J. Biochem, 1988, Vol. 103, No. 6 920-927
© 1988 Japanese Biochemical Society
research-article |
Stability and Transformation of the Glucocorticoid Receptor under Acidic Conditions1
Department of Biochemistry, Iwate Medical University School of Dentistry Morioka, Iwate 020
The [3H]triamcinolone acetonide ([3H]TA)-binding ability of the rat liver glucocorticoid receptor (GR) was investigated under acidic conditions, ranging from pH 2 to 7.3. Both in the presence and absence of 10 mM molybdate, the [3H] TA-binding ability decreased below pH 6.5 and was almost completely lost below pH 5, pH 5.9±0.1 giving 50% [3H] TA-binding. The binding ability was recovered when the pH of the cytosol was reversed to 7.3 or the precipitate obtained on acidification was dissolved in a buffer of pH 7.3. Moreover, in the absence of molybdate, the [3H]TA-GR complexes formed at pH 7.3 remained unchanged until pH 5. Then they decreased, pH 3.9±0.1 giving 50% binding, and completely disappeared at pH 3. [3H]TA-binding activity recovered from the precipitate also decreased in a similar pH region (a 50% decrease in binding being observed at pH 4.2±0.04). These results suggest that rat liver GR is rather resistant under acidic conditions and that it exists in a peculiar state below pH 5.9 to 
4 as to its ligand binding property: unoccupied GR has no [3H]TA-binding ability but [3H]TA-GR complexes once formed at neutral pH do not dissociate. [3H]TA-GR complexes recovered from the precipitate at pH 5 had a Stokes radius of 7.5 nm, little DNA-cellulose-binding ability and sedimented at 8.6S on glycerol gradient centrifugation, indicating that the receptor existed in a nontransformed state. In addition, both occupied and unoccupied GR were transformed at about pH 4, their being 50% transformation. This transformation was accompanied by irreversible denaturation of the receptor. Molybdate prevented the acid-induced transformation and denaturation of both occupied and unoccupied GR. These results suggest that two kinds of dissociable groups, with pK=5.9 and 4, respectively, are involved in the conversion between GR states. The former is related to the binding of [3H]TA, and the latter to the transformation and denaturation of GR. The most probable candidate for the dissociable group with pK = 5.9 is a histidyl residue at or near the steroid binding site, and for those with pK= 4 are acidic amino acid residues on a nonhormone-binding component of the nontransformed GR, i.e., heat shock protein 90.
1This work was supported by Grants-in-Aid for Scientific Research (Nos. 61480392, 61790216, and 62771486) from the Ministry of Education, Science and Culture of Japan, and grants to T.N. from the Keiryokai Research Foundation and to M.O. from the Science Research Promotion Fund of the Japan Private School Promotion Foundation.
2Supported by the Japan Society for the Promotion of Science for Japanese Junior Scientists.