Fluorescent Cellular Sensors of Steroid Receptor Ligands

• Published in: Chembiochem : a European journal of chemical biology Vol. 4; no. 9; pp. 848 - 855
• Main Authors: Muddana, Smita S, Peterson, Blake R
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 05.09.2003; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Animals; Binding, Competitive; biological activity; Biosensing Techniques; biosensors; Flow Cytometry; Fluorescence; green fluorescent protein; high-throughput screening; Humans; Ligands; medicinal chemistry; Rats; Receptors, Steroid - genetics; Receptors, Steroid - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Substrate Specificity; Two-Hybrid System Techniques
• ISSN: 1439-4227; 1439-7633; 1439-4227
• 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 (ERα), estrogen receptor-beta (ERβ), 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.