G protein diffusion states reveal receptor binding dynamics at single-molecule scale.

G protein-coupled receptors (GPCRs) signal through heterotrimeric G proteins, yet the kinetics of receptor-G protein interactions in cells remain poorly understood. Using single-molecule imaging, we tracked individual Gαi1 proteins diffusing in the plasma membrane, expecting that receptor binding should slow these rapidly diffusing lipid-anchored proteins to match the substantially slower transmembrane GPCRs. Gαi1-Halo showed no detectable slowing upon dopamine D2 receptor activation. In contrast, the nucleotide-decoupled Gαi1 4A-Halo mutant exhibited receptor-dependent slowing with a lifetime of ∼0.8 s. Agonist increased the Gαi1 4A-Halo association rate without affecting the lifetime of the receptor-engaged complex, consistent with the idea that agonist efficacy is reflected in an increased probability of productive G protein engagement. These findings suggest that native GPCR-G protein interactions are not resolved at 15 ms time resolution and establish a diffusion-based approach for studying receptor-G protein kinetics in living cells.