Dual Roles of 90-kDa Heat Shock Protein in the Function of the Mineralocorticoid Receptor

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Association of the 90-kDa heat shock protein (HSP90) is required for the high-affinity ligand-binding of the glucocorticoid receptor (GR), but not for that of the androgen receptor [Ohara-Nemoto, Y., Nemoto, T., & Ota, M. (1991) J. Biochem. 109, 113-119]. In the present study, we investigated the ligand- and HSP90-binding characteristics of the mineralocorticoid receptor (MR), which shares to some degree the ligand-binding specificity of the GR. A truncated human MR (designated MR351) starting from Gly-351 was translated in vitro with rabbit reticulocyte lysate. Scatchard analysis revealed the presence of a single class of high-affinity binding sites for [³H] aldosterone (K_d=0.35±0.2nM), comparable to those of the native receptor in target tissues. Glycerol gradient centrifugation and immunoadsorption analyses showed that MR351 associated with rabbit HSP90. Exposure to 0.4M NaCl induced the dissociation of HSP90 from MR351 and simultaneously enhanced binding of MR351 to DNA-cellulose. Moreover, when measured at 10 nM, HSP90-free MR351 showed only 9% of the [³H] aldosterone-binding found in the presence of HSP90. On the other hand, when MR351 was expressed in Escherichia coli as a protein tagged with a histidine hexamer at the N-terminus (designated H₆MR351), specific binding of [³H] aldosterone was detected. The binding affinity (K_d=338±45 nM) was, however, 1, 000-fold lower than that of MR351 translated in vitro. Gel retardation analysis revealed that H₆MR351 specifically interacted with the glucocorticoid response element of the rat tyrosine aminotransferase gene, irrespective of aldosterone-binding. These results indicate that HSP90 has dual roles in the function of the MR, as in that of the GR, i.e., suppression of DNA-binding and maintenance of the high-affinity, steroid-binding state.