J. Biochem, 1990, Vol. 108, No. 2 271-277
© 1990 Japanese Biochemical Society
research-article |
Phosphorylated and Dephosphorylated Types of Non-Activated Glucocorticoid Receptor
*Department of Biochemistry, School of Medicine, Akita Universil Akito Akita 010
**Second Department of Internal Medicine, School of Medicine, Tokyo Medical and Dental Univernty Bunkyo-ku, Tokyo 113
We purified glucocorticoid receptors quickly but very partially using DEAE-resln. [3H]-Triamcinolone acetonide-labeled and non-activated receptors in the quickly purified fraction were found to be separated into two fractions (P-2 and P-3) by hydroxyápatite column chromatography. The P-2 receptor was the main component, and the ratio of P-2/P-3 was around 2. The molecular weights of the two receptors were calculated to be the same, 242,000: R3 = 6.2nm and 820.W=9.0. Treatment of the receptor with catalytic subunits of phosphoprotein phosphatase 2A1 reduced the P-2/P-3 ratio from 2 to 0.5, while treatment with catalytic subunits of cAMP-dependent protein kinase and ATP incresed it to 2.5. The isolated P-3 receptor could be converted Into the P-2 type by the kinase treatment. Tungstate, a phosphatase inhibitor, stabilized the P-2 receptor, and the P-2/P-3 ratio was larger than 3 when the DEAE-fraction was prepared in the presence of tungstate. However, the tungstate effect was not very strong, and the P-2 type tended to change into the P-3. [3H]-Triamcinolone acetonide-labeled and non-activated receptors were purified very highly by using an affinity gel; the precedure required more than 10 h. Only the P-3 form was observed in the preparation of highly purified receptors. Hormone-free receptors were affected by neither the phosphatase nor the kinase. The results indicate that the hormone binding makes the receptor sensitive to phosphatase. The reversibly dephosphorylated receptor is more stable than the non-dephosphorylated one, and can be activated.