Symmetrical TM interfaces in the A 2A R-D 2 R heterotetramer

To identify the arrangement of A 2A R and D 2 R protomers in the heterotetramer (TMs involved in the homo and heterodimerization interfaces), we used synthetic peptides with the amino acid sequence of TMs 1–7 of A 2A R and D 2 R (TMs and TM peptides are abbreviated TM 1, TM 2, … and TM1, TM2, … respectively) fused to the HIV transactivator of transcription (TAT) peptide, which determines the orientation of the peptide when inserted in the plasma membrane (see ref.11 and Methods section). Peptides were first tested in bimolecular fluorescence complementation (BiFC) experiments, in HEK-293T cells expressing receptors fused to two complementary halves of YFP (Venus variant; cYFP and nYFP). Functionality of all fused receptors was shown with cAMP accumulation experiments (Supplementary Fig. 1). Fluorescence was detected when cells were transfected with A 2A R-nYFP and A 2A R-cYFP cDNA (broken lines in Fig. 1a) or with D 2 R-nYFP and D 2 R-cYFP cDNA (broken lines in Fig. 1b), indicating the formation of both A 2A R-A 2A R and D 2 R-D 2 R homodimers. Notably, when BiFC assay was performed in the presence of TM peptides (Fig. 1a, b), fluorescence complementation of A 2A R-nYFP and A 2A R-cYFP was only significantly reduced in the presence of TM6 of A 2A R (Fig. 1a; see Methods and Supplementary Fig. 2 for justification of the optimal concentration and time of incubation of the TM peptides). Similarly, only TM6 of D 2 R reduced fluorescence complementation of D 2 R-nYFP and D 2 R-cYFP (Fig. 1b). These results indicate that TM 6 forms part of a symmetric interface for both A 2A R and D 2 R homodimers when expressed alone. The same results were obtained in cells expressing A 2A R-nYFP and A 2A R-cYFP co-transfected with non-fused D 2 R cDNA (Fig. 1a) or in cells expressing D 2 R-nYFP and D 2 R-cYFP co-transfected with non-fused A 2A R cDNA (Fig. 1b). These results therefore indicate that TM 6 also forms part of a symmetric interface for both A 2A R and D 2 R homodimers in the heterotetramer. Fluorescence was also detected in cells expressing A 2A R-nYFP and D 2 R-cYFP (broken lines in Fig. 1c), indicating the formation of A 2A R–D 2 R heteromers. This fluorescence was only significantly reduced in the presence of TM4 and TM5 of both A 2A R and D 2 R (Fig. 1c), suggesting a TMs 4/5 interface for A 2A R and D 2 R heterodimer in the heterotetramer. Additional evidence of heteromer formation via TMs 4/5 was obtained from proximity ligation assay (PLA). This technique permits the direct detection of molecular interactions between two proteins without the need of fusion proteins. A 2A R–D 2 R heteromers were observed as red punctate staining in HEK-293T cells expressing both A 2A R and D 2 R (Supplementary Fig. 3a–c). Pretreatment of cells with TM4 and TM5 of A 2A R and D 2 R but not with TM6 or TM7 (negative control), significantly decreased PLA staining (Supplementary Fig. 3d), decreasing the number of stained cells and red spots per stained cell (Fig. 2a), supporting TMs 4/5 as the interface of the A 2A R–D 2 R heteromer.

Fig. 1 Quaternary structure of A 2A R-D 2 R heterotetramer coupled to Gs and Gi proteins. a–c BiFC experiments in HEK-293T cells transfected with A 2A R-nYFP (0.5 μg) and A 2A R-cYFP (0.5 μg) cDNA in the absence or presence of D 2 R cDNA (0.5 μg) (a), with D 2 R-nYFP (0.75 μg) and D 2 R-cYFP (0.75 μg) cDNA in the absence or the presence of A 2A R cDNA (0.4 μg) (b) or with A 2A R-nYFP (0.6 μg) and D 2 R-cYFP (0.6 μg) cDNA (c); cells were treated for 4 h with medium (broken lines) or 4 μM of indicated TM peptides (numbered 1–7) of A 2A R (green squares) or D 2 R (orange squares) before addition of medium, CGS21680 (CGS; 100 nM) or quinpirole (Q; 1 μM); fluorescence was detected at 530 nm and values (in means ± SEM) are expressed as fluorescence arbitrary units (n = 8, with triplicates); *, **, and *** represent significantly lower values as compared to control values (p < 0.05, p < 0.01 and p < 0.001, respectively; one-way ANOVA followed by Dunnett’s multiple comparison tests). d Computational model of the A 2A R-D 2 R heterotetramer built using the experimental interfaces predicted in panels (a–c) (TMs 4/5 for heterodimerization and TM 6 for homodimerization) with Gs and Gi binding to the external protomers; schematic slice-representation (left) and the constructed molecular model (right; with the same color code as the schematic slice-representation), viewed from the extracellular side Full size image

