Quantifying effects of ligands on androgen receptor nuclear translocation, intranuclear dynamics, and solubility.
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.
Marcelli
Marco
M
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Stenoien
David L
DL
Szafran
Adam T
AT
Simeoni
Silvia
S
Agoulnik
Irina U
IU
Weigel
Nancy L
NL
Moran
Tim
T
Mikic
Ivana
I
Price
Jeffrey H
JH
Mancini
Michael A
MA
D021581:Active Transport, Cell Nucleus / Q000187:drug effects / Q000502:physiology; D000726:Androgen Antagonists / Q000494:pharmacology*; D059002:Androgen Receptor Antagonists; D000728:Androgens; D000813:Anilides / Q000494:pharmacology*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D015741:Metribolone / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009570:Nitriles; D017434:Protein Structure, Tertiary / Q000235:genetics; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism; D014105:Tosyl Compounds
C410798:AR protein, human; D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D000728:Androgens; D000813:Anilides; D008024:Ligands; D009570:Nitriles; D011944:Receptors, Androgen; D014105:Tosyl Compounds; D015741:Metribolone; C053541:bicalutamide