Muddana Smita S, Peterson Blake R
Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA.
Chembiochem. 2003 Sep 5;4(9):848-55. doi: 10.1002/cbic.200300606.
Steroid hormone receptors comprise a major class of therapeutic drug targets that control gene expression by binding steroid hormone ligands. These small molecule-protein interactions are typically characterized in living cells by quantification of ligand-mediated reporter gene expression. As an alternative, non-transcriptional approach, we constructed fluorescent cellular sensors by expressing yellow fluorescent protein (YFP) fused to the ligand binding domains (LBDs) of estrogen receptor-alpha (ERalpha), estrogen receptor-beta (ERbeta), androgen receptor (AR), and the glucocorticoid receptor (GR). These proteins were tethered through a short two amino acid linker and expressed in S. cerevisiae yeast. Recombinant yeast treated with cognate steroid receptor ligands exhibited dose-dependent fluorescence enhancements that were correlated with known relative receptor binding affinity values. These effects generally paralleled ligand-mediated receptor dimerization quantified with analogous yeast two-hybrid transcriptional assays, suggesting that the majority of the observed fluorescence enhancements were conferred by conformational changes coupled with receptor dimerization, such as ligand-mediated stabilization of protein folding. Remarkably, certain interactions such as the binding of cortisol, progesterone, and dexamethasone to the GR were undetectable with yeast two-hybrid assays. However, these interactions were detected with the fluorescent cellular sensors, indicating the sensitivity of this system to subtle ligand-induced conformational effects. These sensors provide a novel, non-transcriptional, and high-throughput method to identify and analyze ligands of nuclear hormone receptors.
类固醇激素受体构成了一类主要的治疗药物靶点，它们通过结合类固醇激素配体来控制基因表达。这些小分子与蛋白质的相互作用通常在活细胞中通过定量配体介导的报告基因表达来表征。作为一种替代的非转录方法，我们通过表达与雌激素受体α（ERα）、雌激素受体β（ERβ）、雄激素受体（AR）和糖皮质激素受体（GR）的配体结合域（LBD）融合的黄色荧光蛋白（YFP），构建了荧光细胞传感器。这些蛋白质通过一个短的双氨基酸接头连接，并在酿酒酵母中表达。用同源类固醇受体配体处理的重组酵母表现出剂量依赖性的荧光增强，这与已知的相对受体结合亲和力值相关。这些效应通常与用类似的酵母双杂交转录分析定量的配体介导的受体二聚化平行，表明观察到的大部分荧光增强是由与受体二聚化相关的构象变化引起的，例如配体介导的蛋白质折叠稳定。值得注意的是，某些相互作用，如皮质醇、孕酮和地塞米松与GR的结合，用酵母双杂交分析无法检测到。然而，这些相互作用可以用荧光细胞传感器检测到，这表明该系统对微妙的配体诱导的构象效应具有敏感性。这些传感器提供了一种新颖的、非转录的、高通量的方法来鉴定和分析核激素受体的配体。