Fig. 2 Functional A 2A R–D 2 R heterotetramers in transfected cells. a Quantification from PLA experiments (see Supplementary Fig. 1) performed in HEK-293T cells transfected with 0.4 μg of A 2A R and 0.5 μg of D 2 R cDNA treated for 4 h with medium (control) or 4 μM of indicated TM peptides of A 2A R or D 2 R; values are expressed as the ratio between the number of red spots representing heteromers in confocal microscopy images and the number of cells showing spots (r) (30–50 cells from three independent preparations); % values represent the percentage of cells showing one or more red spots; ***p < 0.001, as compared to control (one-way ANOVA followed by Dunnett’s multiple comparison tests). b cAMP production in HEK-293T cells transfected as in (a); cells were incubated overnight with vehicle or pertussis toxin (PTX; 10 ng/ml), or for 2 h with cholera toxin (CTX; 100 ng/ml), and exposed to CGS21680 (CGS; 100 nM) or quinpirole (Q; 1 μM) in the absence or in the presence of forskolin (Fk; 0.5 μM), respectively; values are expressed as percentage over cAMP accumulation in non-treated cells (basal) (n = 5–7, with triplicates); ###p < 0.001, as compared to basal values; ** and ***p < 0.01 and p < 0.001 as compared to Fk, respectively; one-way ANOVA followed by Tukey’s multiple comparison tests. Results are always represented as means ± SEM Full size image

In HEK-293T cells expressing both receptors, the A 2A R agonist CGS21680 (100 nM; minimal concentration with maximal effect) significantly increased basal cAMP and the D 2 R agonist quinpirole (1 μM; minimal concentration with maximal effect) decreased forskolin-induced cAMP (Fig. 2b). Pertussis toxin, by catalyzing ADP-ribosylation of the alpha-subunit of Gi, impeded D 2 R-mediated Gi activation and thus the ability of quinpirole to inhibit forskolin-induced cAMP accumulation (Fig. 2b). Cholera toxin, by selectively catalyzing ADP-ribosylation of the alpha-subunit of Gs and leading to persistent AC stimulation, impeded an additional effect of CGS21680 but left unaltered the Gi-mediated quinpirole-induced inhibition of forskolin-induced cAMP accumulation (Fig. 2b). These results support the coupling of A 2A R and D 2 R to their respective cognate Gs and Gi proteins in the A 2A R–D 2 R heterotetramer. We could then demonstrate that neither A 2A R or D 2 R activation leads to rearrangements of the TM interfaces in the A 2A R–D 2 R heterotetramer, since, in the presence of CGS21680 (100 nM) or quinpirole (1 μM), fluorescence in cells expressing A 2A R-nYFP and D 2 R-cYFP was still selectively reduced by TM4 and TM5 of A 2A R and D 2 R (Fig. 1c). Similarly, A 2A R activation by CGS21680 (Fig. 1a) or D 2 R activation by quinpirole (Fig. 1b) did not modify the corresponding specific homomer TM 6 interface determined in ligand-free experiments.

