Conformational Changes and Coactivator Recruitment by Novel Ligands for Estrogen Receptor- and Estrogen Receptor- : Correlations with Biological Character and Distinct Differences among SRC Coactivator Family Members

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Abstract
Ligands for the estrogen receptor (ER) that have the capacity to selectively bind to or activate the ER subtypes ERa or ERb 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 ERa but is inactive on ERb, and a pair of substituted tetrahydrochrysenes (THC), one enantiomer of which (S,S-THC) is an agonist on both ERa and ERb, the other (R,R-THC) being an agonist on ERa but an antagonist on ERb. To investigate the molecular mechanisms underlying the ER subtype-selective actions of these compounds, we have determined the conformational changes induced in ERa and ERb 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 ERa and ERb 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 stim- ulatory and inhibitory ligands, and that this assay, as well as coac- tivator recruitment, are excellent indicators of their agonist/antago- nist 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 illus- trative 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. (Endocrinology 141: 3534 -3545, 2000)

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