The dynamic basis of G-protein recognition and activation by a GPCR

G-protein-coupled receptor (GPCR) signalling occurs through heterotrimeric G proteins, whose selective activation leads to distinct cellular outcomes¹. Although more than 200 GPCR-G protein complex structures have been determined², these static snapshots provide limited insight into the dynamics of G-protein association and dissociation. Here we present cryo-electron microscopy structures of human neurotensin receptor type 1 (NTSR1) with minimally modified G_o and G_q, showing how the receptor's intracellular surface dynamically rearranges to accommodate each G-protein subtype. Furthermore, time-resolved cryo-electron microscopy analyses of NTSR1-G_i visualized G-protein dissociation processes on GDP/GTP binding. Characterization of more than 20 intermediates, complemented by mutational and computational analyses, identifies four key mechanistic features. First, GDP/GTP induces G_i release from both canonical and non-canonical active conformations with distinct kinetics. Second, NTSR1 uses common intracellular rearrangements to recognize different G-protein subtypes and to promote activation of a single subtype. Third, separation from Gβγ involves stepwise remodelling of the Gα switches I-III. Finally, G_i dissociates from the receptor through a pathway that is distinct from that of G_s, and the canonical and non-canonical NTSR1-G_i complexes further diverge in their dissociation trajectories. These findings provide a comprehensive framework for understanding GPCR signalling dynamics and guiding signal-targeted therapeutic development.