Conformational Changes of Human β1 Thyroid Hormone Receptor Induced by Binding of 3,3′,5-Triiodo-L-thyronine

Bhat, Manoj Kumar; Parkison, Clifford; McPhie, Peter; Cheng, Liang; Cheng, Sheue-yann

doi:10.1006/bbrc.1993.2055

Cited by 35 publications

(16 citation statements)

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“…Another possible explanation for the observed effects of heterodimerization with RXR␣ on the biological activities of T3R␣ is an induced conformational change. By using limited protease digestion, several recent studies have demonstrated that T3 binding has distinct effects on T3R homodimers and T3R:RXR heterodimers in solution (8,9,23,38). By using the mobility shift assay, it was also shown that T3 binding differentially affects homodimer and heterodimer binding to various T3REs (1,31,42,43).…”

Section: Resultsmentioning

confidence: 99%

“…Previous work has shown that ligand binding induces a conformational change in T3Rs in solution (9,24,38), and heterodimerization with RXR enhances this ligand-dependent conformational change (8,24). Such changes in conformation may provide a mechanism for the shift in the response to T3 for both ligand-dependent activation and interference with AP-1 activity.…”

Section: Vol 16 1996 Regulation Of Transcription By T3r:rxr Heterodmentioning

confidence: 96%

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A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Claret

Antakly

Karin

et al. 1996

Molecular and Cellular Biology

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Thyroid hormone (T3) receptors (T3Rs) are ligand-modulated transcription factors that bind to thyroid hormone response elements (T3REs) and mediate either positive or negative transcriptional regulation of target genes. In addition, in response to ligand binding, T3Rs can interfere with AP-1 activity and thereby inhibit transcription of AP-1-responsive genes. T3Rs were recently shown to form heterodimers with retinoid X receptors (RXRs), leading to increased binding to T3REs in vitro and potentiation of transcriptional responses in vivo. Here we demonstrate that T3R␣ forms stable heterodimers with RXR␣ in living cells. Most important, we describe a new role for RXR␣ in modulating ligand-dependent T3R␣ activity: heterodimerization with RXR␣ greatly increases transcriptional interference with AP-1 activity, augments T3-dependent transcriptional activation, and potentiates the reversal of ligand-independent activation by T3R␣. In all three cases, the responses occur at substantially lower T3 concentrations when elicited by T3R␣ plus RXR␣ than by T3R␣ alone. In vitro, the binding of T3 decreases the DNA-binding activity of T3R␣ homodimers but does not affect DNA binding by T3R␣:RXR␣ heterodimers. We provide evidence that increased activities of T3R␣ at lower T3 concentrations are not due to changes in its T3 binding properties. Instead, the altered response could be mediated by either RXR␣-induced conformational changes, increased stability of heterodimers over homodimers, especially following T3 binding, or both.Thyroid hormone 3,5,3Ј-triiodo-L-thyronine (T3) is required for normal growth and development and maintenance of normal metabolism (for a review, see reference 27). The actions of T3 are mediated through binding to T3 receptors (T3Rs), which belong to the nuclear receptor superfamily of transcription factors encoded by the c-erbA␣ and c-erbA␤ genes (36, 41). Nuclear receptors are ligand-activated transcription factors possessing highly conserved DNA-binding domains and moderately conserved ligand-binding domains (7,15). Together with receptors for retinoic acid (RA) and vitamin D, the T3Rs form a subgroup that recognizes the consensus half-site AGGTCA (7). This sequence can be arranged as a direct repeat, a palindrome, or an inverted repeat, and the spacing between the half sites and their relative orientations determine receptor specificity and the nature of the transcriptional response (26,34,39). For example, unliganded T3R␣ can repress promoters that contain palindromic T3 response elements (T3REs), and this repression is reversed upon binding of T3, resulting in net activation (13, 37). Unliganded T3R␣ can also activate transcription from promoters which contain an inverted repeat of a variant half-site, and this activation is reversed upon T3 binding (34). Whereas the ligand-independent repression of basal promoter activity and the ligand-dependent activation functions are mediated by sequences within the Cterminal ligand-binding domain (4-6, 30, 32), the ligand-independent activation function is medi...

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Section: Resultsmentioning

confidence: 99%

Section: Vol 16 1996 Regulation Of Transcription By T3r:rxr Heterodmentioning

confidence: 96%

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Claret

Antakly

Karin

et al. 1996

Molecular and Cellular Biology

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“…At present, it is unknown whether the structure of this A-helix in the context of the intact TR␤1 undergoes changes upon the binding of T 3 to domain E because no crystallographic studies of the entire TR␤1 are yet available. However, it is reasonable to assume that this A-helix in domain D could undergo T 3 -induced conformational changes as it is clearly demonstrated that dramatic structural changes occur in the ligand binding domain E of TR␤1 when bound to T 3 (23,24). The T 3 -induced changes of A-helix in the context of the intact TR␤1 could expose the nuclear localization signal to become more accessible to the receptors that bind the nuclear localization signal (25,26), thereby providing additional regulation of the transcriptional activity of TR␤1.…”

Section: Discussionmentioning

confidence: 99%

Hormone-induced Translocation of Thyroid Hormone Receptors in Living Cells Visualized Using a Receptor Green Fluorescent Protein Chimera

