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

https://doi.org/10.1016/S0960-0760(02)00118-8Get rights and content

Abstract

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.

Introduction

The functions of steroid and thyroid hormones are mediated through the action of specific receptor proteins [1], [2]. Nuclear receptors (NRs) comprise a huge family of ligand-dependent transcription factors that regulate complex gene networks in a wide variety of biological processes, such as growth, development, and differentiation [2]. Members of this superfamily include receptors for steroid hormones, thyroid hormones, Vitamin A and D derivatives, as well as a large group of orphan receptors whose cognate ligands remain to be identified. The functional structure and organization of the NR, as shown in Fig. 1, generally contains six (A–F) domains with a well conserved DNA-binding domain in the central core (C domain) of the protein, which is responsible for DNA recognition and dimerization [1], [2], [3], [4], [5]. Distal to the DNA-binding domain is a variable hinge region (D domain) containing a nuclear localization signal. It is assumed that this region may allow the receptor to bend or alter its conformation [5]. The ligand-binding domain, located in the C-terminal region, is relatively large and functionally complex. It is believed that this domain contains regions important for ligand-binding, dimerization, transactivation, and intramolecular repression [3], [4], [5]. The N-terminal domain is highly variable in amino acid sequence and in length. This domain contains a transactivation function which regulates gene expression by interacting with the core transcriptional machinery, coactivators, or other transactivators [3], [4], [5].

Hormones are synthesized and secreted by endocrine cells [6], [7]. These hormones travel via the blood stream to their target cells, enter these cells by simple or facilitated diffusion, and then bind to specific receptors in the cytoplasm or nucleus (Fig. 2). Upon binding with their respective ligands, the NRs undergo an activation or transformation step. The ligand–receptor complex, serving as a trans-regulator, may specifically bind to a cis-acting DNA sequence, known as a hormone response element (HRE) and thereafter regulate transcription of the target gene. The target gene will be transcribed, translated into protein product, and ultimately alter cellular function [8].

Orphan receptors comprise a novel group of NR-like proteins [2], [3], [9], [10]. Numerous orphan receptors have been isolated from low-stringency hybridization screening, genetic and molecular cloning techniques [2], [9], [11]. Due to distinctive homology in their amino acid sequences, orphan receptors have been classified as members of the NR superfamily. Consequently, they have no known ligand and usually no known function when they are initially identified. Thus far, we estimate approximately 170 orphan receptors have been identified from different species and various tissues [3], [12]. Nevertheless, it is assumed that most of these orphan receptors have important cellular functions based on the following criteria: (1) they are expressed as proteins in cells, sometimes with cell type and developmental specificity; (2) they are members of a highly evolved family of transcriptional factors; (3) several orphan receptors have been shown to function by regulating known specific target genes and developmental processes; (4) certain orphan receptors have been implicated in the mediation of cellular responses to neurotransmitters, retinoic acids (RAs), peroxisome proliferators, or phosphorylation pathways; and (5) some orphan receptors may function as coregulators to modulate signaling pathways.

