Cavanaugh A H, Simons S S
Steroid Hormones Section, Laboratory of Molecular and Cellular Biology, NIDDK/NIH, Bethesda, MD 20892.
J Steroid Biochem Mol Biol. 1994 Apr;48(5-6):433-46. doi: 10.1016/0960-0760(94)90191-0.
We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700-3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.
我们之前报道过，来自大鼠HTC细胞胞质溶胶的活化糖皮质激素受体-类固醇复合物至少以两种亚群形式存在，其中一种亚群需要一种低分子量（700 - 3000道尔顿）的因子才能与DNA结合。这种因子可通过Sephadex G - 50柱层析去除，且主要存在于HTC细胞粗核提取物中。我们现已确定该因子并不局限于HTC细胞，因为在大鼠肾脏和肝脏以及人HeLa和MCF - 7细胞的核提取物中发现了一种明显相同的因子。此外，人糖皮质激素受体亚群的DNA结合依赖于该因子。虽然这些结果是在与激动剂（地塞米松）结合的受体上获得的，但被抗糖皮质激素甲磺酸地塞米松21 - 甲磺酸盐共价标记的HTC细胞受体亚群也表现出因子依赖性DNA结合。这种受体异质性并非无细胞激活的假象，因为甲磺酸地塞米松标记复合物的无细胞核结合，与完整细胞一样，少于地塞米松结合复合物。早期结果表明，因子参与导致的DNA结合增加涉及受体与因子的直接相互作用。我们现在发现，HTC细胞氨基末端截短的（42 kDa）糖皮质激素受体保留了因子诱导的DNA结合能力。因此，受体与因子相互作用的能力由DNA和/或类固醇结合结构域编码。甲磺酸地塞米松标记的98 kDa受体的二维凝胶电泳分析显示，两个亚群均有多个带电荷的异构体，但每个亚群中各种异构体的量没有差异。最后，还发现活化的孕酮和雌激素受体复合物也是异质性的，一个亚群的DNA结合需要一种类似（如果不是相同）的低分子量因子。受体功能异质性并非糖皮质激素受体所特有，无论其与激动剂还是拮抗剂结合，且该因子既无物种特异性也无组织特异性，这些观察结果表明，因子辅助的DNA结合可能是所有类固醇受体的普遍机制。
