Related Experiment Video
Updated: May 12, 2026
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
A multi-parameter imaging assay identifies different stages of ligand-induced androgen receptor activation
Martin E van Royen1, Dennis J van de Wijngaart, Sónia M Cunha
1Department of Pathology, Josephine Nefkens Institute, Erasmus MC, 3000 CA Rotterdam, The Netherlands.
Abstract:
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.
Insights
A new FRET imaging assay quantifies androgen receptor (AR) activity by measuring N/C-domain interactions. This validated assay accurately assesses both agonistic and antagonistic AR ligands for potential cancer therapies.
Area of Science:
- Molecular Biology
- Biochemistry
- Oncology
Background:
- Androgens and the androgen receptor (AR) are crucial for male development and prostate cancer progression.
- Current therapies targeting AR signaling often lead to resistance, necessitating novel therapeutic strategies.
- Understanding AR ligand interactions is key to developing more effective treatments.
Purpose of the Study:
- To validate a fluorescence resonance energy transfer (FRET) based imaging assay for quantifying ligand-induced androgen receptor (AR) activity.
- To assess the correlation between AR N/C-domain interaction and transcriptional activity.
- To explore the assay's utility in understanding ligand mechanisms and developing new therapies.
Main Methods:
- Development and validation of a FRET-based imaging assay measuring AR N/C-terminal domain interaction.
- Testing the assay with known agonistic and antagonistic ligands on wild-type AR and AR mutants.
- Correlating FRET signals with AR transcriptional activity and analyzing additional assay readouts.
Main Results:
- The FRET assay demonstrated a strong correlation between ligand-induced AR N/C-interaction and transcriptional activity.
- The assay effectively measured AR activity in both wild-type AR and mutants with altered ligand responsiveness.
- The assay provided insights into the working mechanisms of various AR ligands.
Conclusions:
- A sensitive and quantitative FRET-based imaging assay for AR activity has been successfully validated.
- This assay can be used to assess the efficacy of agonistic and antagonistic AR ligands.
- The validated assay holds promise for the development of novel anti-cancer therapies targeting AR signaling.
