Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor - PubMed (original) (raw)
. 1989 Mar 25;264(9):4992-7.
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- PMID: 2647745
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Evidence that the 90-kDa heat shock protein is necessary for the steroid binding conformation of the L cell glucocorticoid receptor
E H Bresnick et al. J Biol Chem. 1989.
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Abstract
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.