Structure of the mouse glucocorticoid receptor: rapid analysis by size-exclusion high-performance liquid chromatography

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Abstract
Gel-exclusion high-performance liquid chromatography (HPLC) has been used to separate the untransformed from the transformed glucocorticoid receptor (GC-R) extracted from mouse AtT-20 cells. With 200 mM potassium phosphate as the eluent, an efficient separation of the forms of the GC-R is attained in 15-20 min. The untransformed cytosolic GC-R elutes from the column with a Stokes radius (Rs) of 8.2-8.6 nm, as do the molybdate-stabilized GC-R, the purified untransformed GC-R, and the cross-linked cytosolic GC-R. GC-R transformed in vitro by either ammonium sulfate precipitation, KCl treatment, or G-25 chromatography elutes with an Rs of 5.7-6 nm. Also GC-R extracted from the nucleus with either 0.3 M KCl or 2 mM sodium tungstate, or purified by two cycles of DNA-cellulose chromatography, has an Rs of 5.5-6.3 nm. The data are identical either in the presence or in the absence of 20 mM Na2MoO4, suggesting that molybdate is not causing aggregation to produce a larger Rs value than that of the native receptor. Vertical tube rotor sucrose gradient ultracentrifugation of cytosol produces three forms of the GC-R: 9.1 S, 5.2 S, and 3.8 S. Sequential analysis of the GC-R forms by HPLC and vertical tube rotor ultracentrifugation and vice versa allows for the hydrodynamic determination of molecular weight within a very short time period (2-3 h total). The untransformed 9.1S species of GC-R pooled from MoO42--containing sucrose gradients has an Rs of 8.6 nm (Mr 310K-340K) while both the 5.2S and 3.8S forms have an Rs of 6 nm (Mr 115K-140K and 96K-100K, respectively). Conversely, if the 6-nm form of the receptor, generated either by KCl treatment or by G-25 chromatography, is pooled from HPLC, then it subsequently sediments on MoO42--containing gradients as a 3.5-4.6S species. This probably indicates that the 5.2S transformed GC-R is unstable and dissociates into its constituents during HPLC, despite the rapidity of the process. From the calculated molecular weight for each of the three GC-R forms, it is suggested that the untransformed oligomeric GC-R dissociates into monomeric subunits during transformation and that the intermediate form is not a dimer of identical monomeric subunits.

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