Schulman G, Daniel V, Cooper M, Alnemri E S, Maksymowych A B, Litwack G
Department of Medicine, Temple University School of Medicine, Philadelphia, PA.
Receptor. 1992 Fall;2(3):181-94.
The structures of the unactivated and activated mineralocorticoid receptors have been difficult to characterize because of receptor lability and steroid dissociation. Therefore, human mineralocorticoid receptor mRNA was translated in rabbit reticulocyte lysate in the presence and absence of [35S]methionine to compare the structure of [3H]aldosterone-bound and [35S]labeled receptor. In vitro synthesized receptor was able to specifically bind [3H]aldosterone. Unactivated [3H]aldosterone-bound and 60% of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 250 mM salt and had a Rs of 72A. Forty percent of unactivated [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 54A. SDSPAGE showed intact hMR was present in both DEAE-cellulose eluates as three bands between M(r) 110,000-120,000. However, the low salt eluate contained less intact receptor and more lower MW bands. Neither [3H]aldosterone-bound nor [35S]labeled receptor was activated by incubation at 25 degrees C as readily as glucocorticoid receptor studied under identical conditions. Activated [3H]aldosterone-bound receptor eluted from DEAE-cellulose at 100 mM salt and had a Rs of 37A. After activation, 60% of [35S]labeled receptor eluted from DEAE-cellulose with 100 mM salt and had a Rs of 91A. SDS-PAGE of the high and low salt DEAE-cellulose eluates showed that 50% of intact receptor eluted in the low salt peak after activation. These data indicate that: 1. Some in vitro synthesized mineralocorticoid receptor assembles into the heteromeric unactivated form; 2. The remaining intact receptor remains monomeric and unable to bind steroid; 3. Activation causes dissociation of intact receptor from a larger complex; and 4. Activated receptor tends to aggregate.
由于受体的不稳定性和类固醇的解离，未激活和激活的盐皮质激素受体的结构一直难以表征。因此，在有和没有[35S]甲硫氨酸的情况下，在兔网织红细胞裂解物中翻译人盐皮质激素受体mRNA，以比较[3H]醛固酮结合的和[35S]标记的受体的结构。体外合成的受体能够特异性结合[3H]醛固酮。未激活的[3H]醛固酮结合的受体和60%的未激活的[35S]标记的受体用250 mM盐从DEAE-纤维素上洗脱，Rs为72A。40%的未激活的[35S]标记的受体用100 mM盐从DEAE-纤维素上洗脱，Rs为54A。SDS-PAGE显示完整的hMR存在于两个DEAE-纤维素洗脱物中，为M(r) 110,000-120,000之间的三条带。然而，低盐洗脱物中完整受体较少，低分子量条带较多。在相同条件下研究时，无论是[3H]醛固酮结合的受体还是[35S]标记的受体，在25℃孵育时都不像糖皮质激素受体那样容易被激活。激活的[3H]醛固酮结合的受体用100 mM盐从DEAE-纤维素上洗脱，Rs为37A。激活后，60%的[35S]标记的受体用100 mM盐从DEAE-纤维素上洗脱，Rs为91A。高盐和低盐DEAE-纤维素洗脱物的SDS-PAGE显示，激活后50%的完整受体在低盐峰中洗脱。这些数据表明：1. 一些体外合成的盐皮质激素受体组装成异源未激活形式；2. 其余完整的受体保持单体形式，无法结合类固醇；3. 激活导致完整受体从更大的复合物中解离；4. 激活的受体倾向于聚集。
