Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation

ABSTRACT The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR), which is activated upon ligand binding, and can alter the expression of target genes either by transrepression or transactivation. We have applied FRAP (fluorescence recovery after photobleaching) to quantitatively assess the mobility of the yellow fluorescent protein (YFP)-tagged human GR α-isoform (hGRα) in the nucleus of transiently transfected COS-1 cells and to elucidate determinants of its mobility. Addition of the high-affinity agonist dexamethasone markedly decreases the mobility of the receptor in a concentration-dependent manner, whereas low-affinity ligands like corticosterone decrease the mobility to a much lesser extent. Analysis of other hGRα ligands differing in affinity suggests that it is the affinity of the ligand that is a major determinant of the decrease in mobility. Similar results were observed for two hGRα antagonists, the low-affinity antagonist ZK98299 and the high-affinity antagonist RU486. The effect of ligand affinity on mobility was confirmed with the hGRα mutant Q642V, which has an altered affinity for triamcinolone acetonide, dexamethasone, and corticosterone. Analysis of hGRα deletion mutants indicates that both the DNA-binding domain and the ligand-binding domain of the receptor are required for a maximal ligand-induced decrease in receptor mobility. Interestingly, the mobility of transfected hGRα differs among cell types. Finally, the proteasome inhibitor MG132 immobilizes a subpopulation of unliganded receptors, via a mechanism requiring the DNA-binding domain and the N-terminal part of the ligand-binding domain. Ligand binding makes the GR resistant to the immobilizing effect of MG132, and this effect depends on the affinity of the ligand. Our data suggest that ligand binding induces a conformational change of the receptor which is dependent on the affinity of the ligand. This altered conformation decreases the mobility of the receptor, probably by targeting the receptor to relatively immobile nuclear domains with which it transiently associates. In addition, this conformational change blocks immobilization of the receptor by MG132.

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Evolution of human, chicken, alligator, frog and zebrafish mineralocorticoid receptors: Allosteric influence on steroid specificity

10.1101/151233 ◽

2017 ◽

Cited By ~ 2

Author(s):

Yoshinao Katsu ◽

Kaori Oka ◽

Michael E. Baker

Keyword(s):

Dna Binding ◽

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Full Length ◽

Dna Binding Domain ◽

Mineralocorticoid Receptors ◽

Terminal Domain ◽

Terrestrial Vertebrate ◽

Hinge Domain

AbstractWe studied the response to aldosterone, 11-deoxycorticosterone, 11-deoxycortisol, cortisol, corticosterone, progesterone, 19-norprogesterone and spironolactone of human, chicken, alligator, frog and zebrafish full-length mineralocorticoid receptors (MRs) and truncated MRs, lacking the N-terminal domain (NTD) and DNA-binding domain (DBD), in which the hinge domain and ligand binding domain (LBD) were fused to a GAL4-DBD. Compared to full-length MRs, some vertebrate MRs required higher steroid concentrations to activate GAL4-DBD-MR-hinge/LBD constructs. For example, 11-deoxycortisol activated all full-length vertebrate MRs, but did not activate truncated terrestrial vertebrate MRs and was an agonist for truncated zebrafish MR. Progesterone, 19-norProgesterone and spironolactone did not activate full-length and truncated human, alligator and frog MRs. However, at 10 nM, these steroids activated full-length chicken and zebrafish MRs; at 100 nM, these steroids had little activity for truncated chicken MRs, while retaining activity for truncated zebrafish MRs, evidence that regulation of progestin activation of chicken MR resides in NTD/DBD and of zebrafish MR in hinge-LBD. Zebrafish and chicken MRs contain a serine corresponding to Ser810 in human MR, required for its antagonism by progesterone, suggesting novel regulation of progestin activation of chicken and zebrafish MRs. Progesterone may be a physiological activator of chicken and zebrafish MRs.

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Evidence for DNA-Binding Domain-Ligand-Binding Domain Communications in the Androgen Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.00151-12 ◽

2012 ◽

Vol 32 (15) ◽

pp. 3033-3043 ◽

Cited By ~ 42

Author(s):

C. Helsen ◽

V. Dubois ◽

A. Verfaillie ◽

J. Young ◽

M. Trekels ◽

...

Keyword(s):

Androgen Receptor ◽

Dna Binding ◽

Ligand Binding ◽

Binding Domain ◽

Ligand Binding Domain ◽

Dna Binding Domain

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The nuclear receptor ligand-binding domain: structure and function

Current Opinion in Cell Biology ◽

10.1016/s0955-0674(98)80015-x ◽

1998 ◽

Vol 10 (3) ◽

pp. 384-391 ◽

Cited By ~ 538

Author(s):

Dino Moras ◽

Hinrich Gronemeyer

Keyword(s):

Ligand Binding ◽

Domain Structure ◽

Nuclear Receptor ◽

Structure And Function ◽

Binding Domain ◽

Ligand Binding Domain ◽

Receptor Ligand ◽

And Function

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An Inhibitory Region of the DNA-Binding Domain of Thyroid Hormone Receptor Blocks Hormone-Dependent Transactivation

Molecular Endocrinology ◽

10.1210/mend.12.1.0046 ◽

1998 ◽

Vol 12 (1) ◽

pp. 34-44 ◽

Cited By ~ 3

Author(s):

Ying Liu ◽

Akira Takeshita ◽

Takashi Nagaya ◽

Aria Baniahmad ◽

William W. Chin ◽

...

Keyword(s):

Thyroid Hormone ◽

Dna Binding ◽

Ligand Binding ◽

Transcriptional Activation ◽

Thyroid Hormone Receptor ◽

Binding Domain ◽

Dna Binding Domain ◽

Chimeric Receptor ◽

Receptor System ◽

Inhibitory Region

Abstract We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor β ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-β (TRβ), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRβ derepressed transcription to basal level. Using this system and a battery of TRβ mutants, we found that TRβ/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRβ ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRβ DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRβ from achieving transcriptional activation above basal level in this chimeric receptor system.

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A ligand-entry surface of the nuclear receptor superfamily consists of the helix H3 of the ligand-binding domain

Journal of Molecular Graphics and Modelling ◽

10.1016/j.jmgm.2015.10.002 ◽

2015 ◽

Vol 62 ◽

pp. 262-275 ◽

Cited By ~ 4

Author(s):

Motonori Tsuji

Keyword(s):

Ligand Binding ◽

Nuclear Receptor ◽

Binding Domain ◽

Ligand Binding Domain ◽

Nuclear Receptor Superfamily

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Nuclear receptor phosphorylation in xenobiotic signal transduction

Journal of Biological Chemistry ◽

10.1074/jbc.rev120.007933 ◽

2020 ◽

Vol 295 (45) ◽

pp. 15210-15225 ◽

Cited By ~ 1

Author(s):

Masahiko Negishi ◽

Kaoru Kobayashi ◽

Tsutomu Sakuma ◽

Tatsuya Sueyoshi

Keyword(s):

Dna Binding ◽

Cell Signaling ◽

Nuclear Receptors ◽

Nuclear Receptor ◽

Pregnane X Receptor ◽

Receptor Activation ◽

Binding Domain ◽

Activation Mechanism ◽

Dna Binding Domain ◽

Phosphorylation Motif

Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.

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