title: A multi-parameter imaging assay identifies different stages of ligand-induced androgen receptor activation.
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.
authors.affiliation: Department of Pathology, Josephine Nefkens Institute, Erasmus MC, 3000 CA Rotterdam, The Netherlands.
authors.forename: Martin E
authors.initials: ME
authors.lastname: van Royen
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: van de Wijngaart
authors.forename: Sónia M
authors.initials: SM
authors.lastname: Cunha
authors.forename: Jan
authors.initials: J
authors.lastname: Trapman
authors.forename: Adriaan B
authors.initials: AB
authors.lastname: Houtsmuller
mesh_terms: D059002:Androgen Receptor Antagonists / Q000378:metabolism*; D000728:Androgens / Q000378:metabolism*; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008297:Male; D018613:Microscopy, Confocal; D011471:Prostatic Neoplasms / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000378:metabolism*
chemical_list: C410798:AR protein, human; D059002:Androgen Receptor Antagonists; D000728:Androgens; D011944:Receptors, Androgen
keywords: FRET; N/C-interaction; androgen receptor; ligand; quantitative live cell imaging; recurrent prostate cancer