Elimination and reconstitution of the requirement for hormone in promoting temperature-dependent transformation of cytosolic glucocorticoid receptors to the DNA-binding state.
Cytosols contain a heat-stable, chelatable, anionic, molybdate-like factor that stabilizes glucocorticoid receptors in a heteromeric complex with hsp90 (refers to the 90-kDa heat shock protein) and inhibits their transformation to the DNA-binding state (Meshinchi, S., Grippo, J.F., Sanchez, E.R., Bresnick, E.H., and Pratt, W.B. (1988) J. Biol. Chem. 263, 16809-16817). In this work, we demonstrate that removal of this factor by passage of L cell cytosol through the metal-chelating resin Chelex-100 makes the glucocorticoid receptor unstable, thus markedly facilitating both its dissociation from hsp90 and its transformation to the DNA-binding state. In normal cytosol, both temperature-mediated dissociation of hsp90 and temperature-mediated receptor transformation are hormone-dependent events. In the Chelex-treated, metal-depleted cytosol, however, temperature-mediated dissociation of hsp90 and receptor transformation occur very rapidly in a manner that is no longer hormone-dependent. When boiled L cell cytosol is added to the metal-depleted receptor system, the hormone dependence of both temperature-mediated dissociation of receptor from hsp90 and receptor transformation to the DNA-binding state is reconstituted. Like boiled cytosol, molybdate stabilizes the receptor complex and inhibits its transformation in metal-depleted cytosol, but it does not reconstitute the hormone dependence of the system. These results support the proposal that an endogenous metal anion interacts with the glucocorticoid receptor to stabilize it in the heteromeric, inactive, non-DNA-binding state in cytosol and that binding of the hormone promotes conversion of the receptor to the DNA-binding state through an effect on this metal anion center.
Meshinchi
S
S
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Sanchez
E R
ER
Martell
K J
KJ
Pratt
W B
WB
D000818:Animals; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D051379:Mice; D008982:Molybdenum / Q000494:pharmacology*; D011137:Polystyrenes; D011145:Polyvinyls; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000187:drug effects / Q000378:metabolism*; D013816:Thermodynamics
D002614:Chelating Agents; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D046911:Macromolecular Substances; D011137:Polystyrenes; D011145:Polyvinyls; D011965:Receptors, Glucocorticoid; C044659:molybdate; D003907:Dexamethasone; C006960:chelex; D008982:Molybdenum