Differential recognition of target genes by the NR members, which regulate the transcription of complex gene networks, is determined by at least three properties: protein–DNA interactions, protein–protein interactions, and protein environment. Firstly, protein–DNA interactions are mediated by the highly conserved DNA-binding domain that defines the NR superfamily [13]. The molecular specificity of NRs is achieved by their selective interaction with DNA-binding sites referred to as HREs [14]. The HREs are structurally related but functionally distinct. Based on the finger model, the first zinc finger in the DNA-binding domain of NRs may determine target HRE specificity. As shown in Fig. 3, three amino acids in the C-terminal region of the first zinc finger are marked as the proximal (P) box, which is important in base interaction [14], [15], [16], [17]. Consequently, HREs can be classified into two categories of repeat consensus sequences based on the P box, the glucocorticoid receptor (GR) and estrogen receptor (ER) response element subfamilies [16]. The GR subfamily, which includes GR, androgen receptor (AR), progesterone receptor (PR) and the mineralocorticoid receptor (MR) recognizes two AGAACA core consensus half sites. The ER subfamily, which includes ER, testicular receptor (TR) the 1,25-dihydroxyvitamin D3 receptor (VDR) retinoic acid receptor (RAR), retinoic×receptor (R×R), and many orphan receptors, recognizes two AGGTCA consensus half sites. Based on the P box, TR2 has been grouped with members of the ER subfamily [14]. Therefore, we suspect that TR2 may recognize AGGTCA core consensus motifs. In addition, five amino acids localized to the second zinc finger, referred to as the distal (D) box, are important in dimerization contact formation [14]. Moreover, two elements in the C-terminal region of the second zinc finger of the NGFI-B orphan receptor, referred to as the A and T boxes, are also critical for DNA recognition [18], [19], [20], highlighting the importance of non-zinc-finger regions in the specificity of DNA binding. On the other hand, several features of a HRE determine the specificity of DNA recognition. These include the precise sequence, the orientation of the core recognition motifs, and the spacing between core motifs [13], [14], [21], [22]. The intrinsic DNA-binding properties of NRs cannot be simply ascribed to sequence, orientation, or spacing rules, as exemplified by the NGFI-B response element (NBRE) in which sequences outside of the core recognition motifs also contribute to the specificity of DNA binding [19], [20].

Secondly, protein–protein interactions necessary for the formation of homo and/or heterodimers in solution are mediated by an extensive C-terminal dimerization interface in the ligand-binding domain, an inhibitor of transcription in the ligand-binding domain, and the D box in the DNA-binding domain [13]. The members of the NR superfamily are capable of binding to DNA in monomeric, homodimeric, and heterodimeric modes. A comprehensive analysis of the ligand-binding domain reveals several critical regions for protein–protein interactions. A highly conserved region, referred to as conserved region II or Ti (transcriptional inactivation) plays an important role in the dimerization of TR and other superfamily members [23]. A DNA-supported asymmetric dimerization interface is located within the DNA-binding domain of NRs and selectively promotes DNA binding to cognate direct repeat HREs [24], [25], [26]. In addition, the identity (I) box located in an extensive C-terminal dimerization interface in the ligand-binding domain is structurally similar to the leucine zipper dimerization domain [18], [27], [28], [29], [30]. The combination of this obligatory I box and an optional dimerization interface in the DNA-binding domain increases the diversity of heterodimeric interactions and high affinity DNA binding [29].

Thirdly, the protein environment also influences selectivity of NR recognition of their target genes [14]. For example, ligand specificity is one of the key parameters contributing to DNA-binding and dimerization. Studies of TR and R×Rs suggest that their respective ligands may affect dimerization and diversity of function [13], [31], [32], [33], [34]. Furthermore, nuclear accessory factors (coregulators) including coactivators, corepressors, and cointegrators, are thought to serve as bridging molecules or adaptors between NRs and the basal transcriptional machinery [8], [35]. Finally, the chromatin (nuclear matrix) structure may facilitate or restrict NR action [36], [37], [38]. The nuclear matrix is the structural component of the nucleus that determines nuclear morphology and organizes the DNA in a three-dimensional fashion. The nucleoprotein organization of a variety of hormone responsive regulatory elements and reconstruction of complex chromatin templates are important features in NR action. While the molecular mechanism of the NR regulation of gene expression is currently unclear, evidence suggests that the interplay of the specific three-dimensional organization of the genome, the structural components of the nucleus (nuclear matrix) and histone modification enzymes (histone deacetylases and acetyltransferases) may all contribute to the regulation of gene expression [36], [37], [38], [39].

Our long-term goals are to understand the physiological properties of TR2 and TR4, and their possible roles in cellular responses. In searching for TR2 and TR4 target genes, we may be able to identify new response systems with valuable physiological implications. By revealing the roles of TR2 and TR4 in different physiological pathways, we may ultimately determine their ligand specificity and biological functions.

Section snippets

The TR2 and TR4 orphan receptors and their homologs

The human TR2, is one of the first orphan receptors identified and it shares structural homology with members of the NR superfamily [40], [41]. As is illustrated in Fig. 4, different lengths of human TR2 cDNA variants, TR2-5, -7, -9, and -11 have been isolated [40], [41], [42], [43]. Northern blot analysis suggested that the TR2-11 transcript may represent the major forms among TR2 variants, [44], [45]. Consequently, we have used the TR2-11 in further investigation of TR2 function.

