Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with G_i and G_s

Background

G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown.

Results

We used single-particle tracking experiments in cells expressing functional adenosine A₁-A_2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A₁ receptor in the presence of the A_2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins.

Conclusions

The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (G_i and G_s). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

G-protein-coupled receptor (GPCR) oligomerization is heavily supported by recent biochemical and structural data [1–6]. Optical-based techniques are instrumental in studying the dynamics and organization of receptor complexes in living cells [7]. For instance, total internal reflection fluorescence microscopy shows that 30 % of muscarinic M1 receptors exist as dimers (with no evidence of higher oligomers) that undergo interconversion with monomers on a timescale of seconds [8]. Similarly, the β₁-adrenergic receptors (β₁-AR) are expressed as a mixture of monomers and dimers whereas β₂-adrenergic receptors (β₂-AR) have a tendency to form dimers and higher-order oligomers [9]. Moreover, the monomer-dimer equilibrium of the chemoattractant N-formyl peptide receptor at a physiological level of expression lies within a timescale of milliseconds [10]. Together, these studies in heterologous systems show that a given GPCR is present in a dynamic equilibrium between monomers, dimers, and higher-order oligomers.

Studies in a broad spectrum of GPCRs [11–14] show that these receptors may form heteromers. GPCR heteromers are defined as novel signaling units with functional properties different from homomers and they represent a completely new field of study [15]. Innovative crystallographic techniques have permitted researchers to obtain crystal structures of GPCR families A, B, C, and F, bound to either agonists, antagonists, inverse agonists or allosteric modulators; in the form of monomers or homo-oligomers; and in complex with a G protein or with a ß-arrestin [16]. However, crystal structures of GPCR heteromers have not yet been obtained. Here, we propose a quaternary structure of a heteromer, taking into account the molecular stoichiometry and the interacting G proteins. Adenosine A₁-A_2A receptor (A₁R-A_2AR) complexes constitute a paradigm in the GPCR heteromer field because A₁R is coupled to G_i and A_2AR to G_s; that is, they transduce opposite signals in cyclic adenosine monophosphate (cAMP)-dependent intracellular cascades. First described as a concentration-sensing device in striatal glutamatergic neurons [17], the A₁R-A_2AR heteromer is thought to function as a G_s/G_i-mediated switching mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release [17, 18]. The structural basis of this switch is key to understanding heteromer function and the biological advantage behind the GPCR heteromerization phenomenon. Here, we have devised the molecular architecture of the adenosine A₁R-A_2AR heteromer in complex with G proteins using a combination of microscope-based single-particle tracking, molecular modeling, and energy transfer assays in combination with molecular complementation. The results point to A₁ and A_2A receptors organizing into a rhombus-shaped heterotetramer that couples to G_i and G_s. The overall structure is very compact and provides interacting interfaces for GPCRs and for G proteins.

Reciprocal restriction of adenosine receptor motion in the plasma membrane

To examine the dynamics of A₁R-A_2AR heteromers in the plasma membrane of a living cell, the motion of the receptors tagged with fluorescent proteins (A₁R-green fluorescent protein [GFP] or A_2AR-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 A₁R-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 A₁R-GFP motion. Co-expression of A_2AR-mCherry (Fig. 1b) led to a reduction in the lateral mobility of A₁R-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, A₁R-GFP also decreased the A_2AR-mCherry diffusion coefficient from 0.317 ± 0.002 μm²/s to 0.143 ± 0.005 μm²/s (p < 0.0001) (Fig. 1d–f). A_2AR 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 A₁R-A_2AR receptor-receptor interactions.

