Multiple functions of the TR2-11 orphan receptor in modulating activation of two key cis-acting elements involved in the retinoic acid signal transduction system.

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 beta (RARE beta) (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 beta-CAT gene expression activated by retinoid X receptor alpha (RXR alpha) and retinoic acid receptor alpha (RAR alpha)/RXR alpha heterodimers, respectively. In addition, this suppression may not be mediated by the formation of heterodimers between TR2 orphan receptor and either RXR alpha or RAR alpha. Instead, a minimum of 100-fold higher affinity of the TR2 orphan receptor for CRBPIIp than RXR alpha may explain why the TR2 orphan receptor dominates RXR alpha 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 beta and CRBPIIp, involved in the retinoic acid signal transduction pathway.

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 ligandreceptor complex then interacts with cis-acting DNA hormone response elements (HREs) 1 found mostly at the 5Ј 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) x TGACCT, x ϭ 0 or 3) sequences (10 -13). Lately, investigators have demonstrated that the some of the ER subfamily members also recognize AGGTCA in a direct-repeat (DR) orientation (AGGTCA (n) x AGGTCA, x ϭ 0 -6) or even an AGGTCA hexamer half-site with additional nucleotides at 5Ј end ((n) x 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.

MATERIALS AND METHODS
Construction of Plasmids-The RARE␤-pCATp and CRBPIIp-pCATp reporter plasmids were constructed as follows. Double-stranded RARE␤ and CRBPIIp oligonucleotides (sequences shown in Table I) were inserted into the BglII digestion site located at the 5Ј end of an SV40 basic promoter in the pCAT promoter vector (Promega Co., Madison, WI). The final constructs were confirmed by DNA sequencing (CircumVent thermal cycle sequencing kit, New England Biolabs, Inc., Beverly, MA) to ensure only one copy of RARE␤ or CRBPIIp is inserted in the right orientation.
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 5Ј end polymerase chain reaction primer, TCAG-GACGTCATTATGGAGCAGTAACTTGTGG*AA*GCTGCAAAGT*C*T-TTTTTAAAAGAAGC, was synthesized to cover the unique AatII site (underlined) and fulfill hAR P-box sequences (marked by asterisks). A 3Ј end polymerase chain reaction primer, GGAAGAGTCTAGAGTC-GACC, 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.
In Vitro Transcription and Translation of Receptors-Three expression constructs, pSG5-TR2, pSG5-RAR␣, and pCMX-RXR␣, were utilized to produce in vitro transcribed and translated human TR2 orphan receptor, RAR␣ and RXR␣, respectively. T7 RNA polymerase was used to transcribe these constructs in a rabbit reticulocyte based coupled transcription/translation kit (TnT expression kit, Promega Co.). The yields were analyzed by the incorporation of [ 35 S]methionine, and the molecular weights were confirmed by SDS-PAGE autoradiography. The expression efficiency of pCMX-RXR␣ was about double that of pSG5-TR2 and pSG5-RAR␣. Consequently, to achieve the same concentration of in vitro translated receptors, half the volume of in vitro translated RXR␣ rather than that of TR2, and RAR␣ was applied in the following in vitro experiment.
Electrophoretic Mobility Shift Assay (EMSA)-EMSA analysis was performed according to the methods described by Cooney et al. (15). In brief, each 100-ng double-stranded oligonucleotide probe was either 5Ј end-labeled with 5 Ci of [␥-32 P]ATP or fill-in-labeled with 5 Ci of [␣-32 P]dCTP (DuPont NEN) by using T4 polynucleotide kinase or T4 DNA polymerase Klenow fragment (New England Biolabs, Inc.), respectively. At least 4 ϫ 10 8 dpm/g DNA specific activity was obtained consistently from both labeling methods. Radiolabeled oligonucleotide probes were mixed with unlabeled oligonucleotide competitor, if applicable, and allowed to interact with in vitro translated receptors for 15 min at room temperature before being applied to a 5% TBE (89 mM Tris borate and 2 mM EDTA, pH 8.0) polyacrylamide gel. Protein-DNA complex and free oligonucleotide probe were separated by running the gel at 150 V constant voltage in a cold room. Band shifts were developed and quantified by using ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA). The relative binding affinity (RBA) was calculated from the equation: RBA ϭ specific competitor (I 50 )/test competitor (I 50 ); i.e. the ratio between the concentration of CRBPIIp competitor that inhibits 50% (I 50 ) of the intensity of band shift and the I 50 concentration of tested competitor in the case where 32 P-labeled CRBPIIp is utilized.
