The ligand-binding domain of nuclear receptors contains a transcriptional activation function (AF-2) that mediates hormone-dependent binding of coactivator proteins. Scanning surface mutagenesis on the human thyroid hormone receptor was performed to define the site that binds the coactivators, glucocorticoid receptor-interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1). The residues involved encircle a small surface that contains a hydrophobic cleft. Ligand activation of transcription involves formation of this surface by folding the carboxyl-terminal alpha helix against a scaffold of three other helices. These features may represent general ones for nuclear receptors.

The results of this study show that GC-1 is a member of a new class of thyromimetic compounds that are more synthetically accessible than traditional thyromimetics and have potentially useful receptor binding and activation properties. The TR beta selectivity of GC-1 is particularly interesting and suggests that GC-1 might be a useful in vivo probe for studying the physiological roles of the different thyroid hormone receptor isoforms.

Abstract-Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including ␣-and ␤-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) ␤1, ␣1, and ␣2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid-and hyperthyroid-like changes in the TH target genes, ␣-and ␤-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TR␤1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TR␤1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TR␤1 to transcription of ␤-MyHC, SERCA, and TR␤1, and TR␣1 to ␣-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy. (Circ Res. 2001;89:591-598.) Key Words: thyroid hormone receptor Ⅲ physiological and pathological hypertrophy Ⅲ ␣ 1 -adrenergic receptor Ⅲ cardiac myocyte Ⅲ rat C ardiac hypertrophy is sometimes considered a single process that leads invariably to myocardial dysfunction (pathological hypertrophy). However, physiological hypertrophy exists in which cardiac function is maintained or enhanced, including normal cardiac development, exercise training, and thyroid hormone (TH) treatment. Exercise and TH can reverse molecular and functional abnormalities in pathological hypertrophy without decreasing ventricular mass, indicating that physiological and pathological hypertrophy are qualitatively distinct processes. [1][2][3][4][5][6] TH-responsive genes in cardiac muscle include ␣-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA), which are induced by TH, and ␤-MyHC, which is repressed. 7,8 An intriguing observation is that pathological hypertrophy is characterized by hypothyroid-like changes in these target genes, with decreases in ␣-MyHC and SERCA and increases in ␤-MyHC, a molecular phenotype also called the fetal program. 9 The fact that TH treatment can reverse these genetic changes in some models of pathological hypertrophy is additional evidence for a hypothyroid state, but TH blood levels are usually normal. 3 Conversely, physiological hypertrophy caused by exercise is characterized ...

The tumor necrosis factor-␣ (TNF-␣) promoter was used to explore the molecular mechanisms of estradiol (E 2)-dependent repression of gene transcription. E 2 inhibited basal activity and abolished TNF-␣ activation of the TNF-␣ promoter. The E2-inhibitory element was mapped to the ؊125 to ؊82 region of the TNF-␣ promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) ␤ is more potent than ER␣ at repressing the ؊1044 TNF-␣ promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ER␤, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E2-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

We conclude that decreases in TR-alpha(1) and increases in TR-alpha(2) may lead to local attenuation of thyroid hormone signaling in the failing human heart and that the resulting tissue-specific hypothyroidism is a candidate for the molecular mechanism that induces fetal gene expression in the failing human ventricle.

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1.Thyroid hormone receptors (TR) are expressed from two separate genes (a and p) and belong to the nuclear receptor superfamily, which also contains receptors for steroids, vitamins and prostaglandins.2. Unliganded TR are bound to DNA thyroid hormone response elements (TRE) predominantly as homodimers, or as heterodimers with retinoid X-receptors (RXR), and are associated with a complex of proteins containing corepressor proteins. Ligand binding promotes corepressor dissociation and binding of a coactivator.3. Recent studies from our group have focused on the acquisition and use of X-ray crystallographic structures of ligandbinding domains (LBD) of both the rat (r) TRa and the human (h) TRP bound to several different ligands. We have also developed ligands that bind selectively to the TRP, which may provide ways to explore the differential functions of TRcw compared with TRP isoforms.4. The LBD is comprised mostly of a-helices. The ligand is completely buried in the receptor and forms part of its hydrophobic core. Kinetic studies suggest that the limiting step in formation of high-affinity ligand-receptor complexes is the rate of folding of the receptor around the ligand. Ligands can be fitted tightly in the ligand-binding pocket and small differences in this fitting may explain many structure-activity relationships. Interestingly, analysis of the structures of antagonists suggests that they have chemical groups, 'extensions', that could impair receptor folding around them and, thus, prevent the agonistinduced conformation changes in the receptor.5. The TR structures allowed us to see that the mutations that occur in the syndrome of generalized resistance to thyroid hormone are located in the vicinity of the ligand-binding pocket.6. X-ray structure of the TR has also been used to guide construction of mutations in the TR surface that block binding of various proteins important for receptor function. Studies with these TR mutants reveal that the interfaces for homo-and heterodimerization map to similar residues in helix 10 and 11 and also allow the definition of the surface for binding ofcoactivators, which appears to be general for nuclear receptors. Formation of this surface, which involves packing of helix 12 of the TR into a scaffold formed by helices 3 and 5, appears to be the major change in the receptor structure induced by hormone occupancy.