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/ange.200503185

Cited by 11 publications

(14 citation statements)

References 31 publications

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“…To further seek for the mechanism underlying lack of genomic action by ginsenoside Re, we examined whether ginsenoside Re triggered binding of a coactivator peptide containing a canonical LXXLL-motif (L ϭ leucine, X ϭ any amino acid) to the LBD of ER␣, AR, and PR. We used a FRET indicator, SCCoR, in which agonist-induced recruitment of coactivator to the LBD of receptors was designed to induce FRET signals between enhanced CFP and enhanced YFP (Awais et al, 2004(Awais et al, , 2006. We first confirmed that ginsenoside Re did not change FRET signals for AR-SCCoR, ER␣-SCCoR, or PR-SCCoR (Fig.…”

Section: Downloaded Frommentioning

confidence: 85%

“…Recruitment of coactivator upon agonist binding to ER␣ receptor was assayed using a FRET indicator, SCCoR (single cell coactivator recruitment), as described previously (Awais et al, 2004(Awais et al, , 2006. In brief, an intramolecular FRET-based indicator was constructed to visualize the ligand-dependent recruitment of a coactivator peptide containing a LXXLL motif to the ER␣-LBD connected via a short flexible linker.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, an antagonist inhibits receptor/coactivator interaction. To construct AR-SCCoR and PR-SCCoR, the LBD of ER in the ERSCCoR was replaced with the LBD of AR and PR, respectively (Awais et al, 2004(Awais et al, , 2006. CHO-K1 cells (ATCC) were transfected with indicators for AR-SCCoR, ER␣-SCCoR, or PR-SCCoR in the presence of LipofectAMINE 2000 reagent (Invitrogen) in glass-bottomed dishes.…”

Section: Methodsmentioning

confidence: 99%

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Ginsenoside Re, a Main Phytosterol of Panax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones

Furukawa¹,

Bai²,

Kaihara³

et al. 2006

Molecular Pharmacology

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Ginseng root is one of the most popular herbs throughout the world and is believed to be a panacea and to promote longevity. It has been used as a medicine to protect against cardiac ischemia, a major cause of death in the West. We have previously demonstrated that ginsenoside Re, a main phytosterol of Panax ginseng, inhibits Ca 2ϩ accumulation in mitochondria during cardiac ischemia/reperfusion, which is attributable to nitric oxide (NO)-induced Ca 2ϩ channel inhibition and K ϩ channel activation in cardiac myocytes. In this study, we provide compelling evidence that ginsenoside Re activates endothelial NO synthase (eNOS) to release NO, resulting in activation of the slowly activating delayed rectifier K ϩ current. The eNOS activation occurs via a nongenomic pathway of each of androgen receptor, estrogen receptor-␣, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. However, ginsenoside Re does not stimulate proliferation of androgen-responsive LNCaP cells and estrogen-responsive MCF-7 cells, implying that ginsenoside Re does not activate a genomic pathway of sex hormone receptors. Fluorescence resonance energy transfer experiments with a probe, SCCoR (single cell coactivator recruitment), indicate that the lack of genomic action is attributable to failure of coactivator recruitment. Thus, ginsenoside Re acts as a specific agonist for the nongenomic pathway of sex steroid receptors, and NO released from activated eNOS underlies cardiac K ϩ channel activation and protection against ischemia-reperfusion injury.

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confidence: 85%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Ginsenoside Re, a Main Phytosterol of Panax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones

Furukawa¹,

Bai²,

Kaihara³

et al. 2006

Molecular Pharmacology

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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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“…The resultant protein was inserted between cyan and yellow fluorescent proteins (CFP for the donor, and YFP for the acceptor fluorophore) in such a way that the excitation and emission spectra of CFP and YFP are suitable for fluorescence resonance energy transfer (FRET) from CFP to YFP. [19][20][21] This fusion protein (conpro) functions as a fluorescent indicator in an intramolecular FRET fashion, to visualize the ligand-induced conformational changes in the progesterone receptor that are necessary for the NR to interact with the coactivator peptide in live cells. [19][20][21] Another indicator in which a corepressor peptide is used in place of a coactivator peptide was called conpro1.…”

Section: Introductionmentioning

confidence: 99%

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

2007

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Selective nuclear receptor modulators (SNRMs), which are used clinically for the treatment of NR-related diseases, display mixed agonistic/antagonistic activity in a tissue-selective manner depending on the cellular concentrations of coregulator proteins, that is, coactivators and corepressors. The molecular details of the SNRM function provided us with an idea for a rational method for the high-throughput screening of SNRMs in real time in intact living cells. We have developed genetically encoded fluorescent indicators based on the principle of ligand-induced coactivator and/or corepressor recruitment to NR ligand binding domain in single living cells. We demonstrated that an SNRM induces a distinct conformational change in the NR LBD, which is different from that induced by a full agonist or antagonist, but favorable for the recruitment of a coactivator or corepressor protein to the NR. The molecular details of an SNRM binding to a NR, and the subsequently induced conformational changes and recruitment of coregulator protein(s) are important features for the understanding of SNRM action in the living body. Our fluorescent indicators are capable of distinguishing among agonists, antagonists, and SNRMs, and can therefore serve as versatile molecular sensors that predict the pharmacological character of ligands, which is important for an accurate cure of a disease.

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

Cited by 11 publications

References 31 publications

Ginsenoside Re, a Main Phytosterol of Panax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones

Ginsenoside Re, a Main Phytosterol of Panax ginseng, Activates Cardiac Potassium Channels via a Nongenomic Pathway of Sex Hormones

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

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

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