Cell surface mobility of A₁R-GFP and A_2AR-mCherry. Individual trajectories of particles containing GFP fused to the C-terminus of A₁R (A₁-GFP) (a and b) or mCherry fused to the C-terminus of A_2AR (A_2A-mCherry) (d and e) on HEK-293T cells expressing A₁-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 A₁-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 A₁-GFP (blue line). Data sets were fitted to mathematical models of free and confined diffusion for A₁R and A_2AR respectively. g Co-localization of A₁-GFP and A_2A-mCherry is observed (yellow dots). Scale bar: 100 nm. h Distribution of the fluorescence signal of A₁-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 A₁-GFP and A_2A-mCherry receptor particles co-expressed in HEK-293T cells (yellow dots in g). Green corresponds to A₁-GFP and red to A_2A-mCherry

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Stoichiometry of A₁ 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 A₁R-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_2AR-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 A₁ or A_2A receptors was altered when the partner receptor was also expressed. In cells co-expressing A₁R-GFP and A_2AR-mCherry, the dimer population increased (57 % for A₁R-GFP and 49 % for A_2AR-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 A₁-GFP and A_2A-Cherry (yellow dots in Fig. 1g), we found a similar high amount of dimers of A₁R (75 %, left panel in Fig. 1h and green bar in Fig. 1i) and A_2AR (74 %, right panel in Fig. 1h and red bar in Fig. 1i). Trimers and tetramers of A₁R, and monomers and tetramers of A_2AR, were in the minority or negligible (see Fig. 1h, i). In summary, given that the percentage of dimers of either A₁R-GFP or A_2AR-mCherry in the yellow dots (which show co-localization of the two receptors) was similar and high (~75 %), the heterotetramer containing two A₁Rs and two A_2ARs 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 A₁ 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 A₁R selectively couples to G_i and A_2AR to G_s [22], the working hypothesis was that both G_i and G_s proteins may couple to the A₁R-A_2AR 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 A₁R fused with Renilla luciferase (A₁R-Rluc) or yellow fluorescent protein (A₁R-YFP) (Fig. 2a), A_2AR-Rluc and A_2AR-YFP (Fig. 2b), or A₁R-Rluc and A_2AR-YFP (Fig. 2e). Neither A₁R-Rluc nor A_2AR-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 A₁R-A₁R homodimers (Fig. 2a) and for A₁R-A_2AR heterodimers (Fig. 2e) but not for A_2AR-A_2AR homodimers (Fig. 2b). This indicates that G_i is coupled to A₁R 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_2AR-A_2AR homodimers (Fig. 2b) and for A₁R-A_2AR heterodimers (Fig. 2e) but not for A₁R-A₁R homodimers (Fig. 2a). Interestingly, BRET curves showed sensitivity to both cholera and pertussis toxins in cells expressing either A₁R-Rluc-A₁R-YFP and A_2AR (Fig. 2c) or A_2AR-Rluc-A_2AR-YFP and A₁R (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_2AR 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_2AR-YFP was analyzed in cells co-expressing or not co-expressing A₁R (Fig. 2f). A hyperbolic BRET curve was observed in the presence of A₁R, but not in its absence, indicating that G_i and G_s are bound to their respective receptor homodimers within the A₁R-A_2AR heteromer.

Influence of G proteins on A₁R and A_2AR 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 A₁R-Rluc and increasing amounts of A₁R-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_2AR; (b, d) 0.2 μg of cDNA corresponding to A_2AR-Rluc and increasing amounts of A_2AR-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 A₁R; (e) 0.3 μg of cDNA corresponding to A₁R-Rluc and increasing amounts of A_2AR-YFP (0.1–1.0 μg cDNA); and (f) 0.5 μg of cDNA corresponding to A₁R (except control blue curves that were obtained in cells not expressing A₁R), 2 μg of cDNA corresponding to G_i-Rluc, and increasing amounts of A_2AR-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 A₁ and two A_2A receptors (see “Results”)]

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Further, two complementary BRET experiments were performed to determine the orientation of G_i and G_s within the A₁R-A_2AR 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 A₁R and A_2AR (Fig. 3, bar b) but minimal amounts in negative-control cells (Fig. 3, bars c and d). In cells expressing either A₁R or A_2AR, 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 A₁R-A_2AR heterotetramers.

