Analysis of intranuclear binding process of glucocorticoid receptor using fluorescence correlation spectroscopy.
The diffusion properties of EGFP-hGRalpha and mutants C421G, A458T and I566 in living cells were analyzed. The wild type and mutants C421G and A458T translocated from the cytoplasm to the nucleus after addition of Dex; however, the Brownian motions of the proteins were different. The diffusion constant of wild-type GRalpha after addition of Dex slowed to 15.6% of that in the absence of Dex, whereas those of A458T and C421G slowed to 34.8% and 61.7%, respectively. This is the first report that dimer formation is less important than the binding activity of GRalpha to GRE in the living cell.
Mikuni
Shintaro
S
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, N12W6, Kita-ku, Sapporo 060-0812, Japan.
Tamura
Mamoru
M
Kinjo
Masataka
M
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D019943:Amino Acid Substitution; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D015735:Mifepristone / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry; D014162:Transfection
D017931:DNA Primers; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C411470:glucocorticoid receptor alpha; D049452:Green Fluorescent Proteins; D015735:Mifepristone; D003907:Dexamethasone