(Received for publication, February 17, 1995; and in revised form, October 4, 1995)
From the
The testicular receptor 2 (TR2) orphan receptor binds to hormone
response elements (HREs) consisting of two AGGTCA half-site direct
repeat consensus sequences (DR) with various spacing in the following
order: DR1 > DR2 > DR5 DR4 DR6 > DR3. When binding to natural
HREs, TR2 orphan receptor remains flexible with higher binding
affinities to (a) cellular retinol-binding protein II promoter
region (CRBPIIp) (DR1), SV40 +55 region (DR2), and retinoic acid
response element (RARE
) (DR5) than to (b) NGFI-B
response element (NBRE) and also to (c) the palindromic
thyroid hormone response element (TREpal). This wide spectrum of HRE
recognition sequences suggests possible versatility of the TR2 orphan
receptor in cross-talking with other signal transduction systems.
Chloramphenicol acetyltransferase (CAT) assay demonstrates that the TR2
orphan receptor competes with CRBPIIp- and RARE
-CAT gene
expression activated by retinoid X receptor
(RXR
) and
retinoic acid receptor
(RAR
)/RXR
heterodimers,
respectively. In addition, this suppression may not be mediated by the
formation of heterodimers between TR2 orphan receptor and either
RXR
or RAR
. Instead, a minimum of 100-fold higher affinity of
the TR2 orphan receptor for CRBPIIp than RXR
may explain why the
TR2 orphan receptor dominates RXR
in CRBPIIp-CAT activation.
Together, our data suggest that the TR2 orphan receptor may be a master
regulator in modulating the activation of two key HREs, RARE
and
CRBPIIp, involved in the retinoic acid signal transduction pathway.
Sex steroids, adrenal steroids, thyroid hormones, vitamin A and
D derivatives in vertebrates and ecdysone in Drosophila play
key roles in triggering tissue and embryo development. Their
corresponding intracellular receptors have been revealed to be similar
and belong to a steroid receptor superfamily(1) . These ligands
bind to their specific receptors and the ligand-receptor complex then
interacts with cis-acting DNA hormone response elements (HREs) ()found mostly at the 5` promoter region of the target
genes. As a result, the transcription of these specific target genes is
either activated or suppressed(2, 3, 4) . In
addition to those receptors involved in mediating the specific ligand
signal transduction systems, a large proportion of this gene
superfamily consists of putative receptors, named orphan receptors,
having an unidentified ligand(5) .
TR2 orphan receptor has
been isolated from human prostate and testis cDNA libraries with a
probe designed to select clones encoding the steroid receptor DNA
binding domain. Three TR2 subclasses, TR2-5, TR2-7, and TR2-9, were
isolated from a human testis library; while the fourth clone, TR2-11,
was identified in a human prostate gt11 cDNA library(6) .
An identical DNA binding domain suggests that all TR2 orphan receptor
subtypes may act on the same target HREs. Consequently, we have
selected TR2-11 as the starting point to investigate the function of
TR2 orphan receptors. The TR2-11 orphan receptor is a protein of 603
amino acids with a calculated molecular mass of 67 kDa. The TR2 orphan
receptor has been expressed in many rat tissues with higher abundance
in male reproductive organs(7) . The transcription of TR2
orphan receptor mRNA is negatively regulated by androgen in the human
prostate LNCaP cell line and rat ventral prostate.
