A Fluorescent Indicator To Visualize Activities of the Androgen Receptor Ligands in Single Living Cells

Awais, Muhammad; Sato, Moritoshi; Lee, Xianfen; Umezawa, Yoshio

doi:10.1002/anie.200503185

Cited by 32 publications

(14 citation statements)

References 32 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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“…The sensor displayed a 40% DR, binding E2 with an EC 50 of 80 nM [76]. Similar FRET sensors based on the LBDs of the androgen receptor [78] and the glucocorticoid receptor [79] have also been successfully constructed.…”

Section: Fret Sensors Based On Ligand-induced Domain-domain Interactionsmentioning

confidence: 99%

Engineering Genetically Encoded FRET Sensors

Lindenburg

Merkx

2014

Sensors

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Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening.

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“…We have developed genetically encoded intracellular fluorescent and bioluminescent optical probes, which include second messengers, such as guanosine 3 0 ,5 0 -cyclic monophosphate (cGMP), 1 inositol 1,4,5-trisphosphate (IP 3 ), 2 lipid second messengers, 3,4 and nitric oxide (NO), 5 protein phosphorylations, [6][7][8] protein-protein interactions, [9][10][11][12][13][14] protein localizations, [15][16][17][18][19] and nuclear receptor ligands, 20,21 for monitoring intracellular signaling. These probes are not only for fundamental biological studies, but also for assay and screening of possible pharmaceutical or toxic chemicals that inhibit or facilitate cellular signaling pathways.…”

Section: Illuminating Molecular Processes In Single Living Cellsmentioning

confidence: 99%

Methods of Analysis for Imaging and Detecting Ions and Molecules

UmezawaYoshio¹

2007

Bulletin of the Chemical Society of Japan

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For imaging molecular processes in living cells, we developed novel intracellular fluorescent and bioluminescent indicators for second messengers, protein phosphorylation, protein/protein interactions, and protein localizations that work in single living cells. Key molecules and steps of cellular signaling pathways were visualized in target live cells under fluorescent microscopy using developed fluorescent indicators. A second new approach to molecular imaging is also described. Chemically modified STM tips (molecular tips) were developed for molecular imaging at the interfaces. The molecule-specific imaging is based on an increase in a tunneling current due to the overlap of electronic wave functions induced by the chemical interactions between tip and sample molecules. Chemically selective imaging using STM molecular tips can be described as intermolecular tunneling microscopy.

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A Fluorescent Indicator To Visualize Activities of the Androgen Receptor Ligands in Single Living Cells

Cited by 32 publications

References 32 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

Engineering Genetically Encoded FRET Sensors

Methods of Analysis for Imaging and Detecting Ions and Molecules

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