Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera.
A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the rat glucocorticoid receptor (GR). When GFP-GR is expressed in living mouse cells, it is competent for normal transactivation of the GR-responsive mouse mammary tumor virus promoter. The unliganded GFP-GR resides in the cytoplasm and translocates to the nucleus in a hormone-dependent manner with ligand specificity similar to that of the native GR receptor. Due to the resistance of the mutant GFP to photobleaching, the translocation process can be studied by time-lapse video microscopy. Confocal laser scanning microscopy showed nuclear accumulation in a discrete series of foci, excluding nucleoli. Complete receptor translocation is induced with RU486 (a ligand with little agonist activity), although concentration into nuclear foci is not observed. This reproducible pattern of transactivation-competent GR reveals a previously undescribed intranuclear architecture of GR target sites.
Htun
H
H
Laboratory of Molecular Virology, National Cancer Institute, Bethesda, MD 20892-5055, USA.
Barsony
J
J
Renyi
I
I
Gould
D L
DL
Hager
G L
GL
D000818:Animals; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004734:Energy Metabolism; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007424:Intracellular Fluid / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D008024:Ligands; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium