Photoaffinity labeling identification of thyroid hormone-regulated glucocorticoid-binding peptides in rat liver endoplasmic reticulum: an oligomeric protein with high affinity for 16beta-hydroxylated stanozolol.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic actions of the17alpha-alkylated testosterone derivative (17alpha-AA) stanozolol (ST). We have previously reported that male rat liver endoplasmic reticulum contains two steroid-binding sites associated with high molecular mass oligomeric proteins: (1) the ST-binding protein (STBP); and (2) the low-affinity glucocorticoid-binding protein (LAGS). To further explore the role of LAGS on the mechanism of action of ST, we have now studied: (1) the interaction of ST and its hydroxylated metabolites with solubilized LAGS and the cytosolic glucocorticoid receptor (GR); and (2) the effects of hormones on the capability of STBP to bind ST. We found that, unlike 17alpha-methyltestosterone, neither ST nor its hydroxylated metabolites bind to GR. However, the 16beta-hydroxylation of ST significantly increases the capability of LAGS to bind ST. Interestingly, 3'-hydroxylation of ST abrogates the capability of LAGS to bind ST. ST (k(i)=30 nM) and 16beta-hydroxystanozolol (k(i)=13 nM) bind with high affinity to LAGS, and are capable of accelerating the rate of dissociation of previously bound dexamethasone from the LAGS. STBP and LAGS are strongly induced by ethinylestradiol. However, unlike STBP, LAGS is regulated by thyroid hormones and growth hormone, which proves that these steroid-binding activities are associated with different binding sites. These findings seem to suggest a novel mechanism for ST whereby membrane-associated glucocorticoid-binding activity is targeted by the 16beta-hydroxylated metabolite of ST. ST and its 16beta-hydroxylated metabolite modulate glucocorticoid activity in the liver through negative allosteric modulation of LAGS, with the result of this interaction an effective increase in classical GR-signaling by increasing glucocorticoid availability to the cytosolic GR.

Solubilization and photoaffinity labeling identification of glucocorticoid binding peptides in endoplasmic reticulum from rat liver.

Steroid-binding proteins unrelated to the classical nuclear receptors have been proposed to play a role in non-genomic effects of steroid hormones. We have previously described that the low-affinity glucocorticoid binding protein (LAGS), present in the endoplasmic reticulum of the male rat liver, has pharmacological and biochemical properties different from those of nuclear receptors. The LAGS is under multihormonal regulation and binds glucocorticoids, progestins, and synthetic steroids but is unable to bind either estradiol, testosterone, or triamcinolone acetonide. In this study, we have solubilized the LAGS and investigated their pharmacological and hydrodynamic properties and their peptide composition. We found that LAGS is an integral protein bound to the endoplasmic reticulum. CHAPS provided its optimal solubilization without changes in its pharmacological properties. Hydrodynamic properties of LAGS showed that it has a molecular mass of at least 135 kDa. SDS-PAGE of covalently-labeled LAGS showed that [3H]dexamethasone binds two peptides of 53 and 37 kDa, respectively. Thus, the LAGS appears as an oligomeric protein under multihormonal regulation. The availability of solubilized LAGS and the fact that it can be induced in vivo represent major steps toward purification and understanding the functional significance of this unique steroid-binding protein.