Scatchard Analysis-To determine the dissociation constant (K d ) between receptor and HREs, EMSA was performed with fixed amounts of in vitro translated receptors and increased amounts of [␣-32 P]dCTP fill-in-labeled double-stranded HREs. The specific area corresponding to the receptor-HRE complex and free probe was excised and counted by liquid scintillation. These results were then subtracted from the background of corresponding nonspecific binding lanes, which were done parallel with the nonconditioned reticulocyte lysate (mock). Finally, specific bound and free probe data were processed by EBDA Scatchard analysis software (version 3.0 by G. A. McPherson, 1983).
Cell Culture, Transfection, and CAT Assay-Green monkey kidney CV1 cells were routinely maintained in Dulbecco's modified Eagle's medium/F-12 (Life Technologies, Inc.) supplemented with 5% heatinactivated fetal bovine serum (lot 91203 Bioproducts for Science, Inc., Indianapolis, IN). Cells were seeded in culture medium on 6-cm Petri dishes in a density of 500,000 cells/dish 24 h before transfection. Several hours before transfection, the culture medium was replaced with 2 ml of experimental medium (Dulbecco's modified Eagle's medium without phenol red supplemented with 5% charcoal dextran-treated fetal bovine serum). Serum was treated with charcoal dextran as described in a previous paper (17). Cells were transfected with 2.5 g of reporter CAT plasmids, 1.0 g of ␤-galactosidase expression plasmid, and 3.0 g of receptor expression vector where applicable; the final DNA concentration was brought up to 9.5 g with pSG5 cloning vector (Stratagene Cloning Systems, La Jolla, CA). A modified calcium phosphate method was applied in transfection (18). A ␤-galactosidase activity normalized 2-h CAT reaction product was extracted with ethyl acetate (Mallinckrodt Specialty Chemicals Co., Paris, KT), then applied on thin-layer chromatography plate (TLC, Sigma) and developed in a 95% chloroform, 5% methanol solvent. The CAT activities were quantitated by ImageQuant software.
In Situ Hybridization-In situ hybridization was performed as described with modifications (19). Briefly, the gestation 16.5 day C57BL/6 mouse whole mount sections were hybridized at 50°C for 17 h with 10 6 cpm of TR2 orphan receptor antisense cRNA probes (specific activity: 10 9 cpm/g) per slide in 50% formamide. After high stringency (50% formamide) washing at 65°C, the slides were dipped into Kodak NTB2 emulsion and were exposed for 6 days and developed in Kodak D19 developer, fixed, and counter-stained with hematoxylin.

TR2 Orphan Receptor Binds to Consensus DRs with Different
Affinities-We have tested the preference of the TR2 orphan receptor for binding to consensus AGGTCA tandem repeats with 1-to 6-base pair spacing (DR1 to DR6), sequences shown in Table I, by means of cold competitive EMSA analyses. Specific protein-DNA band shift was identified by comparing EMSA profiles obtained from experiments done with expression vector conditioned reticulocyte lysate and those done with nonconditioned lysate (mock). After EMSA specific band shifts (shown in Fig. 1) were quantified, the 50% competition concentrations (I 50 ) and the RBAs were calculated, as described under "Materials and Methods" and listed in Table I. Different RBAs between various DRs demonstrate clearly that the TR2 orphan receptor can interact with perfect AGGTCA DR type HREs with a preference in the order of DR1 (RBA ϭ 1.00 TR2 Orphan Receptor Binds to Natural HREs in Great Varieties-To search for the potential natural TR2 HREs, several natural HREs (sequences shown in Table I) were synthesized and tested for binding to the TR2 orphan receptor. An RXR response element, CRBPIIp sequence is a DR1 type element (20). A DR2 type HRE was identified in SV40 late promoter in the ϩ55 region which is protected from DNase I digestion by initiator-binding protein (21). RARE␤, a DR5 type HRE, has been demonstrated to respond to the activation of RAR␣/RXR␣ heterodimer upon the addition of retinoic acid (RA).