Tissue distribution

Using Northern and dot blot analyses, the tissue distribution of the human TR2 transcripts was analyzed [40], [41], [42]. The results showed that TR2 mRNA is most abundant (per unit of RNA) in the rat androgen-sensitive prostate, and is least abundant in the estrogen-sensitive uterus, with the following relative amounts in other tissues: ventral prostate as 100%; seminal vesicle, 92%; testes, 42%; submaxillary gland, 18%; uterus, <1%. Additionally, TR2 mRNAs were detected in total RNA isolated

Genomic organization and chromosomal assignment

The human TR2 gene was mapped to human chromosome 12 at band q22 using fluorescence in situ hybridization combined with a high-resolution G-banding technique [71]. The entire TR2 gene features 13 introns and 14 exons joined by the consensus splice sequences (GT-AG) at all intron–exon boundaries. It is noteworthy that genomic structures of all known NRs contain a splice site between the first and second zinc fingers in the DNA-binding domain. In contrast, the TR2 gene has a unique splice site

Androgen

The expression of human TR2 mRNA is negatively regulated by androgen in the rat ventral prostate and in the human prostate cancer LNCaP cell line [40], [42], [43], [44]. Dot blot hybridization showed that TR2 mRNA in the rat ventral prostate increases about two-fold above normal levels two days after castration. This increased mRNA expression could be reversed by the injection of androgen (5α-dihydrotestosterone, DHT). In addition, injection of the antiandrogen (flutamide) into intact rats

Simian virus 40 major late promoter

The first DNA response element (TR2RE–SV40) for human TR2 was identified in the SV40+ 55 region [66]. EMSA analysis showed specific binding with high affinity (dissociation constant=9 nM) between TR2 and this TR2RE–SV40 sequence. DNA-swap experiments using the CAT assay demonstrated that androgen can suppress the transcriptional activity of the SV40 early promoter via the interaction between this TR2RE–SV40 and the chimeric receptor AR/TR2/AR (the DNA-binding domain of TR2 flanked by the

Retinoids

As we described previously, RA can increase the expression of human TR4 at the mRNA and protein levels [81], [83]. On the other hand, TR2/TR4 can repress gene regulation mediated by the retinoid signaling pathway. We hypothesize that the retinoid signaling pathway can be regulated by TR2/TR4 through a negative feedback control mechanism, which may restrict RA signaling in a cell-specific fashion [81]. Thus, RA may have at least two ways to regulate its targets. One way is to bind with RAR/R×R

Functional mechanisms of target gene regulation

There are many studies investigating the role of NRs in the transcriptional activation of eukaryotic promoters [6], [7]. Considerably less is known about the molecular mechanisms of transcriptional repression by NRs [110], [111]. As illustrated in Fig. 6A, a direct repression mechanism has been proposed whereby an activated receptor, a repressor, binds to a HRE at some distance from the target promoter and interferes with the activity of the basal transcription machinery [111]. In the

Future perspectives

Gene regulation by NRs is initiated by binding of their cognate ligands. Ligand activation of orphan receptors remains controversial, since their ligands have not yet been identified. Several ligands or synthetic compounds reportedly activate orphan receptors [112]. The identification of ligands for orphan receptors has basically relied on in vitro biochemical methods, including proteolysis, photocrosslinking, chemical extraction, radiolabeled-ligand binding, affinity chromatography, and the

Acknowledgements

We are grateful to Erik Sampson and Loretta Lynn Collins for critical reading of this manuscript. We would like to apologize for any errors or omissions of important studies. We also greatly appreciate all members in two laboratories for their excellent work and numerous suggestions. This work was supported by National Science Council 89-2320-B-259-002, ROC (to HJL) National Institutes of Health Grants DK47258, and DK56984 (to CC).

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    Authors contributed equally in the production of this work and both should be considered first authors on this paper.

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