Steroid-induced conformational changes at ends of the hormone-binding domain in the rat glucocorticoid receptor are independent of agonist versus antagonist activity
Steroid-induced conformational changes at ends of the hormone-binding domain in the rat glucocorticoid receptor are independent of agonist versus antagonist activity
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DOI：
10.1074/jbc.272.38.23986
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发表时间：
1997-09-19
影响因子：
4.8
通讯作者：
Simons, SS
中科院分区：
文献类型：
--
作者：
Modarress, KJ;Opoku, J;Simons, SS
对于给定的受体-类固醇复合物，激动剂与拮抗剂活性的表达的潜在分子机制仍然未知。一个有吸引力的假设，从孕激素受体的数据的基础上，是激动剂与拮抗剂结合诱导独特的构象在C端的受体，这可以检测到的不同片段产生的部分蛋白水解。我们现在报道，糖皮质激素受体（GR）-拮抗剂复合物活性的决定因素更为复杂。如前所述，类固醇结合确实引起GR中的构象变化，这通过部分胰蛋白酶消化来检测（Simons，S.美国，小的，Sistare，F. D、和Chakraborti，P. K.（1989）J.Biol.Chem.264，14493-14497）。然而，未活化或活化的受体结合的一系列的6个结构不同的拮抗剂，包括亲和标记的抗糖皮质激素地塞米松21-甲磺酸盐的消化模式没有统一的，共观察到四个抗性带的SDS-聚丙烯酰胺凝胶在30-27 kDa的范围内。使用一系列点突变和表位特异性抗体，确定30-kDa种类代表氨基酸518-795的整个C-末端序列，而其他条带来自额外的N-末端和/或C-末端裂解。使用含有各种点和缺失突变的GR的生物测定未能揭示任何可将拮抗剂转化为生物活性激动剂的C-末端改变，因此，GR的C-末端氨基酸的存在或不存在并不能唯一地决定较小的胰蛋白酶抗性片段的外观或受体结合的抗类固醇的生物反应的性质。当与目前的配体结合结构域的模型，这是基于甲状腺和视黄酸受体的可比区域的X-射线结构相比，本研究结果表明，该模型结构以外的序列是相关的GRs的结合和生物活性。
The underlying molecular mechanism for the expression of agonist versus antagonist activity for a given receptor-steroid complex is still not known. One attractive hypothesis, based on data from progesterone receptors, is that agonist versus antagonist binding induces unique conformations at the C terminus of receptors, which can be detected by the different fragments produced by partial proteolysis. We now report that the determinants of glucocorticoid receptor (GR)-antagonist complex activity are more complex, Steroid binding did cause a conformational change in the GR that was detected by partial trypsin digestion, as described previously (Simons, S. S., Jr., Sistare, F. D., and Chakraborti, P. K. (1989) J. Biol. Chem. 264, 14493-14497). However, there was no uniformity in the digestion patterns of unactivated or activated receptors bound by a series of six structurally different antagonists including the affinity labeling antiglucocorticoid dexamethasone 21-mesylate, A total of four resistant bands were observed on SDS-polyacrylamide gels in the range of 30-27 kDa. Using a series of point mutations and epitope-specific antibodies, it was determined that the 30-kDa species represented the entire C-terminal sequence of amino acids 518-795, whereas the other bands arose from additional N-terminal and/or C-terminal cleavages, Bioassays with GRs containing various point and deletion mutations failed to reveal any C-terminal alterations that could convert antagonists into biologically active agonists, Thus, the presence or absence of C-terminal amino acids of the GR did not uniquely determine either the appearance of smaller trypsin-resistant fragments or the nature of the biological response of receptor-bound antisteroids. When compared with the current model of the ligand-binding domain, which is based on the x-ray structures of the comparable region of thyroid and retinoic acid receptors, the present results suggest that sequences outside of the model structure are relevant for the binding and biological activity of GRs.