We then constructed a molecular model of the A 2A R–D 2 R heterotetramer (Fig. 1d), considering: (i) the crystal structures of GPCRs and G proteins, as well as homology models (see Methods section); (ii) the structural details of TM interfaces of GPCR oligomers, observed in crystal structures12 as well as predicted by molecular dynamics simulations (see Methods section); (iii) the results from BiFC experiments with interfering TM peptides; (iv) the general assumption of a common minimal functional unit of GPCRs constituted by a homodimer coupled to its cognate G protein (see Introduction section); (v) the suggested tetrameric structure of the A 2A R–D 2 R heteromer constituted by two interacting homodimers, from previous results obtained with bioluminescence resonance energy transfer (BRET) experiments with complementation of both the donor and the acceptor biosensors10; and (vi) the previously enunciated assumption about the necessity of a simultaneous activation of Gs and Gi coupled to the interacting catalytic domains of the same molecule of AC for a canonical antagonistic interaction8. This resulted in one minimal computational solution that accommodates the TMs 4/5 interface for A 2A R–D 2 R heterodimerization and the TM 6 interface for both A 2A R–A 2A R and D 2 R–D 2 R homodimerization (see Methods and Supplementary Fig. 4). The existence of these interfaces implies two internal interacting A 2A R and D 2 R protomers and two external A 2A R and D 2 R protomers in which the α-subunits of Gi and Gs bind to the corresponding external protomers of the D 2 R or A 2A R homodimers. This would be the only feasible configuration to avoid any steric clash between the two G proteins simultaneously bound to the complex. Finally, the model also predicts a large distance between both βγ-subunits (Fig. 1d).

Asymmetrical TM interfaces of the heterotetramer with AC5

Although several studies have provided direct evidence for pre-coupling between G protein subunits and AC7,13,14,15, specifically with the AC NT7,14, to our knowledge, the existence of pre-coupling between TMs of a GPCR and TMs of AC had not been previously addressed. We first analyzed the ability of AC5 to establish direct intermolecular interactions with A 2A R or D 2 R or with A 2A R–D 2 R heteromers via saturation BRET experiments in the absence of ligands (results are always shown as means ± SEM). Clear-cut saturation BRET curves were obtained with HEK-293T cells transfected with a constant amount of A 2A R fused to Renilla Luciferase (A 2A R-RLuc) cDNA and increasing quantities of AC5 fused to YFP (AC5-YFP) cDNA (Fig. 3a; BRET max = 54 ± 6 mBU and BRET 50 = 42 ± 13) or with cells transfected with a constant amount of D 2 R-RLuc cDNA and increasing amounts of AC5-YFP cDNA (Fig. 3b; BRET max = 38 ± 5 mBU and BRET 50 = 28 ± 14), indicating that AC5 interacts with A 2A R or D 2 R in the absence of ligands. Also, saturation BRET curves were obtained when HEK-293T cells transfected with A 2A R-RLuc and increasing amounts of AC5-YFP cDNAs were co-transfected with D 2 R cDNA (Fig. 3c; BRET max = 39 ± 3 mBU and BRET 50 = 24 ± 8) or when cells transfected with D 2 R-RLuc and increasing amount of AC5-YFP cDNAs were co-transfected with A 2A R cDNA (Fig. 3d; BRET max = 30 ± 2 mBU and BRET 50 = 20 ± 7). All saturation BRET curves were best-fitted to a monophasic model. We also verified that overexpression of AC5 did not alter A 2A R–D 2 R heteromerization with BRET experiments in HEK-293T cells transfected with A 2A R-Rluc (0.4 μg) and D 2 R-YFP (0.6 μg) and increasing amounts of AC5 cDNA. No BRET differences were observed between the results obtained with 0, 0.3, 1.0 and 3.0 μg of AC5 cDNA (56 ± 7, 53 ± 6, 53 ± 3, and 52 ± 4 mBU, respectively). Altogether, these results suggest that AC5 oligomerize with A 2A R-D 2 R heteromers in the absence of ligands.

Fig. 3 Involvement of receptor TMs in A 2A R–D 2 R heterotetramer-AC5 oligomerization. a–d BRET saturation experiments in HEK-293T cells transfected with 0.5 μg of A 2A R-Rluc cDNA and increasing amounts of AC5-YFP cDNA (0.3–2.5 μg) not co-transfected (a) or co-transfected (c) with D 2 R cDNA (0.5 μg), or with 0.75 μg of D 2 R-Rluc cDNA and increasing amounts of AC5-YFP cDNA (0.3–2.5 μg) not co-transfected (b) or co-transfected (d) with A 2A R cDNA (0.4 μg); the relative amount of BRET is given as a function of 1000× the ratio between the fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc) and expressed as milli BRET units (mBU) (6–8 experiments, with duplicates, grouped as a function of the amount of BRET acceptor). e, f BiFC experiments in HEK-293T cells transfected with AC5-nYFP (0.75 μg), A 2A R-cYFP (0.5 μg) and D 2 R (0.75 μg) cDNA (e) or AC5-nYFP (0.75 μg), D 2 R-cYFP (0.75 μg) and A 2A R (0.4 μg) cDNA (f); cells were treated for 4 h with medium (dotted lines) or 4 μM of indicated TM peptides (numbered 1–7) of A 2A R (e) or D 2 R (f) before addition of medium, CGS21680 (CGS; 100 nM; e) or quinpirole (Q; 1 μM; f); fluorescence was detected at 530 nm and values are expressed as arbitrary fluorescent units (n = 8, with triplicates); *, ** and *** represent significantly lower values as compared to control values (p < 0.05, p < 0.01 and p < 0.001, respectively; one-way ANOVA followed by Dunnett’s multiple comparison tests). Results are always represented as means ± SEM Full size image

