Wagner B L, Norris J D, Knotts T A, Weigel N L, McDonnell D P
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Mol Cell Biol. 1998 Mar;18(3):1369-78. doi: 10.1128/MCB.18.3.1369.
Previously, we defined a novel class of ligands for the human progesterone receptor (PR) which function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components which distinguish between different ligand-induced PR conformations, we have determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending upon the class of ligand bound to the receptor. Specifically, we observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner which is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, we show that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis revealed that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, we propose that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology.
此前，我们定义了一类新型的人孕激素受体（PR）配体，其作为混合激动剂发挥作用。这些化合物在与受体结合时会诱导一种构象变化，这种变化不同于激动剂和拮抗剂所诱导的构象变化。这建立了配体-受体复合物的结构与其转录活性之间的相关性。为了确定区分不同配体诱导的PR构象的细胞成分，我们通过哺乳动物双杂交试验确定，核受体共抑制因子（NCoR）和类视黄醇及甲状腺激素受体沉默介质（SMRT）与PR的结合因结合到受体上的配体类别而异。具体而言，我们观察到共抑制因子优先与拮抗剂占据的PR结合，并且这些共抑制因子的过表达会抑制拮抗剂占据的PR的部分激动剂活性。然而，体外进行的结合研究表明，重组SMRT可以以不受结合配体性质影响的方式与PR相互作用。因此，在体内检测时，SMRT或NCoR无法与激动剂激活的PR相互作用，这可能更多地与PR对共激活因子的亲和力增加以及随后共抑制因子的取代有关，而不是与对共抑制因子蛋白的固有低亲和力有关。其他研究小组之前的工作表明，8-溴环磷酸腺苷（8-bromo-cAMP）可以将PR拮抗剂RU486转化为激动剂，此外，还可以增强激动剂结合的PR的转录活性。在这项研究中，我们表明NCoR或SMRT的外源性表达会抑制8-bromo-cAMP介导的PR转录活性增强。进一步分析表明，添加8-bromo-cAMP会降低NCoR和SMRT与PR的结合。因此，我们提出8-bromo-cAMP介导的PR转录活性增强至少部分是由于PR与共抑制因子NCoR和SMRT之间相互作用的破坏。累积起来，这些结果表明NCoR和SMRT的表达可能在PR药理学中起关键作用。
