Wilson E M, Lubahn D B, French F S, Jewell C M, Cidlowski J A
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
Mol Endocrinol. 1988 Nov;2(11):1018-26. doi: 10.1210/mend-2-11-1018.
Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.
利用针对单条合成肽或一组肽混合物产生的高效价多克隆抗体，研究了人糖皮质激素受体DNA结合域的功能特性。这些肽的序列源自类固醇受体蛋白的DNA结合域。七种抗血清中有三种能识别糖皮质激素受体的天然形式和变性形式，不过与天然受体结合所需的抗血清稀释度要低得多。抗体识别天然受体需要将糖皮质激素受体激活为其DNA结合形式。针对DNA结合域第二指状区的抗血清，使一部分活化的4S糖皮质激素受体在含有0.4M氯化钾的蔗糖梯度中沉降为7S或9S，但未改变未转化的8S受体的沉降情况。用肽抗原预吸附后反应性丧失，证明了糖皮质激素受体与抗体相互作用的特异性。与糖皮质激素受体特异性相互作用的抗血清可使活化受体的DNA结合能力降低多达80%。因此，针对DNA结合域序列的抗体探针提供了免疫学证据，表明糖皮质激素受体激活会暴露出该受体的DNA结合区域。
