Reciprocal restriction of adenosine receptor motion in the plasma membrane

To examine the dynamics of A 1 R-A 2A R heteromers in the plasma membrane of a living cell, the motion of the receptors tagged with fluorescent proteins (A 1 R-green fluorescent protein [GFP] or A 2A R-mCherry) was measured by real-time single-particle tracking (SPT) (Fig. 1). Examples of fluorescent images and individual particle trajectories are shown in Additional file 1: Figure S1. Analysis of data corresponding to 500 A 1 R-GFP particles showed a linear relationship between the mean square displacement (MSD) versus time lag in the trajectories of up to 1600 single fluorescent particles (Fig. 1a, c). This is typical for Brownian diffusion, indicating a lack of restrictions in A 1 R-GFP motion. Co-expression of A 2A R-mCherry (Fig. 1b) led to a reduction in the lateral mobility of A 1 R-GFP, which became confined to plasma membrane regions of 0.461 ± 0.004 μm in diameter. Its diffusion coefficient decreased from 0.381 ± 0.002 μm2/s to 0.291 ± 0.003 μm2/s (p = 0.002, one-tailed t-test). Similarly, A 1 R-GFP also decreased the A 2A R-mCherry diffusion coefficient from 0.317 ± 0.002 μm2/s to 0.143 ± 0.005 μm2/s (p < 0.0001) (Fig. 1d–f). A 2A R moved within a confinement zone of 0.941 ± 0.007 μm in diameter that was reduced to 0.360 ± 0.001 μm (p < 0.0001) when both receptors were co-expressed. We conclude from these mobility comparisons that reciprocally restricted motion of the individual receptor particles must be due to A 1 R-A 2A R receptor-receptor interactions.

Fig. 1 Cell surface mobility of A 1 R-GFP and A 2A R-mCherry. Individual trajectories of particles containing GFP fused to the C-terminus of A 1 R (A 1 -GFP) (a and b) or mCherry fused to the C-terminus of A 2A R (A 2A -mCherry) (d and e) on HEK-293T cells expressing A 1 -GFP (a), A 2A -mCherry (d) or both (b and e). The trajectory and the fluorescence intensity of the individual particles were recorded over time using total internal reflection microscopy (TIRFM) and an electron multiplying charged-coupled device (EMCCD) camera recording. Receptor motion was determined by plotting (versus time lag) the mean square displacement (MSD) of A 1 -GFP (c) in the absence (black line) or presence of A 2A -mCherry (blue line), or A 2A -mCherry (f) in the presence (black line) or presence of A 1 -GFP (blue line). Data sets were fitted to mathematical models of free and confined diffusion for A 1 R and A 2A R respectively. g Co-localization of A 1 -GFP and A 2A -mCherry is observed (yellow dots). Scale bar: 100 nm. h Distribution of the fluorescence signal of A 1 -GFP (left) and A 2A -mCherry (right) within co-localized receptors (yellow dots in g). Curves approximately delineate the number of monomers, dimers, or trimers within the co-localized complex. i Stoichiometry analysis performed for co-localized A 1 -GFP and A 2A -mCherry receptor particles co-expressed in HEK-293T cells (yellow dots in g). Green corresponds to A 1 -GFP and red to A 2A -mCherry Full size image

Stoichiometry of A 1 and A 2A receptor heterocomplexes

The stoichiometry of the fluorescent receptors on the cell surface can be calculated from the brightness distribution of the individual particles [19] (see “Methods”). In cells expressing A 1 R-GFP, we found the majority of clusters to consist of either two (~47 %) or four (~34 %) receptors, and clusters with one or three receptors were scarce (~10 % and ~9 %, respectively) (Additional file 2: Figure S2A and black bars in Additional file 2: Figure S2C). In the case of A 2A R-mCherry, the stoichiometry analysis showed that the clusters mostly expressed trimers (45 %), with dimers (29 %) and tetramers (12 %) the second and third most common populations (Additional file 2: Figure S2D and black bars in Additional file 2: Figure S2F). Remarkably, this stoichiometry for either A 1 or A 2A receptors was altered when the partner receptor was also expressed. In cells co-expressing A 1 R-GFP and A 2A R-mCherry, the dimer population increased (57 % for A 1 R-GFP and 49 % for A 2A R-mCherry, blue bars in Additional file 2: Figures S2C, F) and became the predominant species (Additional file 2: Figures S2B, C, E, F).

