title: Evidence that 5 S intermediate state in glucocorticoid receptor transformation contains hsp90 in addition to the steroid-binding protein.
abstract: Previous studies have shown that the exposure of molybdate-stabilized nontransformed glucocorticoid receptor (GR) of the chick embryonic neural retina to 0.4 M KCl dissociated the 9.5 S complex to a 5 S GR complex, which is an intermediate state in GR transformation. The present study was designed to characterize the 5 S GR complex. It shows that molybdate-stabilized nontransformed 9.5 S GR complex and 5 S GR interact with monoclonal antibodies (MAb) directed against 90 kDa heat shock protein (hsp90), as evidenced by the increase in the sedimentation velocity of these GR-complexes. Electrofocusing of the partially purified molybdate-stabilized nontransformed GR, prepared from [32P]-labeled neural retinas, and of the 5 S GR (derived from molybdate-stabilized preparation) showed that nontransformed GR complex, which has an apparent pI (pI') value of 5.0 +/- 0.2, and 5 S GR, which was resolved in a major peak with a pI' value of 5.8, are phosphorylated. Partially purified 5 S GR, cleared of molybdate and exposed to 25 degrees C, was resolved by electrofocusing into two phosphorylated fractions, one with a pI' value of 6.5, representing the monomeric GR form and the other with a pI' value of 5.1, apparently representing the acidic hsp90. The dissociation of hsp90 from the molybdate-cleared 5 S heterodimer seems to account for the decrease in the negative net charge of 5 S GR from pI' 6.5. Monomeric GR, derived from a molybdate-cleared, partially purified GR preparation, by the exposure to 25 degrees C, did not retain glucocorticoid-binding activity. Molybdate-stabilized 5 S GR was apparently re-assembled into the oligomeric nontransformed state when the salt concentration was reduced. This phenomenon was evident under the low-salt conditions of electrofocusing, by the shift in pI' value of GR from 5.8 to 5.0; and in glycerol density gradients containing 0.15 M KCl, by the shift in the sedimentation of the GR complex from 5 S to 9.5 S.
authors.affiliation: Department of Physiology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
authors.forename: S
authors.initials: S
authors.lastname: Ben-Or
mesh_terms: D000818:Animals; D002642:Chick Embryo; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008982:Molybdenum / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D013329:Structure-Activity Relationship; D014222:Triamcinolone Acetonide / Q000378:metabolism
chemical_list: D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011965:Receptors, Glucocorticoid; C044659:molybdate; D008982:Molybdenum; D014222:Triamcinolone Acetonide