Based on the
amino acid sequences, all members of the steroid receptor family share
a high conservation in the DNA binding domain which is predicted to
form two zinc finger motifs. The first zinc finger, containing the P
box sequences, is proposed to be essential for the recognition of a
specific response element, while the second one is involved possibly in
protein-protein interactions, e.g. dimerization with receptors
among the same family(1, 8) . Consequently, these
receptors can be grouped into either the estrogen receptor (ER) or the
glucocorticoid receptor subfamily. The ER subfamily consists of ER,
thyroid hormone receptors (TRs), retinoic acid receptors (RARs),
retinoid X receptors (RXRs), vitamin D receptor, and many orphan
receptors, including the TR2 orphan receptor(9) . They all have
a unique glutamic acid residue following the third cysteine residue
found in the P box, and they are predicted to recognize palindrome
estrogen response element (ERE)/TRE (AGGTCA (n) TGACCT, x = 0 or 3)
sequences(10, 11, 12, 13) . Lately,
investigators have demonstrated that the some of the ER subfamily
members also recognize AGGTCA in a direct-repeat (DR) orientation
(AGGTCA (n)
AGGTCA, x =
0-6) or even an AGGTCA hexamer half-site with additional
nucleotides at 5` end ((n)
AGGTCA). For
instance, RXRs form heterodimers with vitamin D receptor, TRs, and RARs
upon the recognition and activation of response elements consisting of
AGGTCA half-site tandem direct repeats with 3-4-5 base spacing,
respectively(14) . Chicken ovalbumin upstream promoter
transcription factor (COUP-TF) has been shown to recognize not only
AGGTCA direct repeats with various spacing but also AGGTCA palindrome
elements(15) . NGFI-B prefers an octameric HRE, AAAGGTCA, and
functions as a monomeric trans-acting receptor (16) .
Here
we demonstrate that TR2 orphan receptor recognizes not only AGGTCA
direct repeats with various spacing but also TRE palindromic repeat,
albeit at a much lower affinity. The flexibility of TR2 orphan receptor
may allow it to form monomeric binding with single half-site HRE, e.g. NBRE. With the emphasis on the retinoic acid signal
transduction system, we demonstrate that TR2 orphan receptor binds to
RARE and CRBPIIp HREs with high affinities in vitro and
competes for CAT reporter gene expression in cell culture with high
efficiency.
The pSG5-TR2-ARp-TR2 expression vector was designed to swap the hAR P-box sequence into the corresponding TR2 orphan receptor coding region. A 5` end polymerase chain reaction primer, TCAGGACGTCATTATGGAGCAGTAACTTGTGG*AA*GCTGCAAAGT*C*TTTTTTAAAAGAAGC, was synthesized to cover the unique AatII site (underlined) and fulfill hAR P-box sequences (marked by asterisks). A 3` end polymerase chain reaction primer, GGAAGAGTCTAGAGTCGACC, was synthesized according to the very end of the SV40 poly(A) signal found in pSG5 expression vector. The construct was completed by inserting the polymerase chain reaction product, against the pSG5-TR2 plasmid, back to the corresponding AatII and XbaI sites found in pSG5-TR2 plasmid. The construct was confirmed by sequences around ligation sites as well as designated mutated sequence.
Figure 1:
Binding
preference of TR2 orphan receptor to consensus perfect direct repeat
HREs. Cold competitive EMSA analysis was performed with 0.1 ng of P end-labeled DR1 oligomer, 1 µl of in vitro translated TR2 orphan receptor, and applicable cold competitive
oligomer as marked (DR1-DR6). Five quantities (0.00625,
0.025, 0.1, 0.4, and 1.6 ng) of each consensus direct repeat HREs,
sequences listed in Table 1, were allowed to compete for binding
to TR2 orphan receptor. The specific band shifts were quantitated with
PhosphorImager; the I
concentrations were determined and
RBAs calculated as described under ``Materials and Methods.''
The RBAs of these DRs to TR2 orphan receptor are listed in Table 1. Data are shown as a representative autoradiogram of two
independent experiments.
