Subunit dissociation and activation of wild-type and mutant glucocorticoid receptors

Tetrameric structure of the nonactivated glucocorticoid receptor in cell extracts and intact cells

“…In order to confirm the above data we carried out similar experiments with the nt i receptor which has a glucocorticoid-binding polypeptide of about half the wild-type size [15] and a high molecular mass form of about 290 kDa [5][6][7]. This receptor form was separated from nonspecifically labeled material by gel filtration in the presence of molybdate [5].…”

“…Cells were grown and harvested as described [5] and cell pellets were stored frozen. Extracts were prepared from frozen cells [6] and were incubated with 100 nM [3Hldexamethasone mesylate (NEN Research Products; 1.8 TBq/mmol) for 2.5 h at 0°C.…”

“…Extracts were prepared from frozen cells [6] and were incubated with 100 nM [3Hldexamethasone mesylate (NEN Research Products; 1.8 TBq/mmol) for 2.5 h at 0°C. Extracts of wild-type cells were then used for cross-linking while those of nt i cells were first submitted to gel filtration on Sephacryl S-300 in a buffer containing 20 mM sodium molybdate and 150 mM KC1 [5]. Peak fractions corresponding to 70 A Stokes radius were combined and submitted to cross-30- linking.…”

“…During recent years it became clear that high molecular mass glucocorticoid receptors are unable to interact with DNA or chromatin but become activated to a DNA-binding state upon warming or exposure to high ionic strength: this process involves subunit dissociation [3][4][5][6]. The large receptor form of Mr -330000 has recently been shown by chemical cross-linking to exist not only in cell extracts but also in intact cells [6].…”

“…It contains only one hormone-binding potypeptide of Mr -I00000 per complex [7,8]. We now know that the heat shock protein hsp90 is a constituent of the heteromeric gtucocorticoid receptor structure [4,6,9,10]: it may be present as Abbreviations: DMS, dimethyl suberimidate; nt i, receptor of 'increased nuclear transfer'; PAGE, polyacrylamide gel electrophoresis a dimer [5,11,12]. The involvement of other molecular components is less clear.…”