Dual roles of 90-kDa heat shock protein in the function of the mineralocorticoid receptor.
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 (Kd = 0.35 +/- 0.2 nM), 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 0.4 M 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 (Kd = 338 +/- 45 nM) was, however, 1,000-fold lower than that of MR351 translated in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Nemoto
T
T
Department of Biochemistry, Iwate Medical University School of Dentistry.
Ohara-Nemoto
Y
Y
Sato
N
N
Ota
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008901:Mineralocorticoids / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014176:Protein Biosynthesis; D011817:Rabbits; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D006360:Heat-Shock Proteins; D008901:Mineralocorticoids; D018161:Receptors, Mineralocorticoid; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D004247:DNA