Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility
| Autor: | Nancy L. Weigel, Michael A. Mancini, Adam T. Szafran, Jeffrey H. Price, David L. Stenoien, Marco Marcelli, Irina U. Agoulnik, Silvia Simeoni, Timothy J. Moran, I. Mikic |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Agonist
medicine.drug_class Active Transport Cell Nucleus Biology Ligands Biochemistry Green fluorescent protein Tosyl Compounds Nitriles medicine Androgen Receptor Antagonists Humans Anilides Receptor Molecular Biology Cell Nucleus Dose-Response Relationship Drug Fluorescence recovery after photobleaching Androgen Antagonists Cell Biology Transfection Metribolone Molecular biology Protein Structure Tertiary Androgen receptor Nuclear receptor Microscopy Fluorescence Cytoplasm Receptors Androgen Biophysics Androgens HeLa Cells |
| Zdroj: | Journal of cellular biochemistry. 98(4) |
| ISSN: | 0730-2312 |
| Popis: | Using manual and automated high throughput microscopy (HTM), ligand-dependent trafficking of green fluorescent protein-androgen receptor (GFP-AR) was analyzed in fixed and living cells to determine its spatial distribution, solubility, mobility, and co-activator interactions. Within minutes, addition of the agonist R1881 resulted translocation of GFP-AR from the cytoplasm to the nucleus, where it displayed a hyperspeckled pattern and extraction resistance in low expressing cells. AR antagonists (Casodex, hydroxyflutamide) also caused nuclear translocation, however, the antagonist-bound GFP-AR had a more diffuse nuclear distribution, distinct from the agonist-bound GFP-AR, and was completely soluble; overexpressed GFP-AR in treated cells was extraction resistant, independent of ligand type. To more dramatically show the different effects of ligand on AR distribution, we utilized an AR with a mutation in the DNA binding domain (ARC619Y) that forms distinct foci upon exposure to agonists but retains a diffuse nuclear distribution in the presence of antagonists. Live-cell imaging of this mutant demonstrated that cytoplasmic foci formation occurs immediately upon agonist but not antagonist addition. Fluorescence recovery after photobleaching (FRAP) revealed that agonist-bound GFP-AR exhibited reduced mobility relative to unliganded or antagonist-bound GFP-AR. Importantly, agonist-bound GFP-AR mobility was strongly affected by protein expression levels in transiently transfected cells, and displayed reduced mobility even in slightly overexpressing cells. Cyan fluorescent protein-AR (CFP-AR) and yellow fluorescent protein-CREB binding protein (YFP-CBP) in the presence of agonists and antagonists were used to demonstrate that CFP-AR specifically co-localizes with YFP-CBP in an agonist dependent manner. Dual FRAP experiments demonstrated that CBP mobility mirrored AR mobility only in the presence of agonist. HTM enabled simultaneous studies of the sub-cellular distribution of GFP-AR and ARC619Y in response to a range of concentrations of agonists and antagonists (ranging from 10−12 to 10−5) in thousands of cells. These results further support the notion that ligand specific interactions rapidly affect receptor and co-factor organization, solubility, and molecular dynamics, and each can be aberrantly affected by mutation and overexpression. J. Cell. Biochem. 98: 770–788, 2006. © 2006 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text