Evidence That the 90-kDa Heat Shock Protein Is Necessary for the Steroid Binding Conformation of the L Cell Glucocorticoid Receptor

作者：Emery H. Bresnick, Friedrich C. Dalman, Edwin R. Sánchez, William B. Pratt · 发表于：Journal of Biological Chemistry · 年份：1989 · DOI：10.1016/s0021-9258(18)83689-4 · 被引用次数：390 · 研究领域：Heat shock proteins research、Estrogen and related hormone effects、Hormonal Regulation and Hypertension

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...