G_s and G_i coupling to adenosine A₁R-A_2AR 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 A₁R and 0.15 μg of cDNA corresponding to A_2AR; (c, d) 0.2 μg of cDNA corresponding to A₁R or 0.15 μg of cDNA corresponding to A_2AR and 0.4 μg of cDNA corresponding to growth hormone secretagogue receptor GHS1a; (e) 0.2 μg of cDNA corresponding to A₁R; or (f) 0.15 μg of cDNA corresponding to A_2AR. 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

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Molecular model of G_i and G_s bound to the A₁R-A_2AR 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_2AR-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_2AR-YFP indicated close proximity between the N-tail of the α-subunit of G_s and the C-tail of A_2AR. Interestingly, Rluc and YFP in the “monomeric” A_2AR-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_2AR-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 β₁-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_2AR-YFP protomer in a homodimer (Fig. 4c). The homologous A₁R homodimer was built using the same TM4/5 interface as for A_2AR (see Additional file 4: Figure S4 and its legend).

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_2AR-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_2AR-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_2AR (light green) (see Additional file 9: Figure S9 for details). c Arrangement of A_2AR homodimers modeled via the TM4/5 interface as observed in the oligomeric structure of β₁-AR [4]. The A_2AR protomer bound to α_s is shown in light green, whereas the second A_2AR-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_2AR 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_2AR 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_2AR-YFP

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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 A₁R (A₁R-nRluc8) and its C-terminal domain to A_2AR (A_2AR-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 A₁R (A₁R-nVenus) and its C-terminal domain fused to A_2AR (A_2AR-cVenus). When all four receptor constructs were transfected, we obtained a positive and saturable BRET signal (BRET_max of 35 ± 2 mBU and BRET₅₀ of 16 ± 3 mBU) that was not obtained for negative controls (Additional file 5: Figure S5). Figure 5a, b shows that the hemi-donor (A₁R-nRluc8 and A_2AR-cRluc8) and the hemi-acceptor (A₁R-nVenus and A_2AR-cVenus) moieties, placed at the C-terminus of the receptors, can only complement if A₁R-A_2AR 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 A₁R-A_2AR heterotetramer.

Bioluminescence resonance energy transfer (BRET)-aided construction of a model consisting of G_i and G_s bound to an A₁R-A_2AR heterotetramer. a, b A₁R-A_2AR 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 A₁R-cRluc8 and A_2AR-nRluc8, and 1.5 μg of cDNA corresponding to A₁R-nVenus and A_2AR-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_2AR-nRluc8, A₁R-nVenus, and A_2AR-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 A₁R-A_2AR tetramer in complex with G_i and G_s. A₁R bound to G_i is shown in red, G_i-unbound A₁R is shown in orange, A_2AR bound to G_s is shown in dark green, G_s-unbound A_2AR 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

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We next evaluated, using computational tools, whether the proposed A₁R-A_2AR heterotetramer could couple to both G_i and G_s proteins. Clearly, the external protomers of the proposed A₁R-A_2AR heterotetramer can bind to G_i and G_s proteins (Fig. 5d). This model positions the α-subunits of G_i and G_s in close contact, facing the interior of the tetrameric complex, while the N-terminal α-helices of α_i and α_s point outside the complex. The N-terminal α-helices of the γ-subunits are in close proximity, facing the inside (Additional file 6: Figure S6), which explains the significant energy transfer observed between γ-Rluc and γ-YFP (Fig. 3, bar b). The model provides experimental insights into the structural arrangement of heteromers consisting of two GPCRs and coupled to two G proteins, the possibility of which has recently been discussed [25]. We used MD simulations to study the stability of this complex. Additional file 7: Figure S7 shows root-mean-square deviations (rmsd) on protein α-carbons throughout the MD simulation, as well as key intermolecular distances among protomers and G proteins. Clearly, both the A₁R protomer bound to G_i and the A₁R protomer that does not interact with it maintained a close structural similarity (rmsd ≈ 0.3 nm) relative to the initial structures. Similar results were obtained for the A_2AR protomers (bound and unbound to G_s) (Additional file 7: Figure S7A). The fact that rmsd values of the whole system, formed by the A₁R-A_2AR heterotetramer bound to G_i and G_s, are of the order of 0.6 nm indicates that the initial structural model is maintained during the MD simulation (Additional file 7: Figure S7A). As a consequence, selected intermolecular distances among protomers and G proteins remain constant during the MD simulation (Additional file 7: Figure S7B). A key aspect in the assembly of the heterotetramer is the TM interfaces for homodimerization (TM4/5) and heterodimerization (TM5/6). Additional file 8: Figure S8B shows rmsd values of the four-helix bundle forming the TM4/5 and TM5/6 interfaces, the initial and final snapshots of these bundles, and the evolution of the A₁R-A_2AR heterotetramer during the MD simulation. Clearly, the rather small structural variations of these four-helix bundles, also reflected by rmsd <0.3 nm, suggest a stable complex. Notably, the TM5/6 four-helix bundle seems more stable than the TM4/5 bundles, as shown by its lower rmsd value. Additional file 8: Figure S8B, C depicts contact maps of the TM4/5 and TM5/6 interfaces, as well as the evolution of the network of hydrophobic interactions within these interfaces during the MD simulation.

