G-protein-coupled receptors mediate the biological effects of many hormones and neurotransmitters and are important pharmacological targets1. They transmit their signals to the cell interior by interacting with G proteins. However, it is unclear how receptors and G proteins meet, interact and couple. Here we analyse the concerted motion of G-protein-coupled receptors and G proteins on the plasma membrane and provide a quantitative model that reveals the key factors that underlie the high spatiotemporal complexity of their interactions. Using two-colour, single-molecule imaging we visualize interactions between individual receptors and G proteins at the surface of living cells. Under basal conditions, receptors and G proteins form activity-dependent complexes that last for around one second. Agonists specifically regulate the kinetics of receptor–G protein interactions, mainly by increasing their association rate. We find hot spots on the plasma membrane, at least partially defined by the cytoskeleton and clathrin-coated pits, in which receptors and G proteins are confined and preferentially couple. Imaging with the nanobody Nb37 suggests that signalling by G-protein-coupled receptors occurs preferentially at these hot spots. These findings shed new light on the dynamic interactions that control G-protein-coupled receptor signalling.
We thank S. P. Watson, M. Sauer, C. Manzo and E. Cocucci for discussions. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich/Transregio 166–Project C1 to D.C., CA 1014/1-1 to D.C. and FZT82 Rudolf Virchow Center to M.J.L.), the IZKF Würzburg (grant B-281 to D.C.), the European Research Council (Advanced Grant 232944–TOPAS to M.J.L.) and the Polish National Science Center (Maestro Grant No. 2012/06/A/ST1/00258 to K.B. and A.W.). T.S. was in part supported by an Alexander-von-Humboldt/Bayer Foundation fellowship.
Extended data figures
Extended data tables
Shown are individual α2A-ARs (green) and Gαi subunits (magenta), imaged by fast two-colour TIRF microscopy. Frames were acquired every 28 ms.
Shown are individual α2A-AR (green) and Gαi (magenta) trajectories. Frames were acquired every 28 ms.
Shown are individual receptor trajectories (α2A-ARs; different colours) overlaid on the corresponding localization density map. The playback is accelerated to capture receptor accumulation at high density areas.
Shown are individual α2A-AR trajectories (green) over a TIRF image of microtubules (magenta). Frames were acquired every 28 ms.
Shown are individual α2A-AR trajectories (green) over a TIRF image of actin fibres (magenta). Frames were acquired every 28 ms.
Shown are individual α2A-AR trajectories (green) over a TIRF image of CCPs. Frames were acquired every 28 ms.
Shown are individual α2A-AR trajectories (green) over a PALM image of actin fibres (orange). Frames were acquired every 28 ms.
Shown are individual Gαi trajectories (green) over a PALM image of actin fibres (orange). Frames were acquired every 28 ms.
Shown are single-molecule images and corresponding trajectories of an α2A-AR (green) and a Gαi subunit (magenta) diffusing and then stopping during an apparent interaction. The trajectories are coloured in blue during the interaction. Afterwards, the receptor resumes its movement, whereas the G protein remains immobile until it disappears due to fluorophore photobleaching. Frames were acquired every 28 ms.