 behaved
similarly to the hCAR1-328 with respect to the
translocation capability. These results suggested that a regulatory
information for the nuclear translocation might lie within the
20-amino-acid residue segment between positions 319 and 338 of the
receptor. We then examined whether these residues regulated the nuclear
translocation of CAR in mouse liver in vivo following treatment with PB
or other PB-type inducers.
PB-responsive nuclear translocation in livers.
Our previous
immunochemical analyses of mCAR in mouse livers and primary hepatocytes
have shown that mCAR is cytoplasmic and translocates to the nucleus in
response to PB and PB-type inducers such as TCPOBOP and CPZ
(13). To examine whether hCAR was also retained in the
cytoplasm and translocated into the nucleus after treatment with
PB-type inducers, the CFP-tagged hCAR and YFP-tagged hGR were
coexpressed in mouse liver in vivo. After injection with the
corresponding expression plasmids, frozen sections of the livers were
used to visualize these receptors under a fluorescent microscope. The
wild-type hCARwt was expressed in the cytoplasm of liver
cell, while hGR was primarily localized in the nucleus of the same cell
(Fig. 2A), which is opposite from what
happened in the transfected HEK293 cells (Fig. 1A). The cytoplasmic and nuclear localization of hCAR and hGR, respectively, appeared to be a
true reflection of the nucleocytoplasmic localization supposed to be
observed with these receptors in the unexposed livers. To examine
whether the cytoplasmic hCARwt could translocate to the nucleus following treatment with PB-type inducers, the mice bearing livers injected with the expression plasmid were treated with CPZ (Fig.
2B). In fact, none of the 70 cells examined exhibited the exclusive
nuclear localization of CFP-hCARwt in control livers (Fig.
2C). In sharp contrast to the unexposed liver cells, the majority of
the 63 cells examined showed the nuclear localization of the CFP fusion
protein in the CPZ-exposed livers. These results thus indicate that the
expressed hCARwt is capable of translocating to the nucleus
following CPZ treatment in mouse livers.
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FIG. 2.
Nucleocytoplasmic distribution of hCAR and its deletions
in livers in vivo. (A) YFP-hGR and CFP-hCARwt were
coexpressed in the livers of nontreated mice using the in situ DNA
injection and then visualized in the same cell under a fluorescence
microscope. Panels a and b represent YFP-hGR and
CFP-hCARwt, respectively, while panel c shows the stained
nuclei. (B) CFP-hCARwt and YFP-hCAR1-338 were
coexpressed in the livers of the nontreated and CPZ-treated mice using
the in situ DNA injection and then visualized in the same cell under a
fluorescence microscope. Panels a and b represent
YFP-hCAR1-338 and CFP-hCARwt, respectively,
while panel c was stained for nuclei. The three images are imposed in
each panel d. (C) The expression plasmids for various hCARs as
GFP-tagged protein were directly injected into mouse livers, and the
nucleocytoplasmic distribution of the expressed CARs was analyzed in
the nontreated () and CPZ-treated (+) livers as described in
Materials and Methods. The numbers indicate the cell populations as
follows: N, nuclear distribution; N/C, distribution in cytoplasm and
nucleus; and C, cytoplasmic distribution.
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After establishing the in vivo liver system in which the nuclear
translocation of hCAR occurred in response to a PB-type inducer, we
attempted to define the role of the C-terminal region of hCAR in
nuclear translocation. For this, various C-terminal truncated CARs were
coexpressed with the wild-type receptor in the same cell. The
YFP-tagged hCAR1-338, lacking C-terminal 10 residues (i.e., AF2 domain), was sequestered in the cytoplasm of unexposed livers and translocated to the nucleus following CPZ treatment, which
also occurred with the wild-type in same liver cells (Fig. 2B and C).
The expressions of CFP-tagged hCARwt and YFP-tagged hCAR1-338 overlapped exactly in the unexposed and
CPZ-induced liver cells. In fact, the hCAR1-338 responded
not only to CPZ but also to PB and TCPOBOP, indicating that the AF2
domain was nonessential for the nuclear translocation induced by
PB-type inducers.