Cold competitive EMSA experiments
were performed and shown as Fig. 2A. The band shift
intensity versus concentration of competitor results were
plotted out as Fig. 2B and RBAs calculated as listed in Table 1. These RBA data reveal that the affinities of TR2 orphan
receptor to natural HREs are lower than those to perfect consensus HREs (e.g. comparing RBA of DR1 (4.19 ± 1.00) versus that of CRBPIIp (1.00)). And the binding preference of TR2 orphan
receptor to these natural HREs also follows the order of DR1 > DR2
> DR5: TR2 orphan receptor has the highest affinity to DR1 type HRE, i.e. CRBPIIp (1.00), which is followed by DR2, SV40 +55
(RBA = 0.104 ± 0.003), and then by DR5, RARE (RBA
= 0.0101 ± 0.0029). NBRE, consisting of single perfect
half-site(16) , has double the RBA against that of RARE
,
suggesting that TR2 orphan receptor may bind to both direct repeat HREs
and half-site HRE. To test whether TR2 orphan receptor can recognize
palindromic HRE, a TRE perfect palindrome with no space was tested;
however, the significantly lower RBA (0.00369 ± 0.00005) may
suggest that the TR2 orphan receptor prefers interacting with direct
repeat HREs versus palindrome HREs. In addition, a synthetic
SV40 +55 mutant, as illustrated in Table 1, failed to
compete with CRBPIIp even at the highest amount (800 ng) applied. This
result agrees with previous reports (22, 23, 40, 41) and demonstrates
that only one nucleotide mutated from G to C in both half-sites can
abolish the binding to TR2 orphan receptor completely and suggests that
the second G in AGGTCA repeat may be very important in the
determination of potential TR2 HREs. This result also argues against
the possibility that any cold competitive DNA oligomers in such a high
concentration as applied here may compete nonspecifically and
effectively with
P-labeled probe to bind TR2 orphan
receptor.
Figure 2:
Binding preference of TR2 orphan receptor
to natural HREs. A, cold competitive EMSA analysis was
performed with 0.1 ng of P end-labeled CRBPIIp oligomer, 1
µl in vitro translated TR2 orphan receptor, and applicable
cold competitive oligomer as marked. Same five quantities (0.00625,
0.25, 0.1, 0.4, and 1.6 ng) of DR1 cold competitor applied in Fig. 1were utilized here for the purpose of cross-reference
between RBAs of consensus HREs and those of natural HREs. Due to
various competitive abilities among different natural HREs, to obtain
the 50% competition concentration (I
), more excess amounts
of HREs have been applied as marked. For instance, five concentrations
of SV40 +55 (0.125, 0.5, 2, 8, and 32 ng), marked as 20
, used here are 20 times more than the corresponding
five amounts (marked as 1
) of DR1 and also those of
CRBPIIp applied (0.00625, 0.025, 0.1, 0.4, and 1.6 ng). RARE
(0.5,
2, 8, 32, and 128 ng), TREpal (0.625, 2.5, 10, 40, and 160 ng), and
NBRE (0.781, 3.125, 12.5, 50, and 200 ng) cold oligomers applied here
are 80, 100, and 125 times, respectively, the concentrations of those
of CRBPIIp. Data shown here as a representative autoradiogram of five
independent experiments which cover different spectrum of natural HREs
as cold competitors. B, EMSA analysis shown in A was
quantified by using the PhosphorImager. The 100% binding was obtained
as the quantified band shift generated between radiolabeled CRBPIIp and
TR2 orphan receptor without added competitive oligomer. The ratio
between the intensity of competed band shift and that of 100% binding
was calculated as relative binding and plotted as shown here. The
corresponding sequences and RBAs of these six cold competitors (DR1
(
), CRBPIIp(
), SV40 +55 (
), RARE
(
), TREpal (
), and NBRE (
)) applied here are
summarized and shown in Table 1.
Figure 3:
A, TR2 orphan receptor, RAR and
RXR
bind to RARE
with specificity. EMSA analysis was
performed with 0.1 ng of
P end-labeled RARE
oligomer
and applicable in vitro translated TR2 orphan receptor (1
µl), RAR
(1 µl), or RXR
(0.5 µl) as indicated
(
) and with an additional 200-fold excess amount of
nonradiolabeled RARE
oligomer (
). Specific band shifts are
marked as TR2 and RAR
/RXR
next to the autoradiogram. B, two independent binding band shifts are generated from TR2
orphan receptor and RAR
/RXR
heterodimer in the presence of
RARE
oligonucleotide probe. Competitive EMSA analysis was
performed with 50 pg of
P end-labeled RARE
oligomer,
various amounts of TR2 orphan receptor (1.5-6 µl) and
constant amounts of both RAR
(1 µl) and RXR
(0.5 µl)
as marked on top of the autoradiogram (lanes 2-5). As
controls for band shift intensity TR2 orphan receptor (6 µl) and
RAR
(1 µl)/RXR
(0.5 µl) along were applied
simultaneously (lanes 1 and 2, respectively). To
bring up to the same concentration of protein applied in each EMSA
reaction, various amounts of unconditioned reticulocyte lysate were
added accordingly. Autoradiogram shown here represents four independent
experiments. Nonspecific band shifts, marked as open squares
(
), were identified in a parallel experiment done with
nonconditioned (mock) reticulocyte lystate (data not shown). The
migrated position correspond to free probe are marked as F.P. next to the autoradiograms.