In order to focus the analysis on heteromer complexes, we identified clusters containing both receptors (individual yellow dots in Fig. 1g, displaying both GFP and mCherry fluorescence). In ~1000 analyzed co-localized clusters that consisted of a mixture of A 1 -GFP and A 2A -Cherry (yellow dots in Fig. 1g), we found a similar high amount of dimers of A 1 R (75 %, left panel in Fig. 1h and green bar in Fig. 1i) and A 2A R (74 %, right panel in Fig. 1h and red bar in Fig. 1i). Trimers and tetramers of A 1 R, and monomers and tetramers of A 2A R, were in the minority or negligible (see Fig. 1h, i). In summary, given that the percentage of dimers of either A 1 R-GFP or A 2A R-mCherry in the yellow dots (which show co-localization of the two receptors) was similar and high (~75 %), the heterotetramer containing two A 1 Rs and two A 2A Rs must have been the most predominant species. To our knowledge, this is the first stoichiometry data for a GPCR heteromer in living cells.

Arrangement of G proteins interacting with A 1 and A 2A receptors

Monomeric GPCRs are capable of activating G proteins [20]. However, recent findings suggest that one GPCR homodimer bound to a single G protein may be a common functional unit [21]. Thus, an emerging question is how G proteins couple to GPCR heteromers. Because A 1 R selectively couples to G i and A 2A R to G s [22], the working hypothesis was that both G i and G s proteins may couple to the A 1 R-A 2A R heterotetramer. To test this hypothesis, we used bioluminescence resonance energy transfer (BRET) assays [23]. In agreement with the SPT experiments (see above), homodimers and heterodimers were detected by BRET assays in cells expressing A 1 R fused with Renilla luciferase (A 1 R-Rluc) or yellow fluorescent protein (A 1 R-YFP) (Fig. 2a), A 2A R-Rluc and A 2A R-YFP (Fig. 2b), or A 1 R-Rluc and A 2A R-YFP (Fig. 2e). Neither A 1 R-Rluc nor A 2A R-YFP interacted with the ghrelin receptor 1a fused to YFP (GHS1a-YFP), used as a control as a protein unable to directly interact with these adenosine receptors (Fig. 2a, b). In order to test the presence of the two G proteins in the heterotetramer, we transfected cells with minigenes that code for peptides blocking either G i or G s binding to GPCRs [24]. In addition, cells were treated with pertussis or cholera toxins that catalyze ADP-ribosylation of G i or G s . Clearly, treating cells with pertussis toxin, or expressing the minigene-coded peptide that blocks α i coupling, reduced the value of BRET max for A 1 R-A 1 R homodimers (Fig. 2a) and for A 1 R-A 2A R heterodimers (Fig. 2e) but not for A 2A R-A 2A R homodimers (Fig. 2b). This indicates that G i is coupled to A 1 R in both the homodimer and the heterodimer. Similarly, blocking G s -receptor interaction using cholera toxin or a minigene-coded peptide that blocks α S coupling reduced BRET max for A 2A R-A 2A R homodimers (Fig. 2b) and for A 1 R-A 2A R heterodimers (Fig. 2e) but not for A 1 R-A 1 R homodimers (Fig. 2a). Interestingly, BRET curves showed sensitivity to both cholera and pertussis toxins in cells expressing either A 1 R-Rluc-A 1 R-YFP and A 2A R (Fig. 2c) or A 2A R-Rluc-A 2A R-YFP and A 1 R (Fig. 2d). Functionality of constructs and controls in cells expressing minigenes, and in cells expressing the ghrelin GHS1a receptor instead of one of the adenosine receptors, are shown in Additional file 3: Figure S3. To further confirm that G i binds A 2A R in the receptor heteromer, the energy transfer between Rluc fused to the N-terminal domain of the α-subunit of G i (G i -Rluc) and A 2A R-YFP was analyzed in cells co-expressing or not co-expressing A 1 R (Fig. 2f). A hyperbolic BRET curve was observed in the presence of A 1 R, but not in its absence, indicating that G i and G s are bound to their respective receptor homodimers within the A 1 R-A 2A R heteromer.

