Sumida C, Vallette G, Nunez E A
INSERM U224, Faculté de Médecine Xavier Bichat, Paris, France.
Acta Endocrinol (Copenh). 1993 Oct;129(4):348-55. doi: 10.1530/acta.0.1290348.
Polyunsaturated fatty acids have been shown to decrease the binding of [3H]dexamethasone to rat liver glucocorticoid receptors by mixed non-competitive inhibition, suggesting that these fatty acids interact at a site on the receptor different from the hormone binding site. The present study was undertaken to localize the site of interaction of polyunsaturated fatty acids on the receptor by comparing the differential effects of docosahexaenoic acid (a 22-carbon polyunsaturated fatty acid of the series n-3) on antagonist (RU486) and agonist binding, by covalent cross-linking of the hsp 90 and other proteins to the receptor to attempt to mask the site of interaction, by limited trypsinization to cleave the site and by using antibodies against specific epitopes to prevent fatty acid access by steric hindrance. Binding [3H]RU486 was not inhibited by docosahexaenoic acid at a concentration (60 mumol/l) that increases the dissociation constant of [3H]dexamethasone eightfold. Covalent stabilization of the hetero-oligomeric glucocorticoid receptor structure did not keep the fatty acid from inhibiting [3H]dexamethasone binding. The binding to the receptor of monoclonal and polyclonal antibodies against different domains of the receptor did not sterically hinder the fatty acid interaction with the receptor. After limited trypsinization of the receptor, the fatty acid still increased the dissociation rate constant of [3H]dexamethasone binding, indicating that the site of interaction of polyunsaturated fatty acids is on a fragment of the receptor containing the hormone-binding domain and some sequences C-terminal of the DNA-binding domain.
多不饱和脂肪酸已被证明可通过混合非竞争性抑制作用降低[3H]地塞米松与大鼠肝脏糖皮质激素受体的结合，这表明这些脂肪酸在受体上的作用位点不同于激素结合位点。本研究旨在通过比较二十二碳六烯酸（一种n-3系列的22碳多不饱和脂肪酸）对拮抗剂（RU486）和激动剂结合的不同影响、通过将热休克蛋白90和其他蛋白质与受体共价交联以试图掩盖相互作用位点、通过有限的胰蛋白酶消化来切割该位点以及使用针对特定表位的抗体通过空间位阻来阻止脂肪酸进入等方法，来定位多不饱和脂肪酸在受体上的相互作用位点。在使[3H]地塞米松解离常数增加八倍的浓度（60 μmol/L）下，二十二碳六烯酸并未抑制[3H]RU486的结合。糖皮质激素受体异源寡聚体结构的共价稳定并未阻止脂肪酸抑制[3H]地塞米松的结合。针对受体不同结构域的单克隆和多克隆抗体与受体的结合并未在空间上阻碍脂肪酸与受体的相互作用。对受体进行有限的胰蛋白酶消化后，脂肪酸仍增加了[3H]地塞米松结合的解离速率常数，表明多不饱和脂肪酸的相互作用位点位于受体的一个片段上，该片段包含激素结合结构域以及DNA结合结构域C端的一些序列。
