Imaging of Selective Nuclear Receptor Modulator‐Induced Conformational Changes in the Nuclear Receptor to Allow Interaction with Coactivator and Corepressor Proteins in Living Cells

Awais, Muhammad; Sato, Moritoshi; Umezawa, Yoshio

doi:10.1002/cbic.200700001

Cited by 12 publications

(5 citation statements)

References 28 publications

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“…An elegant system directly uses the ligand-induced repositioning of helix 12 in the estrogen receptor (ER) LBD to sense ERligand interactions (44). A whole set of indicators has been designed to detect ligands for the ER, glucocorticoid receptor, progesterone receptor, and AR, but also the orphan receptor peroxisome proliferator-activated receptor c (45)(46)(47)(48)(49). These indicators make use of the conformational change in the LBD that leads to cofactor recruitment to the cofactor groove in the SR-LBD.…”

Section: Introductionmentioning

confidence: 99%

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

et al. 2013

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Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands. ' 2013 International Society for Advancement of Cytometry Key terms androgen receptor; recurrent prostate cancer; ligand; N/C-interaction; quantitative live cell imaging; FRET

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Section: Introductionmentioning

confidence: 99%

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

et al. 2013

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“…Genetically encoded fluorescent sensors provide excellent tools to determine the concentrations and monitor transport of intracellular metabolites in single living cells in real time 8, 9. Here, we report the development of a genetically encoded Förster resonance energy transfer (FRET) bile acid sensor (BAS) based on the ligand‐binding domain (LBD) of FXR.…”

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confidence: 99%

Monitoring Bile Acid Transport in Single Living Cells Using a Genetically Encoded FöRster Resonance Energy Transfer Sensor

et al. 2013

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Bile acids are pivotal for the absorption of dietary lipids and vitamins and function as important signaling molecules in metabolism. Here, we describe a genetically encoded fluorescent bile acid sensor (BAS) that allows for spatiotemporal monitoring of bile acid transport in single living cells. Changes in concentration of multiple physiological and pathophysiological bile acid species were detected as robust changes in F€ orster resonance energy transfer (FRET) in a range of cell types. Specific subcellular targeting of the sensor demonstrated rapid influx of bile acids into the cytoplasm and nucleus, but no FRET changes were observed in the peroxisomes. Furthermore, expression of the liver fatty acid binding protein reduced the availability of bile acids in the nucleus. The sensor allows for single cell visualization of uptake and accumulation of conjugated bile acids, mediated by the Na 1 -taurocholate cotransporting protein (NTCP). In addition, cyprinol sulphate uptake, mediated by the putative zebrafish homologue of the apical sodium bile acid transporter, was visualized using a sensor based on the zebrafish farnesoid X receptor. The reversible nature of the sensor also enabled measurements of bile acid efflux in living cells, and expression of the organic solute transporter ab (OSTab) resulted in influx and efflux of conjugated chenodeoxycholic acid. Finally, combined visualization of bile acid uptake and fluorescent labeling of several NTCP variants indicated that the sensor can also be used to study the functional effect of patient mutations in genes affecting bile acid homeostasis. Conclusion: A genetically encoded fluorescent BAS was developed that allows intracellular imaging of bile acid homeostasis in single living cells in real time. (HEPATOLOGY 2013;57:740-752) B ile acids operate as signaling molecules with systemic endocrine functions that are very important for lipid, glucose, and energy homeostasis and are essential for intestinal absorption of dietary fat and fat-soluble vitamins.

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“…That strategy was used to discriminate among agonists, antagonists, and SNRMs. The authors have developed fluorescent probes for estrogen, 45 androgen, 46 progesterone, 47 glucocorticoid, 48 and peroxisome proliferator-activated gamma receptors. 49 The probes are useful to identify any ligand that activates or inhibits the action of a nuclear receptor through a genomic pathway.…”

Section: Fluorescence Resonance Energy Transfer (Fret)mentioning

confidence: 99%

Illuminating intracellular signaling and molecules for single cell analysis

Awais

Ozawa

2011

Mol. BioSyst.

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Fluorescent and bioluminescent proteins are now widely used for detection of small molecules and various intracellular events ranging from protein conformational change to cell death in living cells. To analyze the dynamics of molecular processes in real time at the level of single cells, engineered protein-based probes with higher sensitivity and selectivity are required. The probes can be entirely genetically encoded and can comprise fusions of different proteins or domains. This review specifically examines basic concepts of designing genetically encoded fluorescent and bioluminescent probes developed in the past decade, highlighting some potential applications for basic research and for drug discovery.

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Imaging of Selective Nuclear Receptor Modulator‐Induced Conformational Changes in the Nuclear Receptor to Allow Interaction with Coactivator and Corepressor Proteins in Living Cells

Cited by 12 publications

References 28 publications

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

A multi‐parameter imaging assay identifies different stages of ligand‐induced androgen receptor activation

Monitoring Bile Acid Transport in Single Living Cells Using a Genetically Encoded FöRster Resonance Energy Transfer Sensor

Illuminating intracellular signaling and molecules for single cell analysis

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