The purified activated glucocorticoid receptor is a homodimer.
The structure of purified preparations of activated (DNA-binding) glucocorticoid receptor (GR) was analyzed in the presence or absence of DNA. A 35-base pair DNA fragment harboring a strong GR-binding site from the mouse mammary tumor virus promoter (-189/-166) was used for stoichiometric analysis of the GR.DNA complex. Glycerol gradient centrifugation was utilized in order to separate the 6 S GR.DNA complex from the 4 S GR and the 3 S DNA fragment. Synthetic glucocorticoid [3H]triamcinolone acetonide bound to GR and 32P-5'-end-labeled DNA fragment were used as probes for quantitation of each component. Such experiments demonstrated that two hormone molecules (two 87.5-kDa GR peptides) are associated with each cognate DNA site. Quantitative DNase I footprinting confirmed this result. The formation of the GR.DNA complex was ligand-dependent, but once formed the complex remained stable after ligand dissociation. Incubation of GR with 0.01-0.1% (w/v) glutaraldehyde resulted in a shift in its sedimentation rate from 4 to 6 S. Gel filtration chromatography of glutaraldehyde-treated GR resulted in a complex of slightly larger size than the gamma-globulin standard (158 kDa). Gel filtration of GR without glutaraldehyde treatment gave the identical result. This suggests that a GR multimer, probably a homodimer, is stable during gel filtration chromatography but needs to be stabilized by glutaraldehyde cross-linking or DNA during glycerol gradient centrifugation. We conclude that the activated GR exists as a homodimer when unbound as well as when bound to DNA.
Wrange
O
O
Department of Molecular Genetics, Karolinska Institutet, Stockholm, Sweden.
Eriksson
P
P
Perlmann
T
T
D000818:Animals; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I; D005976:Glutaral; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D011869:Radioligand Assay; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000302:isolation & purification* / Q000378:metabolism; D014222:Triamcinolone Acetonide
D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D011965:Receptors, Glucocorticoid; D004247:DNA; D003850:Deoxyribonuclease I; D014222:Triamcinolone Acetonide; D005976:Glutaral