Next, we performed BiFC assays in HEK-293T cells expressing AC5-nYFP, A 2A R-cYFP, and D 2 R (Fig. 3e) as well as AC5-nYFP, D 2 R-cYFP and A 2A R (Fig. 3f). Normal functionality of AC5-YFP has been previously reported16. Significant fluorescence was detected in all cases, providing additional support to direct interactions between AC5 and A 2A R–D 2 R heteromers (broken lines in Fig. 3e, f). To determine the possible involvement of receptor TMs in the A 2A R–D 2 R heterotetramer-AC5 interface, we performed BiFC experiments with all different A 2A R (Fig. 3e) or D 2 R (Fig. 3f) TM peptides. In the absence of ligands, pretreatment of cells with TM1, TM5, or TM6 of A 2A R significantly decreased complementation between AC5 and A 2A R (Fig. 3e, top panel). Similarly, pretreatment with TM1, TM4, TM5, or TM6 of D 2 R significantly decreased complementation between AC5 and D 2 R (Fig. 3f, top panel). This suggests a discrete interaction between TM1 of both receptors with AC5. Since TMs 4–5 of the inner receptor protomers and TMs 6 of inner and outer receptor protomers participate in homo- and heterodimerization (see above), respectively, their apparent involvement in the interactions with AC5 must be indirect, implying that the optimal interaction of the A 2A R–D 2 R heterotetramer and AC5 requires the optimal quaternary structure of the heterotetramer. When BiFC experiments were performed in the presence of CGS21680 (100 nM, Fig. 3e, bottom panel) or quinpirole (1 μM, Fig. 3f, bottom panel), the pattern of interfering synthetic peptides changed: In addition to TM5 and TM6 of A 2A R and D 2 R, TM7 of A 2A R and TM2 of D 2 R decreased fluorescence complementation in the presence of CGS21680 and quinpirole, respectively, while TM1 of A 2A R and D 2 R were no longer effective (Fig. 3e, f).

We then investigated the involvement of TMs of AC5 TMs in the oligomerization with A 2A R–D 2 R heteromers. Since the structure of M1 and M2 domains of any AC isoform is unknown, we used five commonly used algorithms to predict their most probable TMs (Supplementary Table 1). All algorithms predicted the same six TMs for the M2 domain (TM 7 to TM 12), but there was discrepancy on the predicted TMs of the M1 domain. Taking into account the orientation of the predicted TM helices, only Uniprot and TOPCONS solutions were compatible with the well-established intracellular N-terminal and C-terminal domains of AC57. First, TM peptides mimicking right-oriented TMs derived from Uniprot predictions (abbreviated TM1 to TM12) were tested for their ability to destabilize complementation in HEK-293T cells expressing AC5-nYFP, A 2A R-cYFP, and D 2 R (Fig. 4a), as well as AC5-nYFP, D 2 R-cYFP and A 2A R (Fig. 4b). In the absence of agonists, pretreatment of cells with TM1 or TM12 significantly decreased complementation between AC5 and A 2A R, while TM5 showed a small but not significant decrease (Fig. 4a, top panel). Similarly, pretreatment with TM6 or TM12 significantly decreased complementation between AC5 and D 2 R while TM5 again showed a small but not significant decrease (Fig. 4b, top panel). Remarkably, when BiFC experiments were performed in the presence of CGS21680 (100 nM, Fig. 4a, bottom panel) or quinpirole (1 μM, Fig. 4b, bottom panel), the pattern of interfering synthetic peptides dramatically changed. When receptors were activated, TM1, TM2, TM3, TM5 and TM6 significantly decreased fluorescence complementation between AC5-nYFP and A 2A R-cYFP and between AC5-nYFP and D 2 R-cYFP. The results imply a major rearrangement of the membrane-spanning domains of the activated pre-coupled complex with an increase in the number of TMs of AC5 directly or indirectly involved in the oligomerization with the A 2A R–D 2 R heterotetramer.

