|
|
| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
|
MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 585 |
Ligand-selective
targeting of the glucocorticoid receptor to nuclear
subdomains is associated with decreased receptor mobility |
Marcel
J.M. Schaaf, Laura J. Lewis-Tuffin and John A. Cidlowski |
glucocorticoid
receptor, nuclear distribution, steroid hormone, FRAP |
One day after transfection, cells
were transferred to 9.6 cm2 dishes containing glass bottoms (MatTek Corp., 1.5 ·105 cells per
dish).
|
time-lapse
confocal microscopy |
|
|
COS-1 |
| Abstract |
| |
The association between nuclear distribution and mobility
of the human glucocorticoid receptor was examined in living COS-1 cells using YFP- and CFP-tagged
receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands
revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide (TA))
receptor distribution. Structure-function analysis revealed that the 9- fluoro and 17-hydroxy
groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microsopy,
the TA-induced receptor distribution did not change significantly over a period of 15 seconds.
However, using Fluorescence Recovery After Photobleaching (FRAP), the individual receptors moved
at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains.
Receptor mobilities for 13 different steroids, measured by FRAP, appeared to correlate with
receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor
mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences
in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed
slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can
be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient
association of receptor with these domains decreases the mobility of the receptor. |
|
|
|
|
