Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling.

G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β 1 -adrenergic receptor (β 1 AR), the β 2 -adrenergic receptor (β 2 AR), and the γ-aminobutyric acid (GABA B ) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β 1 ARs (monomers/dimers) and highest for GABA B receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β 1 -/ β 2 ARs were apparently freely diffusing on the cell surface, GABA B receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA B receptor complexes. These data provide a spatiotemporal characterization of β 1 -/β 2 ARs and GABA B receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.live cell imaging | protein-protein interactions