Multi-Coloured Sequential Resonance Energy Transfer for Simultaneous Ligand Binding at G Protein-Coupled Receptors

Abstract G protein coupled receptors (GPCRs) are the largest family of signalling proteins and highly successful drug targets. To date, most GPCR drugs interact with the binding pocket for the natural ligand, typically near the extracellular part of the transmembrane region. Recent advancements in structural biology have identified additional allosteric binding sites in other parts of these receptors. Allosteric sites provide several theoretical advantages, including the ability to modulate natural ligand function, and there is a need for better ways to study how ligands bind and interact with diVerent GPCR binding sites. We have developed an approach to study multiple ligands binding to the same receptor based on sequential resonance energy transfer between two fluorescent ligands bound simultaneously to a GPCR. We use this approach to gain insight into allosteric ligand interactions for a clinically relevant receptor. This method will provide important information to aid development of new GPCR drugs.