Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor.
Using L cell glucocorticoid receptors that have been immunopurified by adsorption to protein A Sepharose with a monoclonal antireceptor antibody, we have developed an assay to study the requirements for maintenance of steroid-binding capacity. After rapid purification by immunoadsorption, heteromeric receptor complexes retain the ability to bind glucocorticoid hormone. When the receptor complexes are warmed at 20 degrees C, steroid-binding capacity is lost, and the 90-kDa heat shock protein (hsp90) dissociates from the receptor. The rates of both temperature- and salt-dependent dissociation of hsp90 parallel the rates of loss of hormone-binding activity. Molybdate and hydrogen peroxide stabilize the hsp90-receptor complex against temperature-dependent dissociation. Molybdate, however, is much more effective in stabilizing steroid-binding capacity than peroxide. Receptors that have been inactivated in the absence of molybdate or peroxide cannot be reactivated. Inactivation of steroid-binding capacity occurs in the presence or absence of reducing agent, and inactivation is not accompanied by receptor cleavage or dephosphorylation. Under no conditions does an hsp90-free receptor bind steroid. Receptor bound to hsp90 can be cleaved to the 27-kDa meroreceptor in the presence of molybdate with retention of both hsp90 and steroid-binding activity. These observations lead us to propose that hsp90 is necessary but not sufficient for maintaining a competent high affinity glucocorticoid-binding site. Although the 27-kDa meroreceptor fragment is not itself sufficient for a competent binding site, it is sufficient when it is associated with hsp90.
Bresnick
E H
EH
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Dalman
F C
FC
Sanchez
E R
ER
Pratt
W B
WB
D000818:Animals; D000936:Antigen-Antibody Complex; D006360:Heat-Shock Proteins / Q000502:physiology*; D007163:Immunosorbent Techniques; D007739:L Cells / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation; D011965:Receptors, Glucocorticoid / Q000032:analysis / Q000378:metabolism*; D014222:Triamcinolone Acetonide / Q000378:metabolism
D000936:Antigen-Antibody Complex; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D014222:Triamcinolone Acetonide