From a database containing the published nuclear hormone receptor (NR) sequences an alignment of the C, D and E domains of these molecules was constructed. Using this alignment, tree reconstruction was performed using both distance matrix and parsimony analysis. The robustness of each branch was estimated using bootstrap resampling methods. The trees constructed by these two methods gave congruent topologies. From these analyses six NR subfamilies (I - VI) are defined, containing the following:

(I) a large subfamily with clustering thyroid hormone receptors (TRs), retinoic acid receptors (RARs), peroxisome proliferator-activated receptors (PPARs), vitamin D receptors (VDRs) and ecdysone receptors (EcRs) as well as numerous orphan receptors such as RORs or Rev-erbs;
(II) retinoid X receptors (RXRs), together with COUP, HNF4, tailless, TR2 and TR4 orphan receptors;
(III) steroid receptors;
(IV) the NGFIB orphan receptors;
(V) FTZ-F1 orphan receptors, and
(VI), the subfamily that to date has only one gene, the GCNF1 orphan receptor.

TR2, a presumptive homolog of Hormone-receptor-like in 78

A new group of cDNA clones has been isolated from human testis cDNA libraries that might code for new steroid receptors. The cDNA and predicted amino acid sequences of two of these receptors, named TR2-5 and TR2-7, were determined. The nucleotide and deduced amino acid structures of two other receptors are reported that have been named TR2-9 and TR2-11 receptors. The calculated MW of TR2-5 receptor, TR2-7 receptor, TR2-9 receptor and TR2-11 receptor are 52,982, 20,528, 50,849 and 67,223 respectively, which match well with the apparent MW of in vitro translated products. The 26 amino acids involved in the formation ofZn-fingers are conserved. The ligand-binding domain of TR2-9 receptor is 16 amino acids shorter and has 3 different amino acids, as compared with TR2-5 receptor. The TR2-11 receptor has a ligand-binding domain that is longer and quite different when compared with the other TR2 receptors. The multiple ligand-binding domains of TR2 receptor could be the products of different genes or may be due to RNA splicing errors (Chang, 1989).

A cDNA encoding a novel orphan nuclear receptor, aDOR1, from neurulating Ambystoma mexicanum embryos (a salamander) has been isolated. It is closely related to testicular receptor-2 (TR2) orphan receptor family members. The cDNA sequence predicts a protein primary sequence of 416 amino acids with a calculated molecular weight of 45.8 kDa. While the DNA-binding domains of aDOR1 and hTR2-11 share 96% identity, considerable divergence is observed at both extremities of the peptides. At the N-terminus, aDOR1 is 66% identical to hTR2-11 and longer by 37 amino acids. At the C-terminus, despite a greater similarity (69%), aDOR1 is much shorter than the hTR2 isoforms and seems to encode a distinct ligand-binding domain. Expression of aDOR1 was studied by the reverse transcription polymerase chain reaction assay (RT-PCR). High mRNA levels are detected during oogenesis; they remain high during the cleavage stage, and decrease at the mid-blastula transition (MBT). Transcripts increase again at the end of gastrulation, reach a peak level during neurulation, and leveled off after closure of the neural tube. In neurulas dissected along the anteroposterior axis, aDOR1 mRNA is enriched at both extremities of the embryo, while no particular distribution is favored along the dorsoventral axis. Retinoic acid (RA) treatments at the beginning of gastrulation do not affect overall mRNA levels in the neurula nor its distribution along both axes. In the adult, expression is predominant in the brain; lower levels (about 15%) are detectable in all germ layer derivatives, except muscle. These results suggest that aDOR1 may be required for the early determination events occurring during the cleavage stages of development, and may be involved in embryogenesis and in brain function (Wirtanen, 1997).

