scholarly journals A novel approach to investigate the subcellular distribution of nuclear receptors in vivo

2009 ◽  
Vol 7 (1) ◽  
pp. nrs.07004 ◽  
Author(s):  
Marko Matic ◽  
Sarah Nakhel ◽  
Anne M. Lehnert ◽  
Patsie Polly ◽  
Stephen J. Clarke ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Subcellular Distribution ◽  
Mammalian Cells ◽  
Pregnane X Receptor ◽  
Subcellular Localisation ◽  
Regulatory Processes ◽  
Cellular Expression ◽  
Novel Approach

Subcellular compartmentalisation and the intracellular movement of nuclear receptors are major regulatory steps in executing their transcriptional function. Though significant progress has been made in understanding these regulatory processes in cultured mammalian cells, such results have rarely been confirmed within cells of a living mammal. This article describes a simple, time-efficient approach to study the nuclear versus cytoplasmic accumulation of nuclear receptors and the regions of nuclear receptor proteins that govern subcellular trafficking within hepatocytes of live mice. Pregnane X receptor, a xenobiotic-activated member of the nuclear receptor family, was used to exemplify the approach. Using dual-labeled wild-type and mutant PXR expression constructs, we outline their in vivo delivery, simultaneous cellular expression, visualization and categorical classification within hepatocytes of live mice. Using this approach, we identified three mutants that had an altered subcellular distribution in the presence and absence of a PXR ligand. This novel in vivo method complements the current cell culture-based experimental systems in protein subcellular localisation studies.

scholarly journals The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1192 ◽  
Author(s):  
Philip Marx-Stoelting ◽  
Constanze Knebel ◽  
Albert Braeuning
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Liver Tumors ◽  
Current Knowledge ◽  
Constitutive Androstane Receptor ◽  
Pregnane X Receptor ◽  
Receptor Activation ◽  
Azole Fungicides

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

scholarly journals Nuclear receptor phosphorylation in xenobiotic signal transduction

2020 ◽  
Vol 295 (45) ◽  
pp. 15210-15225 ◽  
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.

scholarly journals Stimulation of Innate Immunity byIn VivoCyclic di-GMP Synthesis Using Adenovirus

2014 ◽  
Vol 21 (11) ◽  
pp. 1550-1559 ◽  
Author(s):  
Benjamin J. Koestler ◽  
Sergey S. Seregin ◽  
David P. W. Rastall ◽  
Yasser A. Aldhamen ◽  
Sarah Godbehere ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Mammalian Cells ◽  
Immune Cell ◽  
Innate Immune ◽  
Host Cells ◽  
Content Type ◽  
Cytokines And Chemokines ◽  
Novel Approach

ABSTRACTThe bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesizedin vivoby transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMPin vitroandin vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to aClostridium difficileantigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

scholarly journals The DRIP Complex and SRC-1/p160 Coactivators Share Similar Nuclear Receptor Binding Determinants but Constitute Functionally Distinct Complexes

2000 ◽  
Vol 20 (8) ◽  
pp. 2718-2726 ◽  
Author(s):  
Christophe Rachez ◽  
Matthew Gamble ◽  
Chao-Pei Betty Chang ◽  
G. Brandon Atkins ◽  
Mitchell A. Lazar ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Receptor Binding ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Nuclear Receptor Coactivators ◽  
Nuclear Extracts ◽  
Sequence Requirements

ABSTRACT Transcriptional activation requires both access to DNA assembled as chromatin and functional contact with components of the basal transcription machinery. Using the hormone-bound vitamin D3receptor (VDR) ligand binding domain (LBD) as an affinity matrix, we previously identified a novel multisubunit coactivator complex, DRIP (VDR-interacting proteins), required for transcriptional activation by nuclear receptors and several other transcription factors. In this report, we characterize the nuclear receptor binding features of DRIP205, a key subunit of the DRIP complex, that interacts directly with VDR and thyroid hormone receptor in response to ligand and anchors the other DRIP subunits to the nuclear receptor LBD. In common with other nuclear receptor coactivators, DRIP205 interaction occurs through one of two LXXLL motifs and requires the receptor's AF-2 subdomain. Although the second motif of DRIP205 is required only for VDR binding in vitro, both motifs are used in the context of an retinoid X receptor-VDR heterodimer on DNA and in transactivation in vivo. We demonstrate that both endogenous p160 coactivators and DRIP complexes bind to the VDR LBD from nuclear extracts through similar sequence requirements, but they do so as distinct complexes. Moreover, in contrast to the p160 family of coactivators, the DRIP complex is devoid of any histone acetyltransferase activity. The results demonstrate that different coactivator complexes with distinct functions bind to the same transactivation region of nuclear receptors, suggesting that they are both required for transcription activation by nuclear receptors.

scholarly journals Physical and Functional Interactions between Cellular Retinoic Acid Binding Protein II and the Retinoic Acid-Dependent Nuclear Complex