The nuclear translocation of hCAR, however, began to be affected by an
additional deletion of the C-terminal residues. When the 20 residues
were removed, none of the 32 cells showed the dominant nuclear
localization in the CPZ-induced livers (Fig. 2C). However, the
hCAR1-328 retained some degree of the nuclear
translocation capability in the liver since the receptor was localized
equally in the cytoplasm and nucleus of half of these cells. Given the
differences in the translocation capability of the truncated receptor
in the livers compared with the complete loss in the HEK293 cells, we
removed the 30 residues up to position 318 of the hCAR. The YFP-tagged
hCAR1-318 was always localized in the cytoplasm even after
treatment with CPZ, indicating that the hCAR1-318 lost the
nuclear translocation capability in mouse liver (Fig.
3). These results indicated that, first
of all, the AF2 domain plays no role in the nuclear translocation in
the liver and that the key residue responsible for the hepatic translocation may reside between positions 319 and 328 of the receptor.
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FIG. 3.
Nuclear translocation in response to various PB-type
iducers. The expression vectors CFP-hCARwt and
YFP-hCAR1-318 were simultaneously injected into the livers
of mice, and subsequently the mice were treated with PB, CPZ, or
TCPOBOP as described in Materials and Methods. For each group, panels a
and b display YFP-hCAR1-318 and CFP-hCARwt,
respectively; panel c was stained for nuclei; and panel d shows an
imposition of all three images.
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To search for residue(s) that may regulate the nuclear translocation in
the liver, various amino acid residues between positions 319 and 338 were mutated: S321A, I322A, Y326A, Y328A, I330A, I333A, and L336A.
These mutations, however, did not affect the nuclear translocation of
hCAR (data not shown). Amino acid sequence analysis then revealed a
small region of CAR that contains a cluster of Leu residues starting
with Leu319 toward the N terminus:
L312LGLLAEL319 (Fig.
4). Each of these Leu residues was
mutated to Ala, and the mutated YFP-tagged hCARs were coexpressed with
the CFP-tagged wild-type receptor in the mouse livers using gene
delivery through tail vein injection. Since this delivery system
provided us with high transfection efficiency, the expression of a
given fluorescent protein-tagged CAR could be observed in multiple
cells on same field. The hCAR[L312A] and hCAR[L315A] mutants
translocated to the nucleus after CPZ treatment as the wild-type CAR
did, whereas the mutants hCAR[L313A], hCAR[L316A], and
hCAR[L319A] abrogated the nuclear translocation capability and
remained in the cytoplasm even after CPZ treatment (Fig.
5). Thus, the
L313XXL316XXL319 appeared to
be a motif that regulates the CPZ-inducible nuclear translocation of
hCAR in the mouse livers. Retrospectively, the lack of Leu319 was a
major reason that hCAR1-318 was unable to translocate
to the nucleus in the CPZ-treated livers. Although a role for residues
329 to 338 could not be completely ruled out, any such role might be
secondary to the role played by these Leu residues in the nuclear
translocation. The
L312LGLLAEL319
sequence is conserved in the mouse and rat CARs as
L322MGLLADL329
and
L322MGLLAEL329,
respectively (Leu residues in the motif are underlined). As expected,
the mutation of Leu326 or Leu329 to Ala abolished the nuclear
translocation of mCAR in the mouse livers (Fig.
6). The C-terminal leucine-rich
peptide conserved as L/(M) XXLXXL appeared to be a general
response signal that dictates the receptor CAR to translocate to the
nucleus following CPZ treatment. We have designated this leucine-rich
peptide as the xenochemical response signal (XRS).
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FIG. 4.
Alignment of a carboxy-terminal sequence of hCAR with
the corresponding region of hGR. The hGR sequence and the localization
of the predicted secondary structures are depicted from the Wurtz's
multiple alignments (22). The hCAR sequence was then
aligned underneath of the sequence of hGR. Residues within the putative
LXXLXXL sequence designated the XRS are boxed. The presumed -helices
10, 11, and 12 are indicated by arrows. The numbers indicate the
positions of the residues of each receptor. The sizes of DBD and LBD
are arbitrary.
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FIG. 5.
Nucleocytoplasmic localization of the hCAR mutants. (A)
Various mutated hCARs were coexpressed with the wild-type hCAR using
the gene delivery system through tail vein injection of their
fluorescent protein-tagged expression vectors in the nontreated ()
and CPZ-treated (+) mouse livers as described in Materials and Methods.
All three images of YFP-mutated hCAR, CFP-hCARwt, and
nuclei stained with Hoechst S-33258 are imposed in these pictures. (B)
Semiquantification of the intracellular distribution of various mutated
hCAR in liver cells. The numbers indicate the cell populations as
follows: N, nuclear distribution; N/C, distribution in cytoplasm and
nucleus; and C, cytoplasmic distribution.
