Glycoprotein-glycoprotein receptor binding detection using bioluminescence resonance energy transfer (BRET)

Abstract

The glycoprotein receptors, members of the large G protein-coupled receptors (GPCRs) family, are characterized by a large extracellular domains responsible of binding their glycoprotein hormones. Hormone-receptor interactions are traditionally analyzed by ligand-binding assays most often using radiolabeling but also by thermal shift assays. However, the use of radioisotopes requires appropriate laboratory conditions, and moreover, for this purpose, purified cell membranes are most often used instead of living cells. This in turn poses another challenge due to the altered stability of membrane proteins in detergents used for purification. Here, we overcome such limitations by applying bioluminescence resonance energy transfer (BRET) in living cells to determine hormone-receptor interactions between a Gaussia luciferase (Gluc) luteinizing hormone/chorionic gonadotropin receptor (LHCGR) fusion and its ligands (yoked human chorionic gonadotropin (yhCG) or luteinizing hormone (LH)) fused to the enhanced green fluorescent protein (eGFP). We first show that the Gluc-LHCGR is expressed on the plasma membrane and is fully functional, as well as the chimeric eGFP-hormones that are properly secreted and able to bind and activate the WT LHCGR. Finally, we applied the method to determine the interactions between clinically relevant mutations in the hormone as well as the receptor and show that this assay is fast and effective, plus safer and cost efficient alternative to radioligand-based assays, to screen for mutations in either the receptor or ligand. It enables kinetic measurements in living cells, detection of biosynthesis of the receptor (membrane expression) and it is compatible with downstream cellular assays - including firefly luciferase-based readouts.