DNA binding characteristics of TR2

The mouse orphan nuclear receptor TR2-11-f suppresses the expression of reporters fused to a hormone response elementof the mouse cellular retinoic acid-binding protein I gene promoter. TR2-11-f is able to bind as a homodimer to a direct repeat with four nucleotides in the spacer (5'TGACCTTTGGGGACCT3') located within this hormone response element. The specificity of protein-DNA interactions was demonstrated by competition in gel retardation and antibody-mediated supershift reactions. The residues critical for TR2-11-f binding were mapped to both repeated sequences, whereas the spacer and the flanking sequences are less important. The Kd and Bmax of TR2-11-f homodimer binding to this direct repeat were determined to be 2.6 nM and 0.012 nM, respectively. By using a yeast two-hybrid system, it was demonstrated that dimerization of TR2-11-f is mediated by its ligand-binding domain. The actions of TR2-11-f in regulating the cellular retinoic acid-binding protein I gene will likely influence retinoic action and availability within the cells (Chinpaisal, 1997).

The mouse orphan nuclear receptor TR2-11 functions as a repressor for reporter genes containing a direct repeat-5 or direct repeat-4 hormone response element. The functional domains responsible for TR2-11's suppressive activity are defined, including the DNA-binding domain and the ligand-binding domain. The C-terminal 30 amino acid residues can be deleted without compromising its suppressive activity, whereas a deletion for 40 amino acids completely abolishes the suppressive activity and receptor dimerization, and reduces the DNA-binding affinity. Point mutation at three conserved leucine residues located on the predicted dimer interface abolishes the suppressive activity, receptor dimerization and TR2-11's DNA binding property. However, mutation at two consecutive glutamate residues located within the hinge between the last two helices of the ligand-binding domain (helix 10 and helix 11 according to the human retinoid receptor Xalpha structure) drastically reduces its DNA-binding affinity and abrogates the suppressive activity without compromising its ability to dimerize, indicating that receptor dimerization property can be functionally uncoupled from its suppressive activity. A transferable, active silencing activity is encoded within the DEF segment of the receptor molecule, as evidenced by the suppression of a GAL4 reporter by a chimeric protein containing the DNA-binding domain of GAL4 and the DEF segment of TR2-11. Moreover, the C-terminal 49 amino acid sequence is required for this trans-suppressive activity. It is suggested that TR2-11 functions as a repressor, mediated by mechanisms requiring high affinity DNA binding, receptor dimerization, and active silencing (Chinpaisal, 1998).

Transcriptional targets of TR2-11

A systematic scanning of nucleic acid databases for DNA elements made of combinations of RGGTCA nuclear receptor half sites, has revealed that identical 19 nucleotide-long motifs composed of two inverted RGGTCA sites with a spacing of 7 nucleotides (IR7), are present upstream of the regions coding for the human TR2 and of the sea urchin SpSHR2 orphan receptors. An experimental strategy, based on PCR, has been developed to check if this IR7 could correspond to an unusually long, evolutionarily conserved, cis-element regulating the TR2 genes. Indeed IR7 is present in the 5' untranslated region of TR2 genes from all species tested, including Xenopus, rainbow trout, zebrafish and mouse. The exact conservation throughout the animal kingdom of such a long, non repetitive and non coding genomic region, strongly suggests that it should ensure important biological functions. In addition, this work has allowed the identification of a new, non coding, upstream exon in the mouse TR2 gene present in testicular TR2 mRNAs (Le Jossic, 1998).

Synthesis of the pre-C and pregenomic RNAs of human hepatitis B virus (HBV) is directed by two overlapping yet separate promoters. A binding site for the nuclear receptor hepatocyte nuclear factor 4 (HNF4) spans the TATA box-like sequence of the pre-C promoter. This HNF4-binding site consists of an imperfect direct repeat of the consensus half-site sequence 5'-AGGTCA-3' separated by one nucleotide; i.e., it is a DR1 hormone response element (HRE). Other receptors, including chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), human testicular receptor 2 (TR2), and peroxisome proliferator-activated receptors (PPARs) as heterodimers with retinoid X receptors (RXRs), can also specifically bind this DR1 HRE. Synthesis of the pre-C and pregenomic RNAs is affected both in transfected hepatoma cells and in a cell-free transcription system by the binding of factors to this DR1 HRE. Interestingly, whereas some members of the hormone receptor superfamily differentially repressed synthesis of the pre-C RNA (e.g., HNF4 and TR2) or activated synthesis of the pregenomic RNA (e.g., PPARgamma-RXRalpha), other members (e.g., COUP-TF1) coordinately repressed synthesis of both the pre-C and pregenomic RNAs. Thus, HBV likely regulates its expression and replication in part via this DR1 HRE. These findings indicate that appropriate ligands to nuclear receptors may be useful in the treatment of HBV infection (Yu, 1997).

