Kraichely D M, Sun J, Katzenellenbogen J A, Katzenellenbogen B S
Department of Molecular and Integrative Physiology, University of Illinois and College of Medicine, Urbana 61801, USA.
Endocrinology. 2000 Oct;141(10):3534-45. doi: 10.1210/endo.141.10.7698.
Ligands for the estrogen receptor (ER) that have the capacity to selectively bind to or activate the ER subtypes ERalpha or ERbeta would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. In this study, we examine three compounds of novel structure that act as ER subtype-selective ligands. These are a propyl pyrazole triol (PPT), which is a potent agonist on ERalpha but is inactive on ERbeta, and a pair of substituted tetrahydrochrysenes (THC), one enantiomer of which (S,S-THC) is an agonist on both ERalpha and ERbeta, the other (R,R-THC) being an agonist on ERalpha but an antagonist on ERbeta. To investigate the molecular mechanisms underlying the ER subtype-selective actions of these compounds, we have determined the conformational changes induced in ERalpha and ERbeta by these ligands using protease digestion sensitivity, and we have tested the ability of these ligands to promote the recruitment of representatives of the three SRC/p160 coactivator protein family members (SRC-1, GRIP-1, ACTR, respectively) to ERalpha and ERbeta using yeast two-hybrid and glutathione-S-transferase (GST) pull-down assays. We find that the ligand-ER protease digestion pattern is distinctly different for stimulatory and inhibitory ligands, and that this assay, as well as coactivator recruitment, are excellent indicators of their agonist/antagonist character. Interestingly however, compared with estradiol, the novel agonist ligands show some quantitative differences in their ability to recruit SRC-1, -2, and -3. This implies that while generally similar to estradiol, these ligands induce ER conformations that differ somewhat from that induced by estradiol, differences that are illustrative of the nature of their biological character. The application of methods to characterize the conformations induced in ER subtypes by novel ligands, as done in this study, enables a greater understanding of how ligand-receptor conformations relate to estrogen agonist or antagonist behavior.
能够选择性结合或激活雌激素受体（ER）亚型ERα或ERβ的配体，对于阐明这两种受体的生物学特性将是有用的，并且可能有助于开发具有改善的组织选择性的雌激素药物。在本研究中，我们检测了三种具有新型结构的化合物，它们作为ER亚型选择性配体。这些化合物包括一种丙基吡唑三醇（PPT），它是ERα的强效激动剂，但对ERβ无活性；还有一对取代的四氢 Chrysene（THC），其中一种对映体（S,S-THC）是ERα和ERβ的激动剂，另一种（R,R-THC）是ERα的激动剂但却是ERβ的拮抗剂。为了研究这些化合物的ER亚型选择性作用背后的分子机制，我们使用蛋白酶消化敏感性测定了这些配体在ERα和ERβ中诱导的构象变化，并且我们使用酵母双杂交和谷胱甘肽-S-转移酶（GST）下拉试验测试了这些配体促进三种SRC/p160共激活蛋白家族成员（分别为SRC-1、GRIP-1、ACTR）与ERα和ERβ结合的能力。我们发现，刺激型和抑制型配体的配体-ER蛋白酶消化模式明显不同，并且这种测定以及共激活剂的募集是它们激动剂/拮抗剂特性的良好指标。然而，有趣的是，与雌二醇相比，新型激动剂配体在募集SRC-1、-2和-3的能力上表现出一些定量差异。这意味着虽然这些配体通常与雌二醇相似，但它们诱导的ER构象与雌二醇诱导的构象略有不同，这些差异说明了它们生物学特性的本质。如本研究中所做的那样，应用方法来表征新型配体在ER亚型中诱导的构象，能够更深入地理解配体-受体构象与雌激素激动剂或拮抗剂行为之间的关系。
