Burnstein K L, Jewell C M, Cidlowski J A
Department of Physiology, Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill 27599-7545.
Mol Endocrinol. 1991 Jul;5(7):1013-22. doi: 10.1210/mend-5-7-1013.
We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of human glucocorticoid receptor (hGR), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated hGR was indistinguishable from native hGR, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against hGR. In addition, cell-free synthesized hGR was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these glucocorticoid receptors. In vitro translated hGR was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized GR was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized hGR was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that hGR synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for glucocorticoid receptor present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.
我们使用了一种DNA结合/免疫沉淀测定法来分析在兔网织红细胞裂解物中产生的人糖皮质激素受体（hGR）与DNA结合的能力。通过在十二烷基硫酸钠-聚丙烯酰胺凝胶上的迁移、在蔗糖密度梯度中的沉降以及与针对hGR产生的抗肽抗体的反应性来确定，体外翻译的hGR与天然hGR没有区别。此外，无细胞合成的hGR能够特异性结合含有糖皮质激素反应元件（GRE）的DNA片段。利用这个测定系统，我们评估了配体结合和热激活对这些糖皮质激素受体与DNA结合的贡献。即使在没有糖皮质激素的情况下，体外翻译的hGR也能够选择性地与DNA结合。当受体制剂保持在0℃（无激活）时，用地塞米松或抗糖皮质激素RU486处理对DNA结合能力没有额外影响。相反，将配体或拮抗剂与热激活步骤相结合，促进DNA结合的能力比热激活的未结合配体的受体提高了约3倍。激动剂（地塞米松）在支持特异性DNA结合方面比拮抗剂（RU486）略有效。在添加类固醇和热激活之前，向受体制剂中添加钼酸钠可阻断体外合成的GR与DNA的结合。添加氯化钾导致DNA结合减少，这要么是由于DNA-受体复合物形成受阻，要么是由于复合物的破坏。通过检查各种DNA竞争结合小鼠乳腺肿瘤病毒-长末端重复序列中天然存在的GRE的能力，进一步分析了无细胞合成的hGR与DNA结合的特异性。含有共有GRE的寡核苷酸是最有效的竞争者，含有糖皮质激素可抑制基因调控序列的片段有一定的竞争性，而单链寡核苷酸除了在极高浓度的竞争者存在时，无法竞争与小鼠乳腺肿瘤病毒-长末端重复序列DNA的结合。这些研究共同表明，在兔网织红细胞裂解物中合成的hGR表现出许多与在哺乳动物细胞和组织的胞质提取物中存在的糖皮质激素受体相同的特性，包括GRE特异性DNA结合。地塞米松和RU486作用的相似性表明，RU486的抗糖皮质激素特性并非发生在特异性DNA结合水平。
