TH receptors (TRs) were first cloned in 1986 and belong to the nuclear hormone receptor superfamily that includes the glucocorticoid, estrogen, progesterone, androgen, aldosterone, vitamin D, retinoic acid (RARs), retinoid X (RXRs) and "orphan" (unknown ligand and/or DNA target) receptors (2, 14-16, 31, 32). TRs are the cellular homologs of v-erbA, a viral oncogene product involved in chick erythroblastosis. TRs are encoded at two genomic loci (α and β ) located on human chromosomes 17 and 3, respectively (2,14-16,33, 34) and their gene products result in two major isoforms, TRα and TRβ .
Like other members of the nuclear receptor superfamily (2, 16, 31, 32), the TRs have a central DNA-binding domain (DBD) and a carboxy-terminal ligand-binding domain (LBD) (Figure 3d-1). The two major TR isoforms have high homology in their respective DBDs and LBDs. Dimerization domains also are found in both DBDs and LBDs. The amino-terminal regions are more variable between TRα and TRβ , and contain ligand-independent activation domains. In contrast, multiple sub-regions are located in the LBD for ligand-dependent transcriptional activation and basal repression of target genes.
Figure 1. Functional domains of the TH receptor (TR). The TH receptor (TR) is depicted schematically. The zinc finger DNA-binding domain (DBD) is denoted along with the carboxy terminal ligand-binding domain (LBD). Other functional domains and interaction sites are indicated.
The DNA-binding domains of the nuclear receptors are comprised of two distinct zinc fingers that are separated by a 15-17 amino acid linker sequence. The crystal structure of the DNA-binding domains of the TH receptor and its heterodimeric partner, RXR has been determined. The two heterodimer partners interact with a direct repeat of the receptor binding site in a head-to-tail manner (27,36,37).
A small stretch of amino acids at the base of the first finger (referred to as the P-box) dictates the DNA sequence specificity of the receptor (35, 36, 38). The P-box sequence of the TH receptor is shared by other receptors that bind to similar or identical DNA recognition sites (AGGTCA). The underlined amino acids in the P-box (EGCKG) of the TH receptor are also found in the retinoic acid receptors, the retinoic acid X receptors, the rev-erbA protein, the vitamin D receptor, and NGFI-B. Of note, the steroid hormone receptors have a different P box sequence and bind as homodimers to a different consensus DNA half-site sequence (AGAACA). The region between the DBD and LBD is called the hinge region and contains the nuclear localization signal, typically a basic amino acid‑rich sequence, first described in viral nuclear proteins.
X‑ray crystallographic studies of the liganded rat TRα 1 show that TH is embedded in a hydrophobic "pocket" lined by discontinuous stretches of amino acid sequences within the LBD. Additionally, there are several hydrophobic interfaces within the LBD that contribute to the TR homo- and heterodimerization with RXR (39). There are twelve amphipathic helices in the LBD and specific helices among them provide the critical contact surfaces for protein-protein interactions with co-activators and co-repressors (helices 3,5,6,12 and 3,4,5,6, respectively) (40-43). Ligand-binding to TR causes a major conformational change in the LBD, particularly in helix 12. This, in turn, facilitates TR discrimination between co-activators and co-repressors (see below).
The carboxy-terminal hormone-binding domain of the TRα gene is alternatively-spliced to generate several protein products (Figure 3d-2, below). One variant, referred to as α 2, is identical to TRα 1 through the first 370 amino acids, but then its sequence diverges completely, owing to splicing of alternate exons (44-47). Another splicing variant, referred to as TRvII or α 3, is similar to α 2 except that it lacks the first 39 amino acids found in the unique region of α 2 (45). α 2 cannot bind TH because of the replacement of critical amino acids at the extreme carboxy-terminal end of the protein due to alternative splicing(48), and thus cannot mediate ligand-dependent gene transcription (49 – 51). The amino acid replacements in α 2 also alter its dimerization properties and reduce DNA-binding affinity (52-55). The α 2 splicing variant is highly expressed in many tissues such as brain, testis, kidney, and brown fat, but its function remains poorly understood (56). The α 2 isoform has been proposed to be an endogenous inhibitor of TH receptor function as it inhibits TRα and β activity in transient gene expression assays (44,54). The mechanism by which α 2 antagonizes TR action is controversial. Some studies indicate that α 2 competes for active receptor complexes at DNA target sites (57,58). Other studies indicate that α 2 inhibits TR activity independent of DNA-binding (59). It is likely that the inhibitory effects of α 2 involve more than one mechanism. Amino acid substitutions in the carboxy-terminal region of α 2 also prevent its interactions with transcriptional corepressors (see below) (55), and may provide an explanation as to why α 2 is not a more potent inhibitor of TR activity. Additionally, the phosphorylation state of α 2 may modulate its inhibitory activity (60). Given the foregoing features, the TRα 1 and α 2 system represents one of the few examples in mammals whereby multiple mRNAs generated by alternative splicing encode proteins are antagonistic to each other.
