Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization

Abstract

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.

Fig. 1.

Detection and tracking of individual SNAP-tagged proteins on the surface of living cells. (A) Enlarged view of single SNAP-CD86 particles on the surface of a living cell visualized by TIRF-M. Particles were automatically detected and tracked. The current position (blue circle) and trajectory (blue spline) of each particle are indicated. Apparent merging and splitting events are shown as green and red segments, respectively. (Scale bar, 5 μm.) (B) Same tracks on inverted image (white background). Inset, higher magnification.

Fig. 2.

Analysis of β₁- (A–E) and β₂- (F–J) AR oligomerization and lateral mobility by single-molecule TIRF-M. (A and F) Schematic representation of the used SNAP-tagged constructs. (B and G) Representative intensity distributions of Alexa647-labeled particles. Particle densities were 0.24 (B) and 0.25 (G) particle/μm². Data were fitted with a mixed Gaussian model. A mixed Gaussian fit after partial photobleaching (dashed lines) was used to precisely estimate the intensity of single fluorophores in each image sequence. (G, Inset) Comparison of the fraction of monomeric β₁AR and β₂AR particles at low density (0.15–0.3 particle/μm²). Each data point represents one cell. *P = 0.0003 by Mann–Whitney test. (C and H) Dependency of the distribution of particle components on particle density. Shown is the cumulative distribution of mono-, di-, tri-, and tetramers of Alexa647-labeled receptors, based on mixed Gaussian fitting analyses like those shown in B and G, as a function of particle density. Data were fitted using third-order polynomial functions to provide an indication of their trend. Each data point represents one cell. [n = 6,181 particles from 27 different cells (C) and 7,419 particles from 30 different cells (H)]. (D and I) Distribution of diffusion coefficients of receptor particles calculated from their mean square displacement (MSD). Insets, MSD plots; shown are the mean (red) as well as the 10% and 90% percentiles (shaded area) of particles that were tracked for at least 3 s; black, data of representative individual particles. (E and J) Effect of the size of GPCR complexes on their lateral diffusion. The size of individual particles was estimated on the basis of the number of bleaching steps. Shown are box plots of diffusion coefficients measured for particles of different size. The boxes encompass the 25% and 75% percentiles and median values are indicated by red lines. Differences in E and J are statistically significant by a Kruskal–Wallis test (P < 0.0001) followed by Dunn’s test (*P < 0.001).

Fig. 3.

Dynamic visualization of receptor–receptor interactions. (A) Example of two Alexa647-labeled β₁AR particles showing a transient colocalization. A merging event (green) is followed after some frames by a splitting event (red). Images are centered on the particles’ position. (Scale bar, 1 μm.) (B) Same traces as in A on a white background and without centering. (C) Intensity profiles of the traces in A, showing intensity doubling upon merging. (D) Colocalizations between control particles devoid of true interactions. Black, simulated particles with diffusion coefficients, intensity distribution, and bleaching rate analogous to those of β₁AR particles. Orange, monomeric SNAP-CD86 receptors. The apparent lifetime of particle colocalizations (; 95% confidence intervals in parentheses) was calculated by fitting colocalization time data with an exponential decay function. (E) Lifetime of β₁AR colocalizations. Colocalization time data derived from experiments as in A (green) were fitted to the sum (black) of two exponential decays (blue and red, respectively). The obtained apparent lifetimes of particle colocalizations ( and ; 95% confidence intervals in parentheses) were then used to estimate the true lifetime of receptor–receptor interactions. (F) Same as E with low-density (<0.35 particle/μm²) β₂AR movies. Data in E and F are from 20 and 8 different cells, respectively. Data in E and F were fitted better with two components than with one, as judged by an F-test (P < 1.0 × 10⁻⁸).

Fig. 4.

Selective analysis of GABA_B receptor heterodimers. (A) Schematic representation of the used constructs. Cells were cotransfected with SNAP-GABA_B1 and wild-type GABA_B2 subunits. (B) Representative image of a cell transfected with both constructs, labeled with Alexa647-BG and visualized by TIRF-M. Particle density = 0.43 particle/μm². (C) Intensity distribution of the particles in B. Data were fitted with a mixed Gaussian model. (D) Dependency of the distribution of particle components on particle density. Data are based on mixed Gaussian fitting analyses like those shown in C and are represented as in Fig. 2 C and H. Because only the GABA_B1 subunit is labeled, one fluorophore (n = 1) corresponds to one heterodimer (h.d.) [n = 4,472 particles from 17 different cells]. (E) Distribution of diffusion coefficients of GABA_B heteromeric particles on the cell surface. Inset, MSD plot; shown are the mean (red) as well as the 10% and 90% percentiles (shaded area) of particles that were tracked for at least 3 s; black, representative data of individual mobile and immobile particles. (F) Effect of the size of GABA_B heteromeric particles on their lateral diffusion. The size of individual particles was estimated on the basis of the number of bleaching steps. Shown are box plots of diffusion coefficients measured for particles of different size. The boxes encompass the 25% and 75% percentiles and median values are indicated by red lines. Differences are statistically significant by a Kruskal–Wallis test (P < 0.0001) followed by Dunn’s test (*P < 0.001). (G) Image of a cell with ∼15 times higher receptor density than in B, showing GABA_B receptors arranged in rows. (H) Interaction of GABA_B receptors with the cortical actin cytoskeleton. Cells were cotransfected with SNAP-GABA_B1 and wild-type GABA_B2 subunits, treated or not (control) with latrunculin A and labeled with Alexa647-BG. After fixation, actin filaments were stained with Alexa488-phalloidin. (Scale bars, 5 μm.) (I) Same as D in cells pretreated with latrunculin A [n = 7,748 particles from 26 different cells].

Fig. 5.

Effect of agonists on receptor mobility. (A–C) Cells were transfected with SNAP-β₁AR (A), SNAP-β₂AR (B), or SNAP-GABA_B1 plus wild-type GABA_B2 (C) constructs, labeled with Alexa647-BG, and stimulated with the indicated concentrations of agonists 5–10 min before image acquisition. Shown are the distributions of diffusion coefficients of the analyzed receptor particles compared with control nonstimulated cells. Differences in C are statistically significant by a Mann–Whitney test (P < 0.0001).