Recent advances in the TR2 and TR4 orphan receptors of the nuclear receptor superfamily

The human testicular receptor 2 (TR2) and TR4 orphan receptors are two evolutionarily related proteins belonging to the nuclear receptor superfamily. Numerous TR2 and TR4 variants and homologs have been identified from different species, including vertebrates (e.g. human, murine, rabbit, fish, and amphibian) and invertebrates (e.g. Drosophila, sea urchin, and nematode) since TR2 was initially isolated over a decade ago. Specific tissue distribution, genomic organization, and chromosomal assignment of both orphan receptors have been investigated. In order to reveal the physiological functions played by both TR2 and TR4, upstream modulators of TR2 and TR4 gene expression, their downstream target gene regulation, feedback mechanisms, and differential modulation mediated by the recruitment of other nuclear receptors and coregulators have been investigated. Studies summarized in the present report have provided unexpected insights into the TR2 and TR4 functions in a variety of biological processes. The essential and difficult tasks of identifying orphan receptor ligands, agonist/antagonist assignment, their physiological functions, and mechanisms of action will continue to challenge nuclear receptor researchers in the future.     

Ligand-independent orphan receptor TR2 activation by phosphorylation at the DNA-binding domain

In a previous report we demonstrated protein kinase C (PKC)-mediated phosphorylation of the ligand-binding domain (LBD) of orphan nuclear receptor TR2. In this report, we provide the evidence of PKC-mediated phosphorylation of the DNA-binding domain (DBD) of TR2. Two PKC target sites were predicted within the DBD, at Ser-170 and Ser-185, but only Ser-185 was confirmed by MS. Phosphorylation of DBD facilitated DNA binding of the TR2 receptor and its recruiting of coactivator p300/CBP-associated factor (P/CAF). Ser-185 was required for DNA binding, whereas both Ser-170 and Ser-185 were necessary for receptor interaction with P/CAF. The P/CAF-interacting domain of TR2 was located in its DBD. A double mutant (Ser-170 and Ser-185) of TR2 significantly lowered the activation of its target gene RARbeta2. This study provides the first evidence for ligand-independent activation of TR2 orphan receptor through PTM at the DBD, which enhanced its DNA-binding ability and interaction with coactivator P/CAF.     

Feedback regulation between orphan nuclear receptor TR2 and human papilloma virus type 16

The human TR2 orphan receptor (TR2), initially isolated from testis and prostate cDNA libraries, is a member of the steroid receptor superfamily. TR2 can regulate several target genes via binding to a consensus response element (AGGTCA) in direct repeat orientation (AGGTCAX((n))AGGTCA, n = 0-6). Here we show that TR2 is able to induce the expression of human papilloma virus type 16 (HPV-16) genes via binding to a DR4 response element in the long control region of HPV-16. Additionally, one of the HPV-16 gene products, the E6 oncogene, regulates TR2 gene expression. A likely mechanism for this regulation involves E6-mediated degradation of the tumor suppressor p53, a protein known to suppress TR2 expression. Together our data provide evidence for feedback regulation between TR2 and HPV-16, which represents a novel regulatory pathway involving a member of the steroid receptor superfamily and the HPV-16 DNA tumor virus.     

Identification of a novel human constitutive androstane receptor (CAR) agonist and its use in the identification of CAR target genes

The orphan nuclear constitutive androstane receptor (CAR) is proposed to play a central role in the response to xenochemical stress. Identification of CAR target genes in humans has been limited by the lack of a selective CAR agonist. We report the identification of 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) as a novel human CAR agonist with the following characteristics: (a) potent activity in an in vitro fluorescence-based CAR activation assay; (b) selectivity for CAR over other nuclear receptors, including the xenobiotic pregnane X receptor (PXR); (c) the ability to induce human CAR nuclear translocation; and (d) the ability to induce the prototypical CAR target gene CYP2B6 in primary human hepatocytes. Using primary cultures of human hepatocytes, the effects of CITCO on gene expression were compared with those of the PXR ligand rifampicin. The relative expression of a number of genes encoding proteins involved in various aspects of steroid and xenobiotic metabolism was analyzed. Notably, CAR and PXR activators differentially regulated the expression of several genes, demonstrating that these two nuclear receptors subserve overlapping but distinct biological functions in human hepatocytes.     

Molecular cloning of the human histamine H2 receptor.

We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor.     

Signaling Pathway of Histamine H1 Receptor-Mediated Histamine H1 Receptor Gene Upregulation Induced by Histamine in U-373 MG Cells

Histamine H₁ receptor (H1R) is one of the targets of histamine in the nervous system and the peripheral tissues. Protein kinase Cδ (PKCδ) signaling is involved in histamine-induced upregulation of H1R gene expression in HeLa cells. Histamine also upregulates H1R gene expression in U-373 MG cells. However, the molecular signaling of this upregulation is still unclear. Here, we investigated the molecular mechanism of histamine-induced H1R gene upregulation in U-373 MG cells. Histamine-induced H1R gene upregulation was inhibited by H1R antagonist d-chlorpheniramine, but not by ranitidine, ciproxifan, or JNJ77777120, and H2R, H3R, or H4R antagonists, respectively. Ro-31-8220 and Go6976 also suppressed this upregulation, however, the PKCδ selective inhibitor rottlerin and the PKCβ selective inhibitor {"type":"entrez-nucleotide","attrs":{"text":"Ly333531","term_id":"1257370768"}}Ly333531 did not. Time-course studies showed distinct kinetics of H1R gene upregulation in U-373 MG cells from that in HeLa cells. A promoter assay revealed that the promoter region responsible for H1R gene upregulation in U-373 MG cells was different from that of HeLa cells. These data suggest that the H1R-activated H1R gene expression signaling pathway in U-373 MG cells is different from that in HeLa cells, possibly by using different promoters. The involvement of PKCα also suggests that compounds that target PKCδ could work as peripheral type H1R-selective inhibitors without a sedative effect.