Diffusion analysis of glucocorticoid receptor and antagonist effect in living cell nucleus.
The diffusion properties of hGRalpha in living cells have been analyzed. The hGRalpha translocalized from the cytosol to the nucleus after addition of Dex just as RU486; however, the Brownian motions of the proteins in nucleus were different. In order to analysis microenvironment of the nucleus of living cell, four different tandem EGFPs were constructed. Diffusion of tandem EGFP was dependent on the length of the protein as a rod-like molecule in solution. We found two kinds of mobility, fast diffusional mobility as a major component and much slower diffusional mobility as a major component in living cells nucleoplasm. On the bases of this analysis, we compared the diffusion property of hGRalpha in the nucleus at the presence of Dex or RU486 by distribution of diffusion constants. Our result may suggest that EGFP-hGRalpha is activated by RU486 and kept the stage of binding cofactor, GRE and final complex. Finally this means that dimerization is not required for association with GRE, although it is required for stabilization of a complex of EGFP-hGRalpha.
Mikuni
Shintaro
S
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, N12W6, Sapporo 060-0812, Japan.
Pack
Changi
C
Tamura
Mamoru
M
Kinjo
Masataka
M
D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D004058:Diffusion; D019281:Dimerization; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015735:Mifepristone / Q000494:pharmacology*; D008969:Molecular Sequence Data; D021381:Protein Transport / Q000187:drug effects; D011965:Receptors, Glucocorticoid / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D020218:Response Elements
D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C411470:glucocorticoid receptor alpha; D049452:Green Fluorescent Proteins; D015735:Mifepristone; D003907:Dexamethasone