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FIG. 6.
Nucleocytoplasmic localization of the mCAR mutants.
Expression vectors of the wild-type mCAR and of either mCAR mutants
were simultaneously injected in mice through the tail vein, and the
mice were subsequently treated with CPZ (+CPZ) or dimethyl sulfoxide
(CPZ) as described in Materials and Methods. For each group, panels a
and b display the mutated YFP-mCAR and wild-type CFP-mCAR,
respectively; panel c was stained for nuclei; and panel d shows an
imposition of all three images.
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The XRS region overlaps with the region that could be involved in
various protein-protein interactions of nuclear receptors. The recent
crystal structures of RXR-PPAR and RXR-RAR heterodimers revealed that
N-terminal region of helix 10 is located on the dimerization interface
of these nuclear receptors (3, 7). Site-directed
mutagenesis of residue in the same N-terminal region resulted in the
inhibition of receptor heterodimerization (1). Therefore,
we examined what degree the Leu mutations of XRS could affect the
heterodimerization of hCAR with hRXR. For this, gel shift assays
were performed using 32P-labeled NR1 oligonucleotide as a
probe (Fig. 7A). Although mutations of
each of these Leu residues slightly decreased the band intensities representing the CAR-RXR heterodimer, there was no significant difference in the ability of various mutated hCARs to form RXR heterodimer, regardless of their specific effects on the nuclear translocation of hCAR in mouse livers. We also examined whether the
mutations altered the coactivation of hCAR by SRC-1 in HEK293 cells
(Fig. 7B). However, the coexpression of SRC-1 resulted in an increase
of ca. 70% of all hCAR-mediated transactivations of PBREM in the
cotransfected cells. Our present experiments do not suggest that the
region of XRS is not involved in various heterodimerizations, but they
clearly show that both the RXR heterodimerization and the SRC-1
coactivation are not affected by the Leu mutations that inhibited the
nuclear translocation of hCAR. These results suggest that the Leu
residues regulate the translocation but not activation of the receptor.
Whether the XRS region constitutes and acts as a sequence motif remains
an interesting question for future investigation.
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FIG. 7.
Effect of Leu mutations on the heterodimerization with
RXR and the coactivation by SRC-1. (A) Various mutated hCARs were
prepared by in vitro translation and mixed with the similarly prepared
RXR for gel shift assays using 32P-labeled NR1
oligonucleotide as a probe. (B) pcDNA3.1 plasmids bearing various hCARs
were cotransfected with a PBREM-tk-luciferase reporter plasmid (in
pGL3) into HEK293 cells. After being cultured with or without the
additional transfection of mSRC-1 for 24 h, the cells were lysed
for luciferase activity. The activities were normalized against those
of pRL-CMV, and coactivation was calculated as the percentage of the
increase by coexpression of SRC-1.
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XRS resides on -helix 10 near the C terminus of the CAR molecule, as
predicted from the alignment of multiple nuclear receptor sequences
(25). The C-terminal -helix 12 bears the AF2 domain; ligand-dependent functions are generally associated with the AF2 domain
of nuclear receptors (5, 8). Recent X-ray crystal structures have revealed that -helix 12 is displaced upon binding of
the ligand, allowing its association with a coactivator (15, 22). In addition, AF2 has also regulated the ligand-dependent nuclear translocation of vitamin D receptor, in which deletion of the
AF2 domain (AF-2del-VDR-GFP) abolished the translocation to the nucleus
in response to the vitamin (18). In the case of CAR,
however, our present studies have clearly shown that the receptor does
not require the AF2 for the PB-induced nuclear translocation in liver
in vivo. It is known that the removal of residues within the AF2 domain
abrogated the constitutive transactivation activity of hCAR
(4). Thus, the nuclear translocation and transactivation appear to be regulated by the different mechanism, and only the latter
function is linked to the AF2 domain observed. Instead, the nuclear
translocation function resides in the C-terminal LXXLXXL sequence, XRS.
Nuclear localization of a given protein is determined by a balance of
nuclear import and export (11, 17), which can be actively
regulated by peptide sequences such as the nuclear localization signal
(NLS) and the nuclear export signal (NES) built into the protein. XRS
does not resemble its sequence with so-called monopartite or bipartite
NLSs consisting of a single or repeated cluster of basic amino acids.
