Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic.

Dynamic information is vital to understanding the activation mechanism of G protein-coupled receptors (GPCRs). Despite the availability of high-resolution structures of different conformational states, the dynamics of those states at the molecular level are poorly understood. Here, we used total internal reflection fluorescence microscopy to study the extracellular domain (ECD) of the glucagon receptor (GCGR), a class B family GPCR that controls glucose homeostasis. Single-molecule fluorescence resonance energy transfer was used to observe the ECD dynamics of GCGR molecules expressed and purified from mammalian cells. We observed that for apo-GCGR, the ECD is dynamic and spent time predominantly in a closed conformation. In the presence of glucagon, the ECD is wide open and also shows more dynamic behavior than apo-GCGR, a finding that was not previously reported. These results suggest that both apo-GCGR and glucagon-bound GCGRs show reversible opening and closing of the ECD with respect to the seven-transmembrane (7TM) domain. This work demonstrates a molecular approach to visualizing the dynamics of the GCGR ECD and provides a foundation for understanding the conformational changes underlying GPCR activation, which is critical in the development of new therapeutics.