Conformational dynamics of glucagon receptor domains revealed by single-molecule fluorescence microscopy

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in many cellular signaling processes. Representing the largest family of proteins with approximately 800 members in humans, GPCRs are the target for more than 40% of current pharmaceutical drugs. The structural and dynamic information is vital to understanding the activation mechanism of these receptors. Despite the availability of high-resolution structures of GPCRs at different conformational states, the dynamics of those conformational states at the molecular level are missing. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the glucagon receptor (GCGR), a class B family GPCR involved in glucose homeostasis. We expressed and purified GCGR from mammalian cells and analyzed them at a single molecule level. We used single-molecule Förster resonance energy transfer (smFRET) to observe the conformational dynamics of the donor and acceptor fluorophore-labeled GCGR molecules. We observed ligand-dependent slow and reversible conformational fluctuation of N-terminal extra cellular domain (ECD) without ligand binding. The addition of a saturated concentration of peptide ligand (Glucagon) altered the dynamics towards the lower FRET states. These results provide new insight into the structural dynamics of glucagon receptor and receptor activation.