Fig. 2 Influence of G proteins on A 1 R and A 2A R homodimerization and heterodimerization. B Bioluminescence resonance energy transfer (BRET) saturation curves were performed in HEK-293T cells 48 h post-transfection with (a, c) 0.3 μg of cDNA corresponding to A 1 R-Rluc and increasing amounts of A 1 R-YFP (0.1–1.5 μg cDNA) or GHS1a-YFP (0.25–2 μg cDNA) as negative control (a, purple line), without (a) or with (c) 0.15 μg of cDNA corresponding to A 2A R; (b, d) 0.2 μg of cDNA corresponding to A 2A R-Rluc and increasing amounts of A 2A R-YFP (0.1–1.0 μg cDNA) or GHS1a-YFP (0.25–2 μg cDNA) as negative control (b, purple line), without (b) or with (d) 0.5 μg of cDNA corresponding to A 1 R; (e) 0.3 μg of cDNA corresponding to A 1 R-Rluc and increasing amounts of A 2AR -YFP (0.1–1.0 μg cDNA); and (f) 0.5 μg of cDNA corresponding to A 1 R (except control blue curves that were obtained in cells not expressing A 1 R), 2 μg of cDNA corresponding to G i -Rluc, and increasing amounts of A 2A R-YFP (0.1–0.5 μg cDNA). In panels a, b, and e, cells were also transfected with 0.5 μg of cDNA corresponding to the G i -related (orange curves) or G s -related (blue curves) minigenes. Cells were treated for 16 h with medium (black curves), with 10 ng/ml of pertussis toxin (green curves), or with 100 ng/ml of cholera toxin (red curves) prior to BRET determination. To confirm similar donor expressions (approximately 100,000 bioluminescence units) while monitoring the increase in acceptor expression (1000–40,000 fluorescence units), the fluorescence and luminescence of each sample were measured before energy transfer data acquisition. MiliBRET unit (mBU) values are the mean ± standard error of the mean of four to six different experiments grouped as a function of the amount of BRET acceptor. In each panel (top) a cartoon depicts the proteins to which Rluc and YFP were fused and the presence or not of partner receptors and/or G s or G i proteins [schemes in c to f are not intended to illustrate on stoichiometry because the predominant form in cells expressing the two receptors was the heterotetramer containing two A 1 and two A 2A receptors (see “Results”)] Full size image

Further, two complementary BRET experiments were performed to determine the orientation of G i and G s within the A 1 R-A 2A R heterocomplex. First, Rluc and YFP were respectively fused to the N-terminal domains of the α-subunit of G i (α i -Rluc) and G s (α s -YFP) (Fig. 3, bar a); second, they were fused to the N-terminal domain of the γ-subunit (γ-Rluc and γ-YFP) (Fig. 3, bar b). We observed significant energy transfer between γ-Rluc and γ-YFP in cells co-expressing A 1 R and A 2A R (Fig. 3, bar b) but minimal amounts in negative-control cells (Fig. 3, bars c and d). In cells expressing either A 1 R or A 2A R, the energy transfer between γ-Rluc and γ-YFP was also low (Fig. 3, bars e and f), suggesting that dimers but not tetramers were the most prevalent form of surface receptors in single-transfected cells. These results in co-transfected cells corroborate the 2:2 stoichiometry obtained from analysis of the fluorescence in single particles and are consistent with G i and G s binding to these A 1 R-A 2A R heterotetramers.