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 I. These RBA data reveal that the

TR2 Orphan Receptor in Retinoid Responsive System
affinities of TR2 orphan receptor to natural HREs are lower than those to perfect consensus HREs (e.g. comparing RBA of DR1 (4.19 Ϯ 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 Ͼ DR2 Ͼ DR5: TR2 orphan receptor has the highest affinity to DR1 type HRE, i.e. CRB-PIIp (1.00), which is followed by DR2, SV40 ϩ55 (RBA ϭ 0.104 Ϯ 0.003), and then by DR5, RARE␤ (RBA ϭ 0.0101 Ϯ 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 Ϯ 0.00005) may suggest that the TR2 orphan receptor prefers interacting with direct repeat HREs versus palindrome HREs. In addition, a synthetic SV40 ϩ55 mutant, as illustrated in Table I, 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 32 P-labeled probe to bind TR2 orphan receptor.
TR2 Orphan Receptor Shows No Sign of Forming Heterodimers with RAR␣ or RXR␣-RARE␤ and CRBPIIp, two potential natural TR2 orphan receptor HREs identified by EMSA analyses, are cis-acting regulatory elements for RAR␤ and CRBPII genes, respectively. To investigate whether TR2 orphan receptor may be involved in the RA signaling mechanism via interaction with these two HREs, we have applied the EMSA analyses to study the possible homodimer/heterodimer formation among RAR␣, RXR␣, and TR2 orphan receptor in the presence of RARE␤ HRE. The results, shown in Fig. 3A, demonstrate a clear band shift when TR2 orphan receptor is incubated with 32 P-labeled RARE␤, and this band intensity can be competed with unlabeled RARE␤ completely. We also found a predicted RAR␣/RXR␣ heterodimer, not RAR␣ or RXR␣ homodimers, shifted RARE␤ to a position different from that of the TR2 orphan receptor band shift. Taking advantage of the significant distance between the band shifts, we also tested whether TR2 orphan receptor can interfere in the formation of RAR␣/RXR␣ heterodimer by forming a heterodimer with either RAR␣ or RXR␣ in the presence of RARE␤. Based on the assumption that if there is any significant interaction between TR2 orphan receptor and RAR␣ or RXR␣ upon binding to HREs, one may expect at least one third band shift consisting of either TR2/RAR␣ or TR2/RXR␣ heterodimer when these proteins are made available at the same EMSA reaction. To better demonstrate any possible third band shift, we performed competitive EMSA with limited RARE␤ (50 pg) and fixed amounts of RAR␣ (1 l) and RXR␣ (0.5 l) while varying the amounts of TR2 orphan receptor (from 1.5-6 l). However, the results shown in Fig. 3B failed to demonstrate any significant third band shift. In addition, along with the increased amounts of TR2 orphan receptor applied, the specific RAR␣/RXR␣ heterodimer band shift intensities decreased in a dose-response manner (Fig. 3B, lanes 2-5). Also, after quantitation, we noticed that with increased amounts of RAR␣/RXR␣ protein   Table I, were allowed to compete for binding to TR2 orphan receptor. The specific band shifts were quantitated with PhosphorImager; the I 50 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 I. Data are shown as a representative autoradiogram of two independent experiments. caused a diminishment of TR2 orphan receptor band shift intensity (compare lanes 1 and 5 in Fig. 3B). These results suggest strongly that there is a competition between TR2 orphan receptor and RAR␣/RXR␣ heterodimer upon binding to RARE␤ motif; however, TR2 orphan receptor may not directly interact with either RAR␣ or RXR␣ by forming heterodimers.
