Ligand-specific activation trajectories dictate GPCR signalling in cells

GPCRs signal through multiple active receptor states, and different ligands can drive distinct activation pathways in living cells. Using genetic code expansion and bioorthogonal labeling, researchers engineered a panel of fluorescence-based biosensors on the extracellular surface of the muscarinic acetylcholine receptor M2R (M2 muscarinic acetylcholine receptor). This enables real-time monitoring of agonist-induced conformational changes without disturbing intracellular coupling. Different agonists were found to produce equilibria of at least four distinct active states of the G-protein-bound receptor, each with different abilities to activate G proteins. The formation of these M2R-G-protein complexes occurs over 0.2 to 5 seconds along trajectories that combine shared and ligand-specific conformational changes, and these dynamics appear to determine G-protein selectivity. The findings point toward exploiting ligand-specific activation trajectories and conformational equilibria to improve GPCR drug discovery.