For more than a decade, experimental evidence has supported the occurrence of homo-oligomers and hetero-oligomers of GPCRs [21]. However, our basic understanding of what makes heteromers different from homomers remains unknown. Our results, studying adenosine receptors as a model heteromer, point to three important new findings. First, the predominant stoichiometry in cells expressing A₁R-A_2AR heteromers is 2:2; that is, a dimer of dimers (tetramer). Second, two different heterotrimeric G proteins can couple to heteromers, the overall complex constituting a functional unit. Third, the molecular orientation within the heteromer complex affords various qualitatively different interfaces; the two more relevant are the inter-protomer heteromeric interface and the inter-G-protein interface. Presumably, the two interfaces provide the key characteristic of heteromers: the ability of one protomer/G-protein complex to influence the signaling of the other. Surely, allosteric effects occurring between heteroreceptors and between G_s and G_i proteins are due to conformational changes transmitted along the intimately interacting molecules in the complex. In our controlled cell transfection system, which expressed a low density of receptors, minor species formed by monomers and trimers were found in addition to a predominance of tetramers in the plasma membrane, strongly supporting the occurrence of an in vivo dynamic distribution of receptors.

Adenosine was, from an evolutionary point of view, one of the first extracellular regulators given that it is involved in energy and nucleic acid metabolisms. Adenosine A₁ and A_2A receptors are expressed in almost every mammalian organ and tissue. In the heart, where adenosine plays a key role in both inotropic and chronotropic regulation, A₁R-mediated cardioprotection did not occur in A_2AR knockout mice, suggesting an interaction between A₁ and A_2A receptors. In neurons, A₁ and A_2A receptors show co-localization, leading to inter-receptor interactions unveiled by pharmacological treatments. For instance, Okada et al. [27] showed that cAMP-dependent protein kinase A plays a role in the regulation of hippocampal serotonin release mediated by both A₁ and A_2A receptors. Similarly, the control of γ-amino butyric acid transport in astrocytes was attributed to the expression of A₁R-A_2AR heteromers and to a specific mechanism by which the heteromer signals via G_i or G_s depending on the concentration of adenosine [28]. The structural basis of the differential signaling by the heteromer/G-protein macromolecular complex likely implies communication at the receptor-receptor level but also between G_s and G_i. Because the binding of two G proteins to a heterodimer is not feasible due to steric clashes [25], our finding that the A₁R-A_2AR heterotetramer may bind to both G_s and G_i provides a structural framework to interpret experimental data.

Total internal reflection microscopy and single-particle data analysis

Single-particle imaging and tracking were performed on a Nikon Total Internal Reflection Fluorescence (TIRF) system, as detailed in Additional file 11: Supplementary Methods. Typically, 500 readouts of a 512 × 512-pixel region, the full array of the CCD chip, were acquired. For single-particle data analysis, parameters were calculated by applying the equations described in Additional file 11: Supplementary Methods.