TR2 Orphan Receptor and RAR␣/RXR␣ Heterodimer Bind to RARE␤ with Similar Affinities-To compare binding affinities between TR2 orphan receptor and RAR␣/RXR␣ to RARE␤ HRE, saturation Scatchard analyses were performed as described under "Materials and Methods. " Fig. 4, A and B, show typical patterns of protein-DNA complex formed between the increasing amounts of RARE␤ oligomers (from 0.0625 to 8 ng in a total 20-l reaction volume) and fixed amounts of TR2 orphan receptor and RAR␣/RXR␣, respectively. Specific complex (bound) intensities were quantitated and plotted against unbound (free) probes as saturation curves, illustrated in Fig. 4C. The consequent Scatchard analysis was performed and calculated dissociation constants (K d ) for binding of TR2 orphan receptor and RAR␣/RXR␣ heterodimer to RARE␤ are 5.03 and 2.32 nM, respectively (Fig. 4C, inset). The comparable K d values between these two groups of receptors suggest that TR2 orphan receptor may also express some transcriptional regulatory effects via binding with the RARE␤ cis-acting element.
TR2 Orphan Receptor Down-regulates the Expression of a CAT Reporter Construct Carrying One Copy of RARE␤-To test this possibility, we have constructed an RARE␤-pCATp reporter plasmid. The effect of endogenous RAR␣/RXR␣ on RARE␤-pCATp expression was analyzed with CV1 cells which were transiently transfected with RARE␤-pCATp construct only and exposed to various concentrations of all-trans-retinoic acid (tRA), which may function as an RAR␣ ligand and also as an RXR␣ activator (20,(25)(26)(27). As shown in Fig. 5, with increasing concentrations of tRA, CAT activities increased up to 6.7-fold in a dose-dependent fashion (Fig. 5, lanes 1 -3). In the absence of RA, the co-transfected pSG5-TR2 expression vector caused a 10-fold decrease in CAT activity (compare Fig. 5, lane  1 versus 4). Even in the presence of tRA, significant decreases in CAT activities resulted from co-transfected pSG5-TR2 are  Fig. 1 were 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 50 ), more excess amounts of HREs have been applied as marked. For instance, five concentrations of SV40 ϩ55 (0.125, 0.5, 2, 8, and 32 ng), marked as 20ϫ, used here are 20 times more than the corresponding five amounts (marked as 1ϫ) 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 (Ⅺ), CRBPIIp(q), SV40 ϩ55 (Ç), RARE␤ (É), TREpal (ϫ), and NBRE (f)) applied here are summarized and shown in Table I. FIG. 3. A, TR2 orphan receptor, RAR␣ and RXR␣ bind to RARE␤ with specificity. EMSA analysis was performed with 0.1 ng of 32 P end-labeled RARE␤ oligomer and applicable in vitro translated TR2 orphan receptor (1 l), RAR␣ (1 l), or RXR␣ (0.5 l) as indicated (E) and with an additional 200-fold excess amount of nonradiolabeled RARE␤ oligomer (q). 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 32 P end-labeled RARE␤ oligomer, various amounts of TR2 orphan receptor (1.5-6 l) and constant amounts of both RAR␣ (1 l) and RXR␣ (0.5 l) as marked on top of the autoradiogram (lanes 2-5). As controls for band shift intensity TR2 orphan receptor (6 l) and RAR␣ (1 l)/RXR␣ (0.5 l) 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 (Ⅺ), 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. still observed (compare Fig. 5, lanes 2 versus 5 and 3 versus 6). These results suggest that TR2 orphan receptor may suppress the stimulatory effects of endogenous RAR␣/RXR␣ on the expression of transfected RARE␤-CAT constructs. The exogenous TR2 orphan receptor can effectively compete for RARE␤-CAT activation even in the presence of tRA at a concentration of 10 Ϫ6 M.
TR2 Orphan Receptor Has a Higher Affinity to Bind to CRB-PIIp, an RXR HRE, Than Does RXR␣-Since our data showed that TR2 orphan receptor has the highest affinity to CRBPIIp HRE among tested natural HREs, we are also interested in testing whether CRBPIIp functions as a TR2 orphan receptor natural HRE. We have started analyzing binding affinities. Scatchard analyses demonstrate that at least a 100-fold difference in affinity exists between TR2 orphan receptor (K d ϭ 0.028 nM) and RXR␣ (K d ϭ 4.36 nM) in binding to CRBPIIp in vitro (Fig. 6B). The addition of RXR␣ ligand, 10 Ϫ8 M 9-cisretinoic acid, has failed to enhance affinity between RXR␣ and CRBPIIp (Fig. 6B, inset).