Fig. 4 Involvement of AC5 TMs in A 2A R-D 2 R heterotetramer-AC5 oligomerization. a–d BiFC experiments in HEK-293T cells transfected with AC5-nYFP (0.75 μg), A 2A R-cYFP (0.5 μg) and D 2 R (0.75 μg) cDNA (a, c) or AC5-nYFP (0.75 μg), D 2 R-cYFP (0.75 μg) and A 2A R (0.4 μg) cDNA (b, d); cells were treated for 4 h with medium (dotted lines) or 4 μM of indicated TM peptides predicted from Uniprot algorithm (numbered 1–12) (a, b) or control peptides (numbered 2n−6n and 5b; see text) (c, d), before addition of medium, CGS21680 (CGS; 100 nM) or quinpirole (Q; 1 μM); fluorescence was detected at 530 nm and values (in means ± SEM) are expressed as arbitrary fluorescent units (n = 8, with triplicates); *, ** and *** represent significantly lower values as compared to control values (p < 0.05, p < 0.01, and p < 0.001, respectively; one-way ANOVA followed by Dunnett’s multiple comparison tests). e–g Schematic slice-representations of A 2A R–D 2 R heterotetramer-AC5 models: heterotetramer coupled with two AC5 molecules in the absence (e) and in the presence (f) of agonists; extension of the agonist-bound complex with a second A 2A R–D 2 R heterotetramer, with simultaneous binding of both Gαs and Gαi to the central C1 and C2 domains of AC5 (g). Schematic slice-representation viewed from the extracellular side of the A 2A R–D 2 R heterotetramer in complex with Gs, Gi, and AC5 in the absence and presence of agonists are shown in Supplementary Fig. 6 Full size image

Opposite-oriented TM peptides, abbreviated as TM2n, TM3n, TM4n, TM5n and TM6n, were tested to examine the specificity of their destabilizing effect (see Supplementary Table 2), which should insert in the membrane in the opposite direction and act as scrambled control peptides. The peptides were tested in HEK-293T cells expressing AC5-nYFP, A 2A R-cYFP, and D 2 R (Fig. 4c) as well as AC5-nYFP, D 2 R-cYFP, and A 2A R (Fig. 4d) in the absence or in the presence of agonists. The same as TM4, TM4n did not have a significant effect, and TM2n, TM3n and TM6n did behave as negative controls to their opposite-oriented peptides, since they did not decrease AC5-nYFP-A 2A R-cYFP or AC5-nYFP-D2R-cYFP complementation in the absence (Fig. 4c, d, top panels) or in the presence (Fig. 4c, d, bottom panels) of agonists. Intriguingly, both TM5 and the opposite-oriented TM5n were able to decrease AC5-nYFP-A 2A R-cYFP and AC5-nYFP-D2R-cYFP complementation (Fig. 4c, d). Importantly, TM5 and TM5n had the lowest hydrophobicity as compared to all the other putative TM sequences (Supplementary Table 1), decreasing the probability of being embedded in the membrane bilayer17. This could indicate that the AC5 325–345 amino acid sequence forms part of the second intracellular loop (IL2), which could establish direct or indirect intermolecular interactions with the A 2A R-D 2 R heteromer. Then, the 348–368 aa sequence predicted by the TOPCONS algorithm (TM 5b in Supplementary Table 1), which has the right orientation, becomes a very plausible TM that could interact with the A 2A R–D 2 R heterotetramer. In fact, TM5b peptide significantly decreased AC5-nYFP-A 2A R-cYFP or AC5-nYFP-D2R-cYFP complementation in the absence or in the presence of agonists (Fig. 4c,d). In agreement with this interpretation, a scrambled TM5-TM5n peptide (AC5-TM5s in Supplementary Table 2) did not decrease AC5-nYFP-A 2A R-cYFP or AC5-nYFP-D2R-cYFP complementation in the absence of ligands (93 ± 7, and 95 ± 6%, respectively, in means ± SEM and expressed as percentage of change of fluorescent values without peptide; n = 9, with triplicates). As additional controls, we also tested AC5 TM1 to TM12 peptides on A2AR-nYFP-D2R-cYFP complementation and all the D2R TM and A2AR TM peptides on AC5-nYFP-A2AR-cYFP and AC5-nYFP-D2R-cYFP complementation, respectively, in the absence of ligands; no changes in BiFC were observed under any condition (Supplementary Fig. 5). Considering TM 1, TM 2, TM 3, TM 4, TM 5b, and TM 6 as the six putative TMs of the M1 domain of AC5, altogether these results indicate that TM 1 and TM 6, as well as IL2 and TM 5b, are involved in pre-coupling of A 2A R-D 2 R heterotetramer and AC5 in the absence of agonists. Upon A 2A R or D 2 R activation there is a rearrangement with an apparent participation of almost all TMs of the M1 domain.