Figure 4:
TR2 orphan receptor and RAR/RXR
bind to RARE
with similar affinities. Autoradiogram from EMSA
showing band shifts formed with increasing amounts of
P-labeled RARE
oligomers (0.0625, 0.125, 0.25, 1, 2,
4, and 8 ng, respectively, from left to right) and
fixed amounts of in vitro translated TR2 orphan receptor (2
µl) or RAR
and RXR
(1 and 0.5 µl, respectively).
These specific band shifts and free probes were quantitated as
described under ``Materials and Methods,'' and the data are
used to construct saturation curves, illustrated in C, showing
binding of TR2 orphan receptor (open squares) or
RAR
/RXR
(open diamond) to RARE
. The subsequent
Scatchard analysis (C, inset) reveals similar
dissociation constant (K
) in between: TR2
(5.03 nM) and RAR
/RXR
(2.32
nM).
Figure 5:
Inhibitory effect of TR2 orphan receptor
on RARE-pCATp expression from CV1 cells. Autoradiogram shows CAT
reporter gene analysis performed with monkey kidney CV1 cells. CV1
cells were transfected with RARE
-pCATp reporter gene only (lanes 1-3) or further co-transfected with pSG5 TR2
expression vector (lanes 4-5). The CAT activities were
induced by all-trans-retinoic acid (tRA) in
concentrations of 10
(lanes 2 and 5) and 10
M (lanes 3 and 6). As a negative control pCATp was transfected and CAT
activity shown as lane 7. Chloramphenicol conversion rates
were calculated from PhosphorImager quantifiable intensities. Fold
inductions were normalized according to the chloramphenicol conversion
rate obtained from CV1 cells, transfected with RARE
-pCATp
construct, without further added tRA (lane 1). Data shown here
represents three independent experiments.
Figure 6:
TR2 orphan receptor has higher affinity
binding to CRBPIIp HRE than RXR does. Saturation Scatchard
analysis was performed according to data obtained from an EMSA done
with increasing amounts of
P-labeled CRBPIIp oligomers
(0.0078, 0.031, 0.125, 0.5, and 2 ng) and fixed amounts of in vitro translated TR2 orphan receptor (1 µl). The dissociation
constant (K
), determined as the minus
reciprocal of the Scatchard plot slope, between TR2 orphan receptor and
CRBPIIp was calculated to be 0.028 nM (open square)
While much higher K
value between
RXR
and CRBPIIp were determined as 4.36 nM (open
circle). Additional RXR
ligand, 10
M 9cRA, altered the K
to 4.83 nM (open triangle). Due to lesser affinity between RXR
and CRBPIIp, the Scatchard plots were constructed from EMSA data done
with
P-labeled CRBPIIp in amounts of 0.0625, 0.125, 0.25,
1, 2, 4, and 8 ng and 2 µl of in vitro translated
RXR
.
Figure 7:
TR2 orphan receptor dominates over
RXR in regulating the expression of a CRBPIIp-pCATp construct. A, shows CAT reporter gene assay performed with CV1 cells. CV1
cells were transfected with CRBPIIp-pCATp reporter gene only (lane
1) or further co-transfected with pSG5 TR2 expression vector (lanes 3-8) and/or pCMX RXR
expression vector (lanes 2 and 6-8) in equal amounts (3 µg)
as indicated. The CAT activities were induced by the addition of
all-trans-retinoic acid (tRA) in concentrations of
10
(lanes 4 and 7) and
10
M (lanes 5 and 8). B shows independent CAT reporter gene assay performed with CV1
cells. CV1 cells were transfected the same as A.