When the orphan nuclear receptors TR2 and TR4, the DNA-binding subunits of the DRED repressor complex, are forcibly expressed in erythroid cells of transgenic mice, embryos exhibit a transient mid-gestational anemia as a consequence of a reduction in the number of primitive erythroid cells. GATA-1 mRNA is specifically diminished in the erythroid cells of these TR2/TR4 transgenic embryos as it is in human CD34⁺ progenitor cells transfected with forcibly expressed TR2/TR4. In contrast, in loss-of-function studies analyzing either Tr2- or Tr4-germline-null mutant mice or human CD34⁺ progenitor cells transfected with force-expressed TR2 and TR4 short hairpin RNAs (shRNAs), GATA-1 mRNA is induced. An evolutionarily conserved direct repeat (DR) element, a canonical binding site for nuclear receptors, was identified in the GATA1 hematopoietic enhancer (G1HE), and TR2/TR4 binds to that site in vitro and in vivo. Mutation of that DR element led to elevated Gata1 promoter activity, and reduced promoter responsiveness to cotransfected TR2/TR4. Thus, TR2/TR4 directly represses Gata1/GATA1 transcription in murine and human erythroid progenitor cells through an evolutionarily conserved binding site within a well-characterized, tissue-specific Gata1 enhancer, thereby providing a mechanism by which Gata1 can be directly silenced during terminal erythroid maturation (Tanabe, 2007).

Biological effects of TR2-11

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, these 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 (Lin, 1995).

An alternatively spliced variant of a testis-specific nuclear orphan receptor TR2-11 was identified and designated as TR2-11-t. As a result of retaining intron 5 of this gene, TR2-11-t mRNA encodes a truncated receptor with the complete ligand-binding domain deleted. Protein expression of both isoforms was confirmed using a prokaryotic expression system. In the mouse, the expression of the two TR2 isoforms is elevated in the testis with distinct profiles beginning at puberty. TR2-11 expression increases at postnatal day 18, peaks between day 20 and day 24 and remains at high levels throughout adulthood, whereas TR2-11-t expression is elevated transiently at postnatal day 24. Among separated primary germ cells and established testicular cell lines, TR2-11 is expressed highly in meiotic and postmeiotic germ cells and weakly in a Leydig cell line and a germ cell line, but not expressed in a Sertoli cell line. In contrast, TR2-11-t is expressed at a much lower level in all the testicular cell types examined. In adult testes blocked at germ cell development by vitamin A depletion or hypophysectomy, TR2-11 expression is dramatically reduced, whereas TR2-11-t is highly elevated. Based on the RNA expression patterns of these isoforms, it is suggested that TR2-11 is specific to meiotic and postmeiotic germ cells, whereas TR2-11-t is enriched in early germ cell populations, such as premeiotic cells. The biological activities of TR2-11 and TR2-11-t on a direct repeat 5-type retinoic acid (RA) response element (RARE)-containing reporter gene was examined in Cos cells. TR2-11 represses RA induction of this reporter whereas TR2-11-t enhances RA induction of the same reporter; the opposite biological effects of these isoforms are dose-dependent. Gel-shift experiments provide evidence for a direct interaction of TR2-11, but not TR2-11-t, with DNA fragments containing this RARE. Opposite roles of TR2-11 and TR2-11-t on RA induction of promoters containing this particular RARE are suggested (Lee, 1997).