Optical probes to identify the glucocorticoid receptor ligands in living cells

Abstract

Glucocorticoids act through glucocorticoid receptor (GR) and are used for the treatment of several diseases. Ligand-induced recruitment of coregulator protein(s), coactivator/corepressor, to GR is an initial step in transcriptional activation/inhibition of GR. We describe herein genetically encoded fluorescent probes for screening of glucocorticoids, natural and synthetic, in single living cells. The GR ligand binding domain was connected to the GR interacting peptide sequence from coactivator or corepressor protein via a flexible linker sequence. This fusion protein was sandwiched between cyan and yellow fluorescent proteins (CFP and YFP, respectively) to complete the construct of the probe. This construct functions as an optical probe for imaging ligand-induced interaction between the glucocorticoid receptor and the coregulator protein (GLUCOCOR) in live cells. The interaction between GR LBD and coregulator peptide within GLUCOCOR brings CFP in close proximity of YFP to induce fluorescence resonance energy transfer from CFP to YFP. The GLUCOCORs can identify functionally active GR ligands, rapidly and conveniently, in a high-throughput screen; and are capable of distinguishing GR agonists, antagonists, and selective GR modulators in intact living cells. Therefore, the present method may play a significant role in developing new glucocorticoids for clinical use.