Fig. 3 G s and G i coupling to adenosine A 1 R-A 2A R heterocomplexes. Bioluminescence resonance energy transfer (BRET) experiments were performed in HEK-293T cells 48 h post-transfection with (a, b) 0.2 μg of cDNA corresponding to A 1 R and 0.15 μg of cDNA corresponding to A 2A R; (c, d) 0.2 μg of cDNA corresponding to A 1 R or 0.15 μg of cDNA corresponding to A 2A R and 0.4 μg of cDNA corresponding to growth hormone secretagogue receptor GHS1a; (e) 0.2 μg of cDNA corresponding to A 1 R; or (f) 0.15 μg of cDNA corresponding to A 2A R. Cells were also transfected with 2 μg of cDNA corresponding to the α-subunit of G i fused to Rluc and increasing amounts of cDNA corresponding to the α-subunit of G s fused to YFP (a) or 0.3 μg of cDNA corresponding to the γ-subunit fused to Rluc and increasing amounts of cDNA corresponding to the γ-subunit fused to YFP (b–f). Maximum miliBRET unit (mBU) values are the mean ± standard error of the mean of four different experiments. A scheme showing the protein to which Rluc and YFP were fused is provided (top). ***p < 0.001 by one-way ANOVA with post - hoc Dunnett’s test Full size image

Molecular model of G i and G s bound to the A 1 R-A 2A R heterotetramer

To identify the orientation of the G protein in the receptor homodimer, we combined energy transfer assays between α s -Rluc (Rluc at the N-terminus of the G protein α-subunit) and A 2A R-YFP (Fig. 4a) with information on transmembrane (TM) interfaces based on crystal structures of GPCRs [3, 4], which have been recently summarized [25]. The observed high-energy transfer using α s -Rluc and A 2A R-YFP indicated close proximity between the N-tail of the α-subunit of G s and the C-tail of A 2A R. Interestingly, Rluc and YFP in the “monomeric” A 2A R-G s complex (see “Methods”) point toward distant positions in space (Fig. 4b). Therefore, the observed BRET should occur between Rluc in the G protein α-subunit and a second A 2A R-YFP protomer. Among all described TM interfaces for receptor homodimerization (see Additional file 4: Figure S4), we propose the TM4/5 interface, which is observed in the oligomeric structure of β 1 -AR [4] and in structures derived from coarse-grained molecular dynamics (MD) simulations [26]. In fact, this is the only interface that favors BRET between α s -Rluc and a second A 2A R-YFP protomer in a homodimer (Fig. 4c). The homologous A 1 R homodimer was built using the same TM4/5 interface as for A 2A R (see Additional file 4: Figure S4 and its legend).

Fig. 4 Orientation of a G protein in a receptor homodimer. Bioluminescence resonance energy transfer (BRET) saturation experiments were performed in HEK-293T cells transfected with 2 μg of cDNA corresponding to the α-subunit of G s fused to Rluc and increasing amounts of A 2A R-YFP (0.1–0.5 μg) cDNA. a BRET measurements in cells pretreated for 16 h with medium (black line) or with 100 ng/ml of cholera toxin (red line). Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (approximately 50,000 bioluminescence units) while monitoring the increase in acceptor expression (1000–10,000 fluorescence units). miliBRET unit (mBU) values are the mean ± standard error of the mean of four to five different experiments grouped as a function of the amount of BRET acceptor. A scheme of the placement of donor and acceptor BRET moieties is provided (top). b Molecular model of the A 2A R-G s complex. Rluc (blue) is attached to the N-terminal αN helix of G s (gray), and YFP (yellow) is attached to the C-terminal domain of A 2A R (light green) (see Additional file 9: Figure S9 for details). c Arrangement of A 2A R homodimers modeled via the TM4/5 interface as observed in the oligomeric structure of β 1 -AR [4]. The A 2A R protomer bound to α s is shown in light green, whereas the second A 2A R-YFP protomer is shown in dark green. The molecular model in panel c (BRET between Rluc in G s α subunit and YFP in a second A 2A R protomer; center-to-center distance between Rluc and YFP of 6.5 nm), in contrast to the model shown in panel B (BRET between Rluc in G s α subunit and YFP in the G-protein bound A 2A R protomer; center-to-center distance between Rluc and YFP of 8.3 nm), would favor the observed high-energy transfer (see panel a) between α s -Rluc and A 2A R-YFP Full size image