Cell culture and transient transfection

HEK-293T cells were grow at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fischer Scientific, Madrid, Spain) supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 5 % (v/v) heat-inactivated fetal bovine serum (FBS) (all supplements were from Invitrogen, Paisley, UK). Cells were transiently transfected with cDNA corresponding to receptors, fusion proteins, A_2AR mutants, or G-protein minigene vectors obtained as detailed in an expanded view by the polyethylenimine (PEI; SigmaAldrich, Cerdanyola del Vallès, Spain) method. Sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin dilutions as standards. For single-particle imaging, cells were seeded into six-well plates containing glass coverslips (No. 1, round, 24 mm; Assistent, Sondheim, Germany) or into the Lab-Tek Chambered #1.0 Borosilicate Coverglass System (Nunc, Thermo Fisher Scientific, Schwerte, Germany). Cell transient transfections were performed with Lipofectamine™ 2000 (Invitrogen, Life Technologies, Darmstadt, Germany) or FuGENE 6 (Roche Applied Science, Indianapolis, IN, USA) and the application of 0.1–0.2 μg plasmid DNA per well. Before each experiment, cells were washed three times with 200 μL phenol red-free DMEM.

Plasmids

DNA sequences encoding amino acid residues 1–155 and 155–238 of YFP Venus protein, and amino acids residues 1–229 and 230–311 of RLuc8 protein were subcloned in the pcDNA3.1 vector to obtain the YFP Venus and RLuc8 hemi-truncated proteins. The human cDNAs for adenosine receptors, A_2AR and A₁R, cloned into pcDNA3.1, were amplified without their stop codons using sense and antisense primers harboring unique EcoRI and BamHI sites to clone receptors into the pcDNA3.1RLuc vector (pRLuc-N1; PerkinElmer, Wellesley, MA, USA), and EcoRI and KpnI to clone A_2AR, A₁R, or GHS1a into the pEYFP-N1 vector (enhanced yellow variant of GFP; Clontech, Heidelberg, Germany). G_αs cloned into the SFV1 vector, G_αi cloned into the pcDNA3.1 vector, or G_γ cloned into the pEYFP-C1 vector were amplified without their stop codons using sense and antisense primers harboring unique HindIII and BamHI sites to clone them into the pcDNA3.1-Rluc vector, or EcoRI and KpnI to clone G_αs into the pEYFP-N1 vector. The amplified fragments were subcloned to be in-frame with restriction sites of the pcDNA3.1RLuc or pEYFP-N1 vectors to give plasmids that expressed proteins fused to RLuc or YFP on the N-terminal end (G_αs-RLuc, G_αi-RLuc, G_γ-RLuc, G_αs-YFP, and G_γ-YFP) or the C-terminal end (A₁R-RLuc, A_2AR-RLuc, A₁R-YFP, A_2AR-YFP, and GHS1a-YFP). The human cDNAs for A₁R or GHS1a were subcloned into pcDNA3.1-nRLuc8 or pcDNA3.1-nVenus to give plasmids that expressed A₁R or GHS1a fused to either nRLuc8 or nYFP Venus on the C-terminal end of the receptor (A₁R-nRLuc8 and A₁R-nVenus or GHS1a-nRLuc8 and GHS1a-nVenus). The cDNAs for human A_2A or GHS1a receptors were subcloned into pcDNA3.1-cRLuc8 or pcDNA3.1-cVenus to give plasmids that expressed receptors fused to either cRLuc8 or cYFP Venus on the C-terminal end of the receptor (A_2AR-cRLuc8 and A_2AR-cVenus or GHS1a-cRLuc8 and GHS1a-cVenus). Expression of constructs was tested by confocal microscopy and the receptor-fusion protein functionality by measuring ERK1/2 phosphorylation and cAMP production, as described previously [13, 14, 17, 29].