1999 ◽  
Vol 19 (10) ◽  
pp. 7158-7167 ◽  
Author(s):  
Laurent Delva ◽  
Jean-Noël Bastie ◽  
Cécile Rochette-Egly ◽  
Radhia Kraïba ◽  
Nicole Balitrand ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Nuclear Receptors ◽  
Mammalian Cells ◽  
Specific Response ◽  
Independent Manner ◽  
Control Of Gene Expression ◽  
Steady State Levels ◽  
And Control

ABSTRACT Two sorts of proteins bind to, and mediate the developmental and homeostatic effects of, retinoic acid (RA): the RAR and RXR nuclear receptors, which act as ligand-dependent transcriptional regulators, and the cellular RA binding proteins (CRABPI and CRABPII). CRABPs are generally known to be implicated in the synthesis, degradation, and control of steady-state levels of RA, yet previous and recent data have indicated that they could play a role in the control of gene expression. Here we show for the first time that, both in vitro and in vivo, CRABPII is associated with RARα and RXRα in a ligand-independent manner in mammalian cells (HL-60, NB-4, and MCF-7). In the nucleus, this protein complex binds the RXR-RAR-specific response element of an RA target gene (RARE-DR5). Moreover, in the presence of retinoids that bind both the nuclear receptors and CRABPII, enhancement of transactivation by RXRα-RARα heterodimers is observed in the presence of CRABPII. Thus, CRABPII appears to be a novel transcriptional regulator involved in RA signaling.

scholarly journals Thr176 regulates the activity of the mouse nuclear receptor CAR and is conserved in the NR1I subfamily members PXR and VDR

2005 ◽  
Vol 388 (2) ◽  
pp. 623-630 ◽  
Author(s):  
Akiko UEDA ◽  
Kenji MATSUI ◽  
Yukio YAMAMOTO ◽  
Lars C. PEDERSEN ◽  
Tatsuya SUEYOSHI ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Receptor ◽  
Pregnane X Receptor ◽  
Activation Function ◽  
Constitutive Activity ◽  
Charged Amino Acids ◽  
Binding Surface ◽  
Hydrophobic Amino Acids ◽  
Constitutively Active

The mouse nuclear receptor CAR (constitutively active receptor) is a transcription factor that is activated by phenobarbital-type inducers such as TCPOBOP {1,4 bis[2-(3,5-dichloropyridyloxy)]benzene} in liver in vivo. However, CAR is constitutively active in cell-based transfection assays, the molecular mechanism for which has not been elucidated yet. In the model structure of CAR, Thr176 constitutes a part of the ligand-binding surface, but its side chain is not directed toward the surface, instead it forms a hydrogen bond with Thr350 in the AF2 (activation function 2) domain of CAR. Thr350 is known to regulate CAR activity [Ueda, Kakizaki, Negishi, and Sueyoshi (2002) Mol. Pharmacol. 61, 1284–1288]. Thr176 was mutated to various amino acids to examine whether this interaction played a role in conferring the constitutive activity. Hydrophobic and positively charged amino acids at position 176 abrogated the constitutive activity, whereas polar and negatively charged amino acids retained it. When one of the small hydrophobic amino acids, such as alanine or valine, was substituted for threonine, the mutants were fully activated by TCPOBOP. The co-activator SRC-1 (steroid receptor co-activator-1) regulated the activity changes associated with the mutations. Thr248 and Ser230 are the Thr176-corresponding residues in human pregnane X receptor and mouse vitamin D3 receptor respectively, interacting directly with the conserved threonine in the AF2 domains. Thr248 and Ser230 also regulated the ligand-dependent activity of these receptors by augmenting binding of the receptors to SRC-1. Thr176, Thr248 and Ser230 are conserved residues in the NR1I (nuclear receptor 1I) subfamily members and determine their activity.

scholarly journals Alternative splicing determines the interaction of SMRT isoforms with nuclear receptor–DNA complexes

2009 ◽  
Vol 29 (3) ◽  
pp. 143-149 ◽  
Author(s):  
Flavie Faist ◽  
Stephen Short ◽  
G. Geoff Kneale ◽  
Colinb R. Sharpe
Keyword(s):  
Retinoic Acid ◽  
Alternative Splicing ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Thyroid Hormone Receptor ◽  
Retinoic Acid Receptor ◽  
Class Ii ◽  
Dna Complexes ◽  
Response Elements

Signalling by small molecules, such as retinoic acid, is mediated by heterodimers comprising a class II nuclear receptor and an RXR (retinoid X receptor) subunit. The receptors bind to DNA response elements and act as ligand-dependent transcription factors, but, in the absence of signal, the receptors bind the co-repressors SMRT [silencing mediator for RAR (retinoic acid receptor) and TR (thyroid hormone receptor)] and NCoR (nuclear receptor co-repressor) and repress gene expression. Alternative splicing of the SMRT transcript in mammals generates six isoforms containing 1, 2 or 3 CoRNR (co-repressor for nuclear receptor) box motifs which are responsible for the interactions with nuclear receptors. We show that human cell lines express all six SMRT isoforms and then determine the binding affinity of mouse SMRT isoforms for RAR/RXR and three additional class II nuclear receptor–DNA complexes. This approach demonstrates the importance of the full complement of CoRNR boxes within each SMRT protein, rather than the identity of individual CoRNR boxes, in directing the interaction of SMRT with nuclear receptors. Each class of SMRT isoform displays a distinct feature, as the 1-box isoform discriminates between DNA response elements, the 2-box isoforms promote high-affinity binding to TR complexes and the 3-box isoforms show differential binding to nuclear receptors. Consequently, the differential deployment of SMRT isoforms observed in vivo could significantly expand the regulatory capacity of nuclear receptor signalling.

RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal co-integrator-2

2008 ◽  
Vol 410 (2) ◽  
pp. 319-330 ◽  
Author(s):  
You Lee Son ◽  
Ok Gu Park ◽  
Gwang Sik Kim ◽  
Jae Woon Lee ◽  
Young Chul Lee
Keyword(s):  
Nuclear Receptors ◽  
Mammalian Cells ◽  
Specific Interaction ◽  
Critical Determinant ◽  
Interaction Domains ◽  
Interaction Surface ◽  
Unique Mechanism ◽  
Allosteric Activator

ASC-2 (activating signal co-integrator-2) is a transcriptional co-activator that mediates the transactivation of NRs (nuclear receptors) via direct interactions with these receptors. ASC-2 contains two separate NR-interaction domains harbouring a core signature motif, LXXLL (where X is any amino acid), named the NR box. Although the first NR box (NR box-1) of ASC-2 interacts with many different NRs, the second NR box (NR box-2) specifically interacts with only LXR (liver X receptor), whose transactivation in vivo requires heterodimerization with RXR (retinoid X receptor). Interestingly, RXR has been shown to enhance the LXR transactivation, even in the absence of LXR ligand via a unique mechanism of allosteric regulation. In the present study we demonstrate that LXR binding to an ASC-2 fragment containing NR box-2 (Co4aN) is enhanced by RXR and even further by liganded RXR. We also identified specific residues in Co4aN involved in its interaction with LXR that were also required for the ASC-2-mediated transactivation of LXR in mammalian cells. Using these mutants, we found that the Co4aN–LXR interaction surface is not altered by the presence of RXR and RXR ligand and that the Ser1490 residue is the critical determinant for the LXR-specific interaction of Co4aN. Notably the NR box-2, but not the NR box-1, is essential for ASC-2-mediated transactivation of LXR in vivo and for the interaction between LXR–RXR and ASC-2 in vitro. These results indicate that RXR does not interact directly with NR box-1 of ASC-2, but functions as an allosteric activator of LXR binding to NR box-2 of ASC-2.

scholarly journals Use of Differential Scanning Fluorimetry as a High-Throughput Assay to Identify Nuclear Receptor Ligands

2012 ◽  
Vol 10 (1) ◽  
pp. nrs.10002 ◽  
Author(s):  
Kara DeSantis ◽  
Aaron Reed ◽  
Raneen Rahhal ◽  
Jeff Reinking
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
High Throughput ◽  
Initial Step ◽  
Estrogen Receptor Α ◽  
Chemical Library ◽  
Differential Scanning Fluorimetry ◽  
Nuclear Receptor Ligands

Identification of ligands that interact with nuclear receptors is both a major biological problem and an important initial step in drug discovery. Several in vitro and in vivo techniques are commonly used to screen ligand candidates against nuclear receptors; however, none of the current assays allow screening without modification of either the protein and/or the ligand in a high-throughput fashion. Differential scanning fluorimetry (DSF) allows unmodified potential ligands to be screened as 10μL reactions in 96-well format against partially purified protein, revealing specific interactors. As a proof of principle, we used a commercially-available nuclear receptor ligand candidate chemical library to identify interactors of the human estrogen receptor α ligand binding domain (ERα LBD). Compounds that interact specifically with ERα LBD stabilize the protein and result in an elevation of the thermal denaturation point, as monitored by the environmentally-sensitive dye SYPRO orange. We successfully identified all three compounds in the library that have previously been identified to interact with ERα, with no false positive results.

scholarly journals Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4α1

1999 ◽  
Vol 19 (10) ◽  
pp. 6509-6522 ◽  
Author(s):  
Frances M. Sladek ◽  
Michael D. Ruse ◽  
Luviminda Nepomuceno ◽  
Shih-Ming Huang ◽  
Michael R. Stallcup
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Regulatory Region ◽  
Physical Contact ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Splicing Variants

ABSTRACT Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 α1 (HNF4α1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4α2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4α1 and HNF4α2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4α1 and -α2 and that that contact is different in the HNF4α1 and HNF4α2 isoforms. Finally, we propose a model in which the F domain of HNF4α1 acts as a negative regulatory region for transactivation and in which the α2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4α1 and -α2 suggests that this model may be relevant to other nuclear receptors as well.

Sign in / Sign up

Export Citation Format

Share Document