Figure 2. TH receptor isoforms. The TH receptors (TR) β and α are expressed from separate genes. Each TR gene can be expressed as distinct isoforms, reflecting the use of alternate promoters and exons. The central zinc finger DNA-binding region is indicated and unique domains are shown by distinct patterns of shading. The TRβ 2 isoform, which is expressed predominantly in the pituitary and hypothalamus, contains a unique amino-terminus. The TRα 2 isoform contains unique carboxy-terminal sequences that eliminate hormone binding. The DNA- and T3-binding properties and transcriptional activity of the various isoforms are shown at the right.
A receptor-like molecule, Rev-erbA, is, surprisingly, encoded on the opposite strand of the TRα gene locus (61, 62). Rev-erbA mRNA contains a 269-nucleotide stretch which is complementary to the α 2 mRNA due to its transcription from the DNA strand opposite of that used to generate TRα 1 and α 2. This protein also is a member of the nuclear hormone receptor superfamily, and is highly expressed in adipocytes and muscle cells. Rev-erbA, contains a DBD that is homologous to the TR DBD. However, Rev-erbA does not bind TH and its putative LBD has minimal homology with other nuclear hormone receptors. Since no cognate ligand has been identified for Rev-erbA, it is categorized as an "orphan nuclear receptor. " It can act as a transcriptional repressor for nuclear hormone receptors and other transcription factors (63-65). Since Rev-erbA shares an exonic segment of the bidirectionally transcribed TRα gene, it is possible that it modulates the expression or splicing of TRα 1 and α 2 (66, 67) as parallel increases in rev-erbA mRNA and TRα 1mRNA expression occur relative to α 2 mRNA expression.
The major variant of the TRβ gene, TRβ 2, has a different amino-terminus than TRβ 1 (Figure 3d-2, above) (68). The distinct amino-terminal region of the TRβ 2 is due to transcription from a tissue-specific promoter. The function of the amino-terminus of the TH receptor is not known, but it likely plays a role in transcriptional control (69,70). The β 1 and β 2 isoforms function similarly in most transient gene expression assays (69, 71), although differences in the transcriptional activities of the TRβ 1 and TRβ 2 isoforms have been noted with respect to certain target genes (69-72). It is likely that tissue-restricted expression of the TRβ 2 isoform contributes to unique patterns of TR expression, which in turn, may modulate target gene regulation.
Recently, short isoforms of TRα and TRβ have been described (73, 74). The novel TRα isoforms arise from translational start sites in the 7th intron and yield shortened TRα 1 and α 2 isoforms that have dominant negative activity on WT TR. Novel short TRβ isoforms arise from alternative splicing of TRβ . It is possible that these isoforms may modulate T3- responsiveness in a tissue- and/or development-stage-specific manner.
Most studies of TR isoform expression have employed mRNA analyses rather than protein measurement (75). In general, the α and β receptor isoforms are distributed widely and exhibit overlapping patterns of expression (56, 75, 76). TRα 1 mRNA is expressed in skeletal and cardiac muscle whereas TRβ 1 mRNA is predominant in liver, kidney, and brain. α 2 mRNA is most prevalent in brain and testis. In contrast, TRβ 2 mRNA has the most tissue-restricted expression, and is present in the anterior pituitary gland, hypothalamus, and cochlea (77-79).
The TRs also are expressed in specific stages during development, and are subject to regulation by hormones and other factors (80, 81). For instance, TRα 1 mRNA is expressed early whereas TRβ 1 mRNA is expressed later during embryonic brain development. In the rat pituitary gland, TH decreases TRβ 2, TRα 1, and α 2 mRNAs while slightly increasing TRβ 1 mRNA. However, in most other tissues, TH decreases TRα 1 and α 2, but not TRβ ‑1 mRNA (56). Isoform-specific knockout mice of each of the TR isoforms that display distinct phenotypes (17,18). However, lack of significant TR isoform-specific gene expression was recently observed in cDNA microarrays of hepatic genes in TR isoform knockout mice (82). Given the apparent redundancy in TR isoform function, it is possible the different KO phenotypes may be due to absolute TR expression levels in critical tissues and developmental stages.