Fluorescent cellular sensors of steroid receptor ligands.

Abstract

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.