TR2 Orphan Receptor Competes with RXR␣ in Activating the Same CRBPIIp cis-acting Element-To determine whether the interaction between TR2 orphan receptor and CRBPIIp may result in possible transacting activities in vivo, we have constructed a CRBPIIp-pCATp reporter plasmid. In the presence of 10 Ϫ6 M tRA, an RXR␣ activator, our results showed no significant increase in CAT activities obtained from CV1 cells transfected only with CRBPIIp-pCATp (data not shown). However, tRA could induce CAT activities up to 11.5 fold when CV1 cells were co-transfected with CRBPIIp-pCATp and pCMX-hRXR␣ expression vectors (Fig. 7, lanes 1 and 3-5). In addition, the increase of CAT activity induced by exogenous RXR␣ can be suppressed (from 11.5-to 3.5-fold increases of TR2 orphan receptor basal induction activity) by co-transfected pSG5-TR2 expression vector even in the presence of 10 Ϫ6 M tRA (Fig. 7,  lane 5 versus 8). These results suggest that the high binding affinity of exogenous TR2 orphan receptor to CRBPIIp may dominate the effect of RXR␣ on CRBPIIp-CAT transcription activities in CV1 cells. To demonstrate further that the competitive effect of TR2 orphan receptor on CRBPIIp transactivation is not due to any titration of co-activator, we have constructed a mutated TR2 orphan receptor, pSG5-TR2-AR p -TR2. This mutated TR2 expression vector has been generated by swapping TR2 orphan receptor P-box with that found in human androgen receptor gene. As a result, this mutated TR2 orphan receptor may reserve all the properties of wild type TR2 orphan receptor except the binding affinity to CRBPIIp HRE. The K d changes from 0.028 nM to 1.44 M (data not shown) support this argument. Furthermore, the CAT reporter gene assay, shown in Fig. 7, lanes 9 -11, demonstrates that this mutated TR2 orphan receptor cannot effectively repress CAT activities induced by RXR␣ in the presence of tRA. Together, these results show that the inhibitory effect of the TR2 orphan receptor on CRBPIIp requires direct receptor HRE interaction.
TR2 Orphan Receptor Expresses during Late Fetal Development Stage-Using in situ hybridization techniques, investigators were able to localize RAR␣/RXR␣ in many tissues with higher expression in kidney and intestine (28 -30). We, therefore, are also interested to see whether TR2 orphan receptor expresses in these two organs. As shown in Fig. 8, our results clearly demonstrated that TR2 orphan receptor expresses in many tissues, including kidney and intestine, when the mouse  (lanes 3 and 6). As a negative control pCATp was transfected and CAT activity shown as lane 7. Chloramphenicol conversion rates were calculated from Phos-phorImager 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. embryo is at the age of gestation 16.5 day. This co-expression of TR2 orphan receptor and RAR␣/RXR␣ in kidney and intestine provides a strong argument supporting the possible role of TR2 orphan receptor in RAR␣/RXR␣ signal pathway. DISCUSSION 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. 2 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 Ͼ DR2 Ͼ DR5 DR4 DR6 Ͼ DR3. Three natural HREs that bind to the TR2 orphan receptor effectively were also identified: CRBPIIp (an RXRE), SV40 ϩ55 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 Ͼ 2 Ͼ 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,  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. 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 halfsite sequences like AGtTCA (i.e. CRBPIIp and RARE␤), AG-GTtc (i.e. SV40 ϩ55), 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 ϩ55 abolishes the competitive ability completely (compare RBAs of SV40 ϩ55 and SV40 ϩ55 m in Table  I). 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 crosstalk with various signal transduction pathways. RXRs are effective co-activators for vitamin D 3 , 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 -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 con-FIG. 8. Tissue-specific distribution of TR2 orphan receptor in mouse fetus. A gestation 16.5 day mouse fetus sagital section was hybridized with 32 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. structs 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.