Ronacher Katharina, Hadley Katie, Avenant Chanel, Stubsrud Elisabeth, Simons S Stoney, Louw Ann, Hapgood Janet P
Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa.
Mol Cell Endocrinol. 2009 Feb 27;299(2):219-31. doi: 10.1016/j.mce.2008.10.008. Epub 2008 Oct 19.
The mechanisms that determine ligand-selective transcriptional responses by the glucocorticoid receptor (GR) are not fully understood. Using a wide panel of GR ligands, we investigated the relationships between the potency and maximal response for transactivation via a glucocorticoid response element (GRE) and transrepression via both nuclear factor small ka, CyrillicB (NFsmall ka, CyrillicB) and activator protein-1 (AP-1) sites, relative binding affinity for the GR, as well as interaction with both coactivators and corepressors. The results showed ligand-selective differences in potency and efficacy for each promoter, as well as for a particular ligand between the three promoters. Ligand potency correlated with relative affinity for the GR for agonists and partial agonists in transactivation but not for transrepression. Maximal response was unrelated to relative affinity of ligand for GR for both transactivation and transrepression. A good and significant correlation between full length coactivator binding in two-hybrid assays and efficacy as well as potency of different receptor-steroid complexes for both transactivation and transrepression supports a major role for coactivator recruitment in determination of ligand-selective transcriptional activity. Furthermore, ligand-selective GR binding to GRIP-1, as determined by both two-hybrid and DNA pull down assays, correlated positively with ligand-selective efficacy for transactivation of both a synthetic GRE reporter with expressed GR as well as of an endogenous gene via endogenous GR. The receptor interacting domain of the corepressor SMRT exhibited strong interaction with both agonists and partial agonists, similar to the results for coactivators, suggesting a possible role for SMRT in activation of transcription. However, there was no correlation between ligand affinity for the GR and cofactor interaction. These results provide strong quantitative biochemical support for a model in which GR-mediated ligand-selective differential interaction with GRIP-1, SRC-1A, NCoR and SMRT is a major determinant of ligand-selective and promoter-specific differences in potency and efficacy, for both transactivation and transrepression.
糖皮质激素受体（GR）决定配体选择性转录反应的机制尚未完全明确。我们使用了多种GR配体，研究了通过糖皮质激素反应元件（GRE）进行反式激活以及通过核因子小κB（NFκB）和活化蛋白-1（AP-1）位点进行反式抑制的效力与最大反应之间的关系、配体与GR的相对结合亲和力，以及与共激活因子和共抑制因子的相互作用。结果显示，每种启动子以及三个启动子中特定配体的效力和功效存在配体选择性差异。配体效力与激动剂和部分激动剂在反式激活中与GR的相对亲和力相关，但在反式抑制中不相关。最大反应与配体对GR在反式激活和反式抑制中的相对亲和力均无关。双杂交实验中全长共激活因子结合与不同受体-类固醇复合物在反式激活和反式抑制中的效力及功效之间存在良好且显著的相关性，这支持了共激活因子募集在决定配体选择性转录活性中起主要作用的观点。此外，通过双杂交和DNA下拉实验确定的配体选择性GR与GRIP-1的结合，与合成GRE报告基因表达GR时以及内源性基因通过内源性GR进行反式激活的配体选择性功效呈正相关。共抑制因子SMRT的受体相互作用结构域与激动剂和部分激动剂均表现出强烈相互作用，与共激活因子的结果相似，表明SMRT在转录激活中可能发挥作用。然而，配体对GR的亲和力与辅因子相互作用之间没有相关性。这些结果为一个模型提供了强有力的定量生化支持，该模型认为GR介导的与GRIP-1、SRC-1A、NCoR和SMRT的配体选择性差异相互作用是反式激活和反式抑制中配体选择性及启动子特异性效力和功效差异的主要决定因素。