The remaining possible TMs able to form heteromeric interfaces are TM1 and TM5/6 (Fig. 5). Both are possible inter-GPCR interfaces as observed in the structure of the μ-opioid receptor (μ-OR) [3]. To discern between these two possibilities, a bimolecular fluorescence complementation strategy was undertaken. For this purpose, the N-terminal fragment of Rluc8 was fused to A 1 R (A 1 R-nRluc8) and its C-terminal domain to A 2A R (A 2A R-cRluc8), which only upon complementation can act as a BRET donor (Rluc8). The BRET acceptor protein was obtained upon complementation of the N-terminal fragment of YFP Venus protein fused to A 1 R (A 1 R-nVenus) and its C-terminal domain fused to A 2A R (A 2A R-cVenus). When all four receptor constructs were transfected, we obtained a positive and saturable BRET signal (BRET max of 35 ± 2 mBU and BRET 50 of 16 ± 3 mBU) that was not obtained for negative controls (Additional file 5: Figure S5). Figure 5a, b shows that the hemi-donor (A 1 R-nRluc8 and A 2A R-cRluc8) and the hemi-acceptor (A 1 R-nVenus and A 2A R-cVenus) moieties, placed at the C-terminus of the receptors, can only complement if A 1 R-A 2A R heterodimerization occurs via the TM5/6 interface. The TM4/5 interface for homodimerization and the TM5/6 interface for heterodimerization give a rhombus-shaped tetramer organization (Fig. 5a). Remarkably, cell pre-incubation with either pertussis or cholera toxins decreased the BRET max by 35 % (Fig. 5c), further suggesting that both G s and G i proteins bind to the A 1 R-A 2A R heterotetramer.

Fig. 5. Bioluminescence resonance energy transfer (BRET)-aided construction of a model consisting of G i and G s bound to an A 1 R-A 2A R heterotetramer. a, b A 1 R-A 2A R tetramer built using TM5/6 (a) or TM1 (b) inter-receptor interfaces modeled as in the structure of the μ opioid receptor [3]. TM helices 1, 4, and 5, involved in receptor dimerization, are highlighted in dark blue, light blue, and gray, respectively. nRluc8 and cRluc8 are shown in blue and nVenus and cVenus in dark yellow. c BRET and bimolecular fluorescence complementation experiments were performed in HEK-293T cells transfected with 1.5 μg of cDNA corresponding to A 1 R-cRluc8 and A 2A R-nRluc8, and 1.5 μg of cDNA corresponding to A 1 R-nVenus and A 2A R-cVenus. As the negative control, cells were transfected with 1 μg of cDNA corresponding to nRluc8 and 1.5 μg of cDNA corresponding to A 2A R-nRluc8, A 1 R-nVenus, and A 2A R-cVenus. Cells were treated for 16 h with medium (– toxins), 10 ng/ml of pertussis toxin (+ pertussis), or 100 ng/ml of cholera toxin (+ cholera) prior to BRET determination. The relative amount of BRET is given as in Fig. 4 and values are the mean ± standard error of the mean of three different experiments. Student’s t-test showed statistically significant differences with respect to the control (# p < 0.05, ## p < 0.01) and a significant effect in the presence of either toxin over BRET in the absence of toxins (*p < 0.05). A schematic representation at the top shows the protein to which the hemi luminescent or fluorescent proteins were fused. d Molecular model of the A 1 R-A 2A R tetramer in complex with G i and G s . A 1 R bound to G i is shown in red, G i -unbound A 1 R is shown in orange, A 2A R bound to G s is shown in dark green, G s -unbound A 2A R is shown in light green, and the α, β-, and γ-subunits of G i and G s are shown in dark gray, light gray, and purple, respectively. Transmembrane helices 4 and 5 are highlighted in light blue and gray, respectively Full size image