Stability of the androgen receptor from rat submandibular gland under acidic conditions

Abstract

The[³H]miborelone ([³H]MI) -binding ability of the androgen receptor (AR) from rat submandibular gland has been studied under acidic conditions. Both in the presence and absence of 10 mM molybdate, the[³H]MI-binding ability was decreased as the pH of the cytosol decreased with the addition of acetic acid and HCl, with pH 5.9 giving 50% binding. This loss of the binding ability was recovered when the acidic precipitate obtained by centrifugation was dissolved in a buffer at pH 7.3. In the presence of molybdate, this recovery of the binding clearly correlated with the protein content of the precipitate. In the absence of molybdate, however, the maximum binding recovered in the precipitate was achieved at pH 4.9, and rapidly decreased at lower pH values (1/2 value obtain-ed at pH 4.1), even though the protein content in the precipitate was still rather high. When the cytosol was first labeled with[³H]MI and then the pH was decreased in the absence of molybdate, [³H]MI-AR complexes remained unchanged until reaching pH 5 and the amount was then decreased, with pH 4.2 giving 50% binding. These results indicate that both unoccupied and liganded AR denatures irreversibly at pH 4.1-4.2. On the other hand, in the presence of molybdate, [³H] MI-AR complexes were more resistent under acidic conditions and a considerable amount of the complexes could still be observed until pH 3. In addition, under acidic conditions in the presence of 0.5 M KCl, AR was transformed to a state which could bind to DNA-cellulose. In conclusion, AR from rat submandibular gland exists in a specific state between pH 4.1-5.9; AR at this state has no ligand-binding ability, but once formed, [³H]MI-AR complexes do not dissociate. At lower pH values, AR is transformed, and subsequently denatures. Moreover, similar results were obtained from AR from rat prostate, glucocorticoid receptor from rat liver, and estrogen receptor from rat uterus, indicating that these are the common characteristics among these receptors.