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The thyroid hormone receptors (TR) are nuclear proteins that include TR alpha and TR beta subtypes, each encoded by a separate gene. Both TR alpha and TR beta give rise to several isoforms of which three, TR alpha 1, TR beta 1, and TR beta 2 bind T3 and mediate the action of thyroid hormone. Although TR beta 2 was initially thought to be confined to the anterior pituitary, we recently observed small quantities of TR beta 2 messenger RNA (mRNA) by polymerase chain reaction analysis of discrete hypothalamic regions. To further examine the distribution of TR beta 2 in the brain, we performed immunocytochemical studies using a highly specific antiserum to TR beta 2, raised against a unique amino acid sequence (TR beta 2[131-145]) that is not present in the other known TRs. This antiserum immunoprecipitated TR beta 2 but not TR alpha 1 or TR beta 1. Immunoreactive TR beta 2 was widely distributed throughout the brain and primarily localized to the cell nucleus. Particularly intense immunostaining was present in the cerebral cortex, cerebellum, and hypothalamus, including regions where TR beta 2 mRNA had not previously been identified. In addition, immunoprecipitation of nuclear extracts with anti-TR beta 2 reduced total T3 binding capacity by approximately 20%, suggesting that immunoreactive TR beta 2 comprises a substantial portion of the total content of nuclear thyroid hormone binding proteins. These studies demonstrate that immunoreactive TR beta 2 is more widely represented in the central nervous system than previously suspected and may play an important role in mediating the action of T3 in many different regions of the brain. The finding of TR beta 2-like material could be due to a disproportionately high ratio of the TR beta 2 translation product and its mRNA in certain regions of the brain, or could indicate the existence of a novel TR beta 2-related protein that is important for T3 binding.

Thyroid hormone receptor (TR) binds to DNA as a monomer, homodimer, and heterodimer with nuclear proteins. We have confirmed that the TR can heterodimerize with retinoid X receptors (RXRs)-alpha and -beta, and have found that another member of the nuclear receptor superfamily, chicken ovalbumin upstream promoter transcription factor (COUP-TF), also formed heterodimers with the TR in the context of binding to a palindromic thyroid hormone-responsive element (TREp). The interaction between COUP-TF and the TR was confirmed using specific antibodies which supershifted the COUP-TF/TR DNA complexes. The complex between the TR and the major TR heterodimerization partner in liver was unaffected by antibodies to COUP-TF and RXR beta, but was supershifted by an anti-RXR alpha antibody, indicating that the liver protein is highly related to RXR alpha. Indeed, the TR/RXR and TR/liver protein heterodimers contact the same guanidine residues in TREp. The retinoic acid receptor (RAR) also heterodimerized with COUP-TF as well as with RXR alpha, RXR beta, and the TR heterodimerization partner in liver. In contrast to its ability to heterodimerize with the TR and RAR, we did not detect heterodimers between COUP-TF and either RXR alpha, RXR beta, or the liver nuclear protein in the context of binding to the TREp. These results show that the major TR heterodimerization partner in liver is highly related to RXR alpha, but that other nuclear receptors such as COUP-TF can heterodimerize with the TR and RAR, suggesting that selective protein-protein interactions may be involved in the tissue and target gene specificities of hormone action.

Thyroid hormone receptors (TRs) mediate the regulation of gene transcription by thyroid hormone (T3) by binding to T3-responsive elements (TREs) in target genes. c-erbA alpha 2 is a C-terminal TR variant which does not bind T3 and is a dominant inhibitor of T3 action. When synthesized in Escherichia Coli, alpha 2 formed two TRE-binding complexes similar to the monomeric and homodimeric forms of TR alpha 1. However, alpha 2 did not bind nearly as well as TR alpha 1. Furthermore, alpha 2 failed to bind DNA with proteins that heterodimerized with TR alpha 1. TR alpha 1 and alpha 2 also did not bind DNA as heterodimers with one another. The differences between TR alpha 1 and alpha 2 were further analyzed by studying a variety of C-terminal mutants synthesized in reticulocyte lysates. Deletion of the last 20 of the 122 unique amino acids (aa) of alpha 2 increased its DNA binding to approximately the level of TR alpha 1, indicating that the C-terminus of alpha 2 is an inhibitory domain. This alpha 2 mutant (alpha 2 delta C) was still unable to heterodimerize with nuclear proteins, as were C-terminal deletion mutants of TR alpha 1. We hypothesized that fusion of TR alpha 1-specific sequences to the C-terminus of alpha 2 delta C would transfer the property of heterodimerization. Indeed, although alpha 2/alpha 1 chimeras containing the last 40 and 70 aa of TR alpha 1 failed to heterodimerize with nuclear proteins, addition of the last 100 or 150 aa of TR alpha 1 did render alpha 2 delta C heterodimerization competent. Thus, TR alpha 1 contains a C-terminal structure which is necessary for heterodimerization and can confer this property on alpha 2, which lacks this domain. The effects of the unique C-termini of TR alpha 1 and alpha 2 on their in vitro DNA binding have important implications for their mechanisms of action in vivo.

We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)