The androgen receptor ligand-binding domain stabilizes DNA binding in living cells.
The androgen receptor (AR) is a member of the steroid receptor family, a group of transcription factors that activate steroid-regulated genes. Live cell studies of several steroid receptors have shown that the mobility of the liganded receptor is strongly reduced compared to the unliganded receptor. To investigate the nature of this reduced mobility, we generated Hep3B cells stably expressing green fluorescent protein (GFP)-AR at physiological levels. Computer-aided analysis of photobleaching experiments showed that in the presence of ligand on average one out of five ARs is immobilized, each individual AR being immobile for 1-2 min. This immobilization depended on DNA binding since GFP-ARs mutated in the DNA-binding domain were not immobilized. Interestingly, a truncated AR lacking the ligand-binding domain (LBD) displayed substantially shorter immobilizations, in the order of seconds, although its transcriptional activation function was stronger. Our data suggest the LBD has a role in maintaining the stability of AR-DNA complexes.
Farla
Pascal
P
Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.
Hersmus
Remko
R
Geverts
Bart
B
Mari
Pierre O
PO
Nigg
Alex L
AL
Dubbink
Hendrikus J
HJ
Trapman
Jan
J
Houtsmuller
Adriaan B
AB
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002460:Cell Line; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D015741:Metribolone / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011944:Receptors, Androgen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D008024:Ligands; D046911:Macromolecular Substances; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D015741:Metribolone; D004247:DNA