Two A 2A R–D 2 R heterotetramers and two AC5 molecules

It seems reasonable to hypothesize that the membrane-spanning domains of AC5 are formed by two interacting antiparallel six-helix-bundle domains (M1–M2) with an elliptical ring shape7. In the absence of ligands, since it is not feasible that TM 1 from both A 2A R and D 2 R interact simultaneously with the same TM 5 or TM 12 or the same IL2 of a single AC5 molecule, this suggests the presence of two AC5 molecules simultaneously binding to the A 2A R–D 2 R heterotetramer in complex with Gi and Gs, possibly with TM1 of D 2 R and TM 1 of A 2A R interacting specifically with TM 1 and TM 6 of AC5, respectively (Fig. 4e). The ability of peptides that mimic TM 5, TM 12, and IL2 of AC to destabilize oligomerization between AC5 and the A 2A R–D 2 R heterotetramer might depend on an indirect modification of their discrete asymmetrical interfaces.

It is well established that the Gα binding site for Gβγ overlaps with the Gα binding sites for the effector, the cytoplasmic domains C1 and C2 of AC. During G protein activation, Gβγ relative movement promotes Gα binding to AC18,19. These swapping interactions can take place within the frame of the A 2A R–D 2 R heterotetramer with two AC5 molecules binding simultaneously to Gs and Gi in the complex (Fig. 4e, f). The rearrangement of TM interfaces between the A 2A R–D 2 R heterotetramer and AC5 upon receptor activation occurs simultaneously with the rearrangement of the Gβγ subunit, by its established stable coupling with the NT of AC516, which facilitates the interaction between the Gα subunit and its corresponding catalytic AC5 domain. This rearrangement in the frame of the heteromer gives a computational molecular model of activated complex schematized in Fig. 4f. Details about the model are shown in Supplementary Fig. 6. However, within the frame of the constraints imposed by a pre-coupled A 2A R–D 2 R heterotetramer-Gs-Gi-AC5 complex, a single A 2A R–D 2 R heterotetramer cannot accomplish the model proposed by Dessauer et al.8, in which one Gs and one Gi bind simultaneously to one single AC5 (see below). Therefore, we propose that AC5 should oligomerize with an additional A 2A R–D 2 R heterotetramer (Fig. 4g). The results with interfering peptides, together with the proposed simultaneous binding of Gs and Gi to AC5, suggest a minimal functional complex composed of two A 2A R–D 2 R heterotetramers and two AC5 molecules (Fig. 4g).

The canonical Gs–Gi antagonistic interaction

To corroborate the proposed model we studied the functional characteristics of the A 2A R–D 2 R heterotetramer-AC5 complex in rat striatal neuronal primary cultures, which express endogenous A 2A R–D 2 R heteromer complexes20. Furthermore, AC5 is the predominant AC subtype in striatal neurons21. First, we analyzed by PLA the expression of A 2A R–D 2 R heteromers, as well as the ability of the synthetic peptides mimicking the TMs of A 2A R and D 2 R to modify the quaternary structure of the endogenous A 2A R–D 2 R heterotetramer. A 2A R–D 2 R heteromers were observed as red punctate staining in neuronal cells (Fig. 5a, b). As expected, pretreatment of cells with TM4 and TM5 of A 2A R and D 2 R, but not with TM6 or with TM7, produced a significant decrease in the number of red spots per cell (Fig. 5b, c). These results mirrored those obtained in HEK-293T cells (see Fig. 2a and Supplementary Fig. 3), confirming the same TMs 4/5 interface of A 2A R–D 2 R heteromers in striatal cells and that TM6 does not destabilize heterodimerization. PLA experiments were also performed with a recently characterized AC5 antibody22. A 2A R-AC5 and D 2 R-AC5 complexes could be revealed as red punctate staining in neuronal cells (Supplementary Fig. 7). Next, we measured cAMP production to analyze the functional characteristics of the A 2A R–D 2 R heteromer and the effect of the interfering peptides. As expected, CGS21680 (100 nM) increased the synthesis of cAMP (Fig. 6a) and quinpirole (1 μM) decreased forskolin-induced cAMP accumulation (Fig. 6a). Pertussis toxin, selectively counteracted the ability of quinpirole to inhibit forskolin-induced cAMP (Fig. 6b), while cholera toxin impeded the activating effect of CGS21680 while leaving unaltered quinpirole-induced inhibition of forskolin-induced AC5 activation (Fig. 6b). Simultaneous exposure to both agonists demonstrated the ability of quinpirole to inhibit the effect of CGS21680, revealing the canonical Gs–Gi interaction at AC5 (Fig. 6a).