“Minigene” plasmid vectors are constructs designed to express relatively short polypeptide sequences following their transfection into mammalian cells. Here, we used minigene constructs encoding the carboxyl-terminal 11-amino acid residues from G_α subunits of G_i1/2 (G_i minigene) or G_s (G_s minigene) G proteins; the resulting peptides inhibit G-protein coupling to the receptor and consequently inhibit the receptor-mediated cellular responses as previously described [24]. The cDNA encoding the last 11 amino acids of human G_α subunit corresponding to G_i1/2 (I K N N L K D C G L F) or G_s (Q R M H L R Q Y E L L), inserted in a pcDNA3.1 plasmid vector, were generously provided by Dr Heidi Hamm.

Energy transfer assays

For BRET and complementation BRET assays, HEK-293T cells were transiently cotransfected with a constant amount of cDNA encoding for proteins fused to RLuc, nRLuc8, or cRLuc8, and with increasing amounts of the cDNA corresponding to proteins fused to YFP, nYFP Venus, or cYFP Venus (see figure legends). To quantify protein-YFP expression or protein-reconstituted YFP Venus expression, cells (20 μg protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read in a FLUOstar OPTIMA Fluorimeter (BMG Labtechnologies, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 400 nm reading. Protein fluorescence expression was determined as the fluorescence of the sample minus the fluorescence of cells expressing the BRET donor alone. For BRET measurements, the equivalent of 20 μg of cell suspension were distributed in 96-well microplates (Corning 3600, white plates; Sigma) and 5 μM coelenterazine h (Molecular Probes, Eugene, OR, USA) was added. After 1 min for BRET or after 5 min for BRET with bimolecular fluorescence complementation, the readings were collected using a Mithras LB 940 that allows the integration of the signals detected in the short-wavelength filter at 485 nm (440–500 nm) and the long-wavelength filter at 530 nm (510–590 nm). To quantify protein-RLuc or protein-reconstituted RLuc8 expression, luminescence readings were also performed 10 min after adding 5 μM coelenterazine h. The net BRET was defined as [(long-wavelength emission)/(short-wavelength emission)] – Cf, where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the donor construct expressed alone in the same experiment. BRET is expressed as miliBRET units (mBU; net BRET × 1000).

Computational model of the A₁R-A_2AR tetramer in complex with G_i and G_s

The crystal structure of inactive A_2AR [PDB:4EIY] [30] was used for the construction of human A_2AR [UniProt:P29274] and A₁R [UniProt:P30542] homology models using Modeller 9.12 [31]. These receptors share 51 % of sequence identity and 62 % of sequence similarity, excluding the C-terminal after helix 8. Intracellular loop 3 (ICL3) of A_2AR (Lys209–Gly218) and A₁R (Asn212–Ser219) were modeled using Modeller 9.12 [31] using ICL3 of squid rhodopsin [PDB:2Z73] as a template. The C-terminus tails of A₁R, containing 16 amino acids (Pro311–Asp326), and of A_2AR, containing 102 amino acids (Gln311–Ser412), were modeled as suggested for the oxoeicosanoid receptor (OXER) [32] (see Additional file 9: Figure S9 for details). The N-terminus of A₁R and A_2AR were not included in the model. The “active” conformations of A₁R bound to G_i and A_2AR bound to G_s were modeled using the crystal structure of β₂-AR in complex with G_s [PDB:3SN6] [33]. The globular α-helical domain of the α-subunit was modeled in the “closed” conformation [34], using the crystal structure of [AlF₄ ⁻]-activated G_i [PDB:1AGR]. The location of YFP [PDB:2RH7] attached to the C-tail of A_2AR was determined as suggested for the OXER [32] (see Additional file 9: Figure S9 for details). Rluc [PDB:2PSD] and YFP were fused to the to the N-terminus of the α-subunits and γ-subunits of G_i and G_s by a covalent bond. The structures of adenosine receptor oligomers were modeled via the TM4/5 interface for homodimerization, using the oligomeric structure of the β₁-AR [PDB:4GPO] [4], or via the TM5/6 interface for heterodimerization, using the structure of the μ-OR [PDB:4DKL] [3]. The G_i-bound A₁R and G_s-bound A_2AR protomers were rotated 10° to avoid the steric clash of the N-terminal helix of G_i and G_s with the C-terminal helix (Hx8) of G_s-unbound A_2AR and G_i-unbound A₁R, respectively. This computational model, without Rluc and YFP, was placed in a rectangular box containing a lipid bilayer (814 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - POPC -) with explicit solvent (102,973 water molecules) and a 0.15 M concentration of Na⁺ and Cl⁻ (1762 ions). This initial complex was energy-minimized and subsequently subjected to a 10 ns MD equilibration, with positional restraints on protein coordinates. These restraints were released and 500 ns of MD trajectory were produced at constant pressure and temperature (see Additional file 10: Movie M1). Computer simulations were performed with the GROMACS 4.6.3 simulation package [35], using the AMBER99SB force field as implemented in GROMACS and Berger parameters for POPC lipids. This procedure has been previously validated [36].