The NLS associates with specific factors such as importins that carry
NLS-bearing proteins into nucleus. Based on its sequence, XRS is not a
typical NLS, although the possibility of XRS being a novel NLS still
remains an interesting question for future research. The sequence of
XRS is somewhat similar to those of the NESs, occurring in the Ah
receptor, heat-stable inhibitor of cyclic AMP-dependent protein kinase
and human immunodeficiency virus type 1 Rev (6, 12, 24).
The XRS acting as an NES may be an alternative possibility: in the
liver treated with PB-type inducers, the XRS activity as NES is masked
by the heterodimerization with RXR, for example, resulting in the
accumulation of CAR in the nucleus. This is a less likely possibility
since the CAR without XRS is retained in cytoplasm and is not
accumulated in the nucleus of both livers as well as HEK293 cells.
Moreover, the Leu mutations of XRS did not abolish the
heterodimerization of hCAR with RXR. Thus, the XRS as an NLS is
still an interesting possibility.
The molecular and cellular mechanism of how XRS regulates the nuclear
translocation of CAR in response to PB-type inducers remains a major
interest in future research. Is direct PB binding to CAR essential for
the receptor's nuclear translocation? The most potent PB-type inducer
TCPOBOP induces the Cyp2b10 gene at a concentration of 10 to
50 nM in mouse primary hepatocytes compared with other PB-type inducers
that require micromolar to millimolar concentrations to induce the
gene, making it the best candidate for demonstrating its binding. Using
sensitive but indirect in vitro binding assays, it has recently been
shown that TCPOBOP can bind to mCAR but not to hCAR (14,
23). PB did not exhibit a meaningful binding to either mCAR or
hCAR. Under the present circumstances, in which the direct binding is
not firmly established, it is difficult to speculate how PB-type
inducers activate XRS and translocate the receptor into the nucleus.
Our finding of the translocation of hCAR as well as mCAR in the mouse
livers following treatment with TCPOBOP, however, is insightful. CAR may translocate to the nucleus in the absence of the direct binding of
TCPOBOP to the receptor. The protein phosphatase inhibitor okadaic acid
is known to suppress the PB-induced nuclear translocation of mCAR in
mouse primary hepatocytes (13). PB-type inducers may
elicit a phosphorylation/dephosphorylation pathway that regulates the
nuclear translocation of CAR in mouse livers. Although the direct
binding mechanism should be considered, the indirect regulation via a
protein dephosphorylation offers an alternative direction for future research.
Steroid hormone receptors are sequestered in the cytoplasm; as a
multimeric complex with the hsp90 chaperonic system, a detailed hypothesis of the hormone-dependent release of the receptors has been
proposed (2). GR is retained in the cytoplasm of
transfected HEK293 cells prior to dexamethasone treatment. However,
hCAR could not be retained in the cytoplasm and accumulated in the
nucleus of the cells. The ability of HEK293 cells to retain GR but not hCAR in the cytoplasm leads us to think that these two receptors are
regulated distinctly with respect to their translocation. In fact, the
CAR also appeared to exist in the complex with hsp90 in liver cytoplasm
(K. Yoshinari et al., unpublished observation), suggesting the presence
of factors specific to CAR that regulate the receptor translocation.
Since the -helix 10 contains multiple sequences responsible for
intramolecular and intermolecular interactions of the nuclear receptors
(15), XRS may be one of these sequences and may bind to
the specific factors regulating the PB-induced nuclear translocation of
CAR in liver in vivo. Identifications of the intramolecular interaction
with XRS and of proteins that associate with XRS would help us to
uncover the mechanism by which XRS regulates the receptor CAR for the
nuclear translocation. To this end, the purification and
characterization of CAR as a large complex from the liver cytosol of
untreated mice is now under way in this laboratory. Nevertheless,
further characterization of XRS should provide insight into the nuclear
translocation of CAR and the induction of various genes following
exposure to PB-type inducers.
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ACKNOWLEDGMENTS |
I.Z. and T.S. contributed equally to this work.
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FOOTNOTES |
*
Corresponding author. Mailing address: Pharmacogenetics
Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. Phone: (919)
541-2404. Fax: (919) 541-0696. E-mail:
negishi{at}niehs.nih.gov.
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Molecular and Cellular Biology, April 2001, p. 2838-2846, Vol. 21, No. 8
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.8.2838-2846.2001
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Abstract
Introduction