Chloramphenicol conversion rates were calculated from PhosphorImager
quantifiable intensities. Fold inductions were normalized according to
the chloramphenicol conversion rate obtained from CV1 cells,
transfected with CRBPIIp-pCATp construct, without further added tRA (lane 1). Data shown here represents three independent
experiments.
Figure 8:
Tissue-specific distribution of TR2 orphan
receptor in mouse fetus. A gestation 16.5 day mouse fetus sagital
section was hybridized with S-labeled antisense TR2 orphan
receptor cRNA probe and processed as described under ``Materials
and Methods.'' The section was photographed under dark-field
illumination so that the autoradiography signal grain appears white.
TR2 orphan receptor expresses in various tissues with our interest on
its expression in kidney (K) and intestine (I). The inserted bar represents 1.5 mm in
length.
The biological significance of the human TR2 orphan receptor
subfamily was not clearly demonstrated shortly after its identification (6, 7) . The distribution of TR2 orphan receptor in
many tissues was recently reconfirmed by in situ hybridization
approaches done in mouse fetus. ()Also, recent studies have
manifested the importance of this orphan receptor subfamily. The TR2
orphan receptor family has now expanded to include the TR4 orphan
receptor(31) , and the spatial difference in distribution among
the members suggests an important role of the TR2 orphan receptor in
physiological events. It has been demonstrated recently that the TR2
orphan receptor may repress transcription activities of the SV40 major
late promoter(22) . In prostate, the expression of TR2 orphan
receptor can also be repressed by androgens (32) . The
identification of potential HREs that are recognized by the TR2 orphan
receptor may expand our understanding of the function of the TR2 orphan
receptor.
From amino acid and nucleotide sequences encoding the
P-box of the DNA binding domain, one can place TR2 orphan receptor into
the ER subfamily of the steroid receptor superfamily. Like other
members in the ER subfamily, we have demonstrated in this paper that
TR2 orphan receptor binds to all tested consensus HREs with the
following preferences in terms of nucleotide spacing: DR1 > DR2 >
DR5 DR4 DR6 > DR3. Three natural HREs that bind to the TR2 orphan
receptor effectively were also identified: CRBPIIp (an RXRE), SV40
+55 region, and RARE (an RAR
/RXR
RE), respectively.
In accord to the RBAs of consensus HREs, these three potential TR2 HREs
followed the same 1 > 2 > 5 space order in the RBAs.
Thyroid
hormone receptors are known to bind as homodimers and heterodimers with
RXR to response elements consisting of AGGTCA hexamer half-sites
in direct repeat, palindromic, and inverted palindromic
orientations(14, 33, 34, 35) .
Similar to NGFI-B and FTZ-F1, thyroid hormone receptor has also been
demonstrated to recognize octameric half-site HREs, NNAGGTCA consensus,
and function as a monomeric transacting factor(36) . Here we
also demonstrate that NBRE, an AAAGGTCA half-site HRE, with an RBA
double that of RARE
competes with CRBPIIp in binding to TR2 orphan
receptor. However, whether this half-site HRE operates as a TR2
response element remains to be elucidated by at least a reporter assay
in cell culture. On the other hand, to show effective competition
against CRBPIIp binding to the TR2 orphan receptor, approximately 140
times more concentrated TREpal is required, suggesting that TR2 orphan
receptor binds to palindromic HRE weakly. However, whether or not
palindromic repeats with various spacing alters the efficiencies in the
competition has not been tested yet. Thus far we have not yet explored
any putative TR2 HREs in an inverted palindromic orientation.