Availability of data and materials

The crystal structures 4EIY, 2Z73, 3SN6, 1AGR, 2RH7, 2PSD, 4GPO, and 4DKL are available from PDB (http://www.rcsb.org). All other relevant data are within the paper and its Additional files.

We acknowledge the technical help provided by Jasmina Jiménez (CIBERNED, University of Barcelona). This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnología (SAF2009-07276, SAF2010-18472, SAF2011-23813, SAF2013-48271-C2-2-R; those grants may include FEDER funds), the Max Planck Society, the German Cancer Research Center, and the German Ministry for Education and Research (BMBF). PJM and LP participate in the European COST Action CM1207 (GLISTEN). Authors gratefully acknowledge the computer resources provided by the Barcelona Supercomputing Center - Centro Nacional de Supercomputación.

Author notes

Affiliations

1. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain

   • Gemma Navarro
   • , Marc Brugarolas
   • , Estefania Moreno
   • , David Aguinaga
   • , Antoni Cortés
   • , Vicent Casadó
   • , Josefa Mallol
   • , Enric I. Canela
   • , Carme Lluís
   • , Peter J. McCormick
   •  & Rafael Franco

2. Institute of Biomedicine of the University of Barcelona (IBUB), Barcelona, Spain

   • Gemma Navarro
   • , Marc Brugarolas
   • , Estefania Moreno
   • , David Aguinaga
   • , Antoni Cortés
   • , Vicent Casadó
   • , Josefa Mallol
   • , Enric I. Canela
   • , Carme Lluís
   • , Peter J. McCormick
   •  & Rafael Franco

3. Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona, Barcelona, 08028, Spain

   • Gemma Navarro
   • , Marc Brugarolas
   • , Estefania Moreno
   • , David Aguinaga
   • , Antoni Cortés
   • , Vicent Casadó
   • , Josefa Mallol
   • , Enric I. Canela
   • , Carme Lluís
   • , Peter J. McCormick
   •  & Rafael Franco

4. Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain

   • Arnau Cordomí
   • , Laura Perez-Benito
   •  & Leonardo Pardo

5. Max Planck Institute for Intelligent Systems, Heisenbergstrasse 3, 70569, Stuttgart, Germany

   • Monika Zelman-Femiak
   •  & Ana J. García-Sáez

6. German Cancer Research Center, Bioquant, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany

   • Monika Zelman-Femiak
   •  & Ana J. García-Sáez

7. Interfaculty Institute of Biochemistry, Hoppe-Seyler-Strasse 4, 72076, Tübingen, Germany

   • Ana J. García-Sáez

8. School of Pharmacy, University of East Anglia, Norwich, NR4 7TJ, UK

   • Peter J. McCormick

Corresponding authors

Correspondence to Peter J. McCormick or Rafael Franco.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

GN performed the molecular biology. GN, MB, EM, and DA perfomed BRET experiments. MZ-F performed single-particle tracking experiments. AC and LP-B performed molecular modeling studies. AC, VC, JM, and EIC analyzed the data. CL, LP, AJG-S, PJM, and RF designed the experiments, supervised the work in the respective laboratories and wrote the manuscript.. All authors read and approved the final manuscript.

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