Zhu

Hanover

Hager³

et al. 1998

Journal of Biological Chemistry

112

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Thyroid hormone nuclear receptors (TRs) are liganddependent transcription factors that regulate growth, differentiation, and development. To understand the role of the hormone, 3,3,5-triiodo-L-thyronine (T 3 ), in the nuclear translocation and targeting of TRs to the regulatory sites in chromatin, we appended green fluorescent protein (GFP) to the human TR subtype ␤1 (TR␤1). The fusion of GFP to the amino terminus of TR␤1 protein did not alter T 3 binding or transcriptional activities of the receptor. The subcellular localization of GFP-TR␤1 in living cells was visualized by laser-scanning confocal microscopy. In the presence of T 3 , the expressed GFP-TR␤1 was predominately localized in the nucleus, exhibiting a nuclear/cytoplasmic ratio of ϳ5.5. No GFP-TR␤1 was detected in the nucleolus. In the absence of T 3 , more GFP-TR␤1 was present in the cytoplasm, exhibiting a nuclear/cytoplasmic ratio of ϳ1.5. In these cells, cytoplasmic GFP-TR␤1 could be induced to enter the nucleus by T 3 . The T 3 -induced translocation was blocked when Lys 184 -Arg 185 in domain D of TR␤1 was mutated to Ala 184 -Ala 185 . Furthermore, the inability of the mutant TR to translocate to the nucleus correlated with the loss of most of its transcriptional activity. These results suggest that TR functions may, in part, be regulated by T 3 -induced nuclear entry.Thyroid hormone receptors (TRs) 1 are ligand-dependent transcription factors, which are members of the steroid hormone/retinoic acid receptor superfamily. Two TR genes, TR␣ and TR␤, located on chromosomes 17 and 3, respectively, give rise to four TR isoforms, ␣1, ␣2, ␤1, and ␤2, by alternative splicing of the primary transcripts (1, 2). TRs mediate the biological activities of the thyroid hormone, 3,3Ј,5-triiodo-Lthyronine (T 3 ), by binding to the specific DNA sequences, known as the thyroid hormone response elements (TREs), in the promoter regions of T 3 target genes (1, 2). The transcriptional regulatory activity of TRs not only depends on T 3 and the types of TREs but also on a host of co-regulatory proteins including co-repressors, co-activators, and the tumor suppressor p53 (3, 4).To function as transcription factors, TRs have to interact with transcriptional machinery in the nucleus. However, the process by which TRs are targeted to the nucleus is poorly understood. Before the genes encoding TRs were isolated, high affinity, low capacity T 3 binding sites were detected in the nuclear fractions of tissues and cultured cells by subcellular fractionation (5-9). Subsequently, when anti-TR antibodies became available, TRs were found only in the nuclei of fixed cells by immunocytochemistry and immunohistochemistry (10 -12). However, in these studies, dynamic cytoplasmic nuclear trafficking of TRs and the effect of T 3 on nuclear trafficking were not addressed. In the present study, we appended the green fluorescence protein (GFP) to the human TR subtype ␤1 (TR␤1) allowing direct examination of the nuclear transport of TRs in living cells. TR exhibited both constitutive nuclear l...

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“…fragment became resistant to high concentration trypsin digestion, as described previously (21). The mutants, L428R, L428Q indicated a similar proteolysis to the wild-type in the absence of T 3 , because they were completely digested by incubation of 50 ng/mL of trypsin.…”

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confidence: 99%

Leucine at Codon 428 in the Ninth Heptad of Thyroid Hormone Receptor β₁is Necessary for Interactions with the Transcriptional Cofactors and Functions Regardless of Dimer Formations

Monden

Yamada

Ishii

et al. 2003

Thyroid

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Structure/function studies of the thyroid hormone receptor (TR) beta(1) have demonstrated that single amino acid substitutions in either position 428 or 429 in the ligand-binding domain (LBD) can alter heterodimerizations and homodimerizations, respectively. A leucine at 428 is located in a highly conserved region corresponding to the putative ninth heptad repeat of a leucine-zipper-like motif in the LBD of TRbeta(1). To investigate how the side chain of amino acids at 428 affect receptor characteristics, gel-shift mobility shift assays and yeast two-hybrid assays were analyzed. The neutral status amino acids such as a leucine (wild-type) or a glutamine at 428 preferred heterodimerization with RXR. Furthermore, a positively charged side chain of amino acids at 428 such as an arginine or a lysine, preserved homodimer formation. Irrespective of charge, ninth heptad mutant receptors did not bind the ligand and were not able to interact with either corepressor or coactivating proteins. Limited trypsinization assays revealed no major conformational change in the ninth heptad mutant receptors. Together, these findings suggested that a leucine at 428 was a critical amino acid for both interaction with the thyroid hormone receptor associated proteins and ligand-independent and -dependent functions regardless of dimer formations.

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Conformational Changes of Human β1 Thyroid Hormone Receptor Induced by Binding of 3,3′,5-Triiodo-L-thyronine

Cited by 35 publications

References 0 publications

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Hormone-induced Translocation of Thyroid Hormone Receptors in Living Cells Visualized Using a Receptor Green Fluorescent Protein Chimera

Leucine at Codon 428 in the Ninth Heptad of Thyroid Hormone Receptor β₁is Necessary for Interactions with the Transcriptional Cofactors and Functions Regardless of Dimer Formations

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Conformational Changes of Human β1 Thyroid Hormone Receptor Induced by Binding of 3,3′,5-Triiodo-L-thyronine

Cited by 35 publications

References 0 publications

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

A shift in the Ligand Responsiveness of Thyroid Hormone Receptor α Induced by Heterodimerization with Retinoid X Receptor α

Hormone-induced Translocation of Thyroid Hormone Receptors in Living Cells Visualized Using a Receptor Green Fluorescent Protein Chimera

Leucine at Codon 428 in the Ninth Heptad of Thyroid Hormone Receptor β1is Necessary for Interactions with the Transcriptional Cofactors and Functions Regardless of Dimer Formations

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Leucine at Codon 428 in the Ninth Heptad of Thyroid Hormone Receptor β₁is Necessary for Interactions with the Transcriptional Cofactors and Functions Regardless of Dimer Formations