Study
of HREs sequences has revealed that the TR2 orphan receptor recognizes
various naturally available imperfect half-site sequences like AGtTCA (i.e. CRBPIIp and RARE), AGGTtc (i.e. SV40
+55), and gGtTCA (i.e. RARE
). These findings
indicate that TR2 orphan receptor has great flexibility in recognizing
various DNA sequences, and thus significant protein conformational
changes may be required to accommodate this phenomena. However, just
one nucleotide change of the second G, in AGGTtc/AGGTCA, into c, in
AGcTtc/AGcTCA, in both half-sites of SV40 +55 abolishes the
competitive ability completely (compare RBAs of SV40 +55 and SV40
+55 m in Table 1). This result provides indirect evidence
that the third position of the hexamer half-site may be a key
determinate on which the TR2 orphan receptor relies for binding.
Protein crystallographic or nuclear magnetic resonance approaches may
provide further direct evidence of this flexibility(37) . These
results support the idea that TR2 orphan receptor may interact with a
broad range of HREs involved in various signal transduction pathways.
The data presented here also suggest an important biological role of
TR2 orphan receptors in retinoid response pathways. The retinoic acids
(RAs) and retinoids are key elements in regulating embryogenesis,
tissue differentiation, and teratogenesis. Several gene products have
been demonstrated to be involved in this signal transduction system
directly. For example, nuclear RARs and RXRs can be activated by RAs
and operate as trans-acting factors that result in direct target gene
transcription activation. Two groups of cytoplasmic proteins, cellular
retinoic acid- and retinol-binding proteins (CRABPs and CRBPs,
respectively) were characterized to bind with RAs and retinoids and may
be involved in fine-tuning the concentrations of free intracellular
RAs. Functionally, RA can cross-talk with various signal transduction
pathways. RXRs are effective co-activators for vitamin D,
thyroid hormone, and retinoic acid receptors by forming stable
heterodimers and thus can promote a greater level of gene
activation(38) .
In this report we demonstrate clearly that
the TR2 orphan receptor down-regulates the stimulatory effect of
RAR/RXR
on RARE
CAT expression from monkey kidney CV1
cells in the presence of tRA. These results suggest a possibility that
this TR2 orphan receptor is a strong suppressor for RARE
activation and may result in the interruption of the RA gene regulation
cascade. The tissue distribution and spatial expression pattern of
RAR
and RXR
gene during mouse fetal development are well
documented using an in situ hybridization
technique(28, 29, 30) . Comparing these
results and ours in Fig. 8, we conclude that TR2 orphan
receptor, RAR
, and RXR
are expressed abundantly in at least
kidney and intestine during mouse embryo development. All these
expression patterns from in situ hybridization experiments may
manifest the potential physiological significance of this study.
COUP-TF orphan receptors have also been demonstrated to be negative
regulators of the RA response pathways(39) . Lately, this
negative regulatory effect of COUP-TF has been expanded to vitamin D
and thyroid hormone responding systems(15, 24) .
COUP-TF may activate the silencing of transcription by competing for
cis-acting response elements and/or forming heterodimer with
RXR(15) . Unlike COUP-TF, our EMSA experiments do not
support the possibility that TR2 orphan receptor forms heterodimers
with either RAR
or RXR
receptors directly. Our observations
shown here favor the hypothesis that this suppressive effect results
from competition between TR2 orphan receptor and RAR
/RXR
heterodimer by recognizing the same RARE
sequences. RARE
bound with TR2 orphan receptor may fail to activate the expression of
RAR
gene.
It is worth noting that TR2 orphan receptor showed at
least 100-fold higher affinity in binding to CRBPIIp response element
than RXR. Furthermore, CAT activities driven by the interaction
between RXR
and CRBPIIp could be suppressed when exogenous TR2
orphan receptors were made available. This high affinity of TR2 orphan
receptor to CRBPIIp in vitro suggests the possibility that the
transfected CRBPIIp-CAT constructs could be occupied completely by
exogenous TR2 orphan receptor and CAT activities driven accordingly.
This argument is strengthened further by the observation that a
P-box-mutated TR2 orphan receptor has failed to bind to CRBPIIp
effectively, and as a result, this mutant also did not interfere with
CRBPIIp-mediated CAT induction. In summary, our data suggest that TR2
orphan receptor may offer another regulatory mechanism involved in the
RA signal transduction system.