Fig. 5 A 2A R-D 2 R heterotetramer expression in striatal neurons in culture. Proximity ligation assay (PLA) in rat striatal primary cultures. a, c Confocal microscopy images (superimposed sections) are shown in which A 2A R-D 2 R heteromers appear as red spots. Primary cultures were treated for 4 h with medium (a) or 4 μM of indicated TM peptides (numbered 1–7) of A 2A R or D 2 R (c); cell nuclei were stained with DAPI (blue); scale bars: 20 μm. b Quantification from PLA experiments: values (in means ± SEM) are expressed as the ratio between the number of red spots and the number of cells showing spots (r) (20–30 neurons from three independent preparations); % values represent the percentage of cells showing one or more red spots; ***p < 0.001, as compared to control (one-way ANOVA followed by Dunnett’s multiple comparison tests) Full size image

Fig. 6 Canonical Gs–Gi antagonistic interaction in striatal neurons in culture. a, b cAMP production determined in rat striatal primary cultures incubated overnight with vehicle (a) or with pertussis toxin (PTX; 10 ng/ml), or for 2 h with cholera toxin (CTX; 100 ng/ml) (b), and exposed to CGS21680 (CGS; 100 nM), quinpirole (Q; 1 μM) or both in the absence or in the presence of forskolin (Fk; 0.5 μM), respectively. c–e cAMP production determined in rat striatal primary cultures incubated 4 h with 4 μM of indicated TM peptides of A 2A R (c), D 2 R (d), or AC5 (e) and exposed to agonists as in a, b. Values (in means ± SEM) are expressed as percentage of cAMP accumulation in non-treated cells (basal) (n = 5–7, with triplicates); ###p < 0.001, as compared to basal values; ** and ***p < 0.01 and p < 0.001 as compared to Fk, respectively; &, &&&p < 0.05 and p < 0.001 as compared to CGS, respectively; one-way ANOVA followed by Tukey’s multiple comparison tests Full size image

Accumulation of cAMP was also determined in striatal cultures upon exposure to ligands and interfering TM peptides. Pretreatment with TM7 (as negative control) or with TM6 of A 2A R or D 2 R did not modify receptor signaling or the canonical interaction (Fig. 6c, d). In contrast, although pretreatment with TM4 and TM5 of A 2A R (Fig. 6c) or D 2 R (Fig. 6d) did not modify receptor signaling, it blocked the canonical interaction (Fig. 6c, d). These results indicate that TMs that destabilize receptor heteromerization do not disrupt the individual functional interactions between the receptors and AC5, most probably because of stable pre-coupling between the G proteins and AC5, as recently demonstrated for the specific Gα olf β 2 γ 7 -AC5 complex in the rodent striatum22. Nevertheless, the peptides that destabilize receptor heteromerization alter the correct coupling of AC5 to the complex that allows the simultaneous binding of Gαs and Gαi subunit to AC, impeding the canonical interaction. In conclusion, A 2A R–D 2 R heteromerization is a necessary condition for the canonical antagonistic interaction between Gs-coupled A 2A R and Gi-coupled D 2 R at AC in striatal neurons in culture. In agreement with this conclusion, cAMP accumulation induced by CGS21680 was not counteracted by an agonist of dopamine D 4 R, which does not heteromerize with A 2A R23 (Supplementary Fig. 8). Finally, pretreatment of striatal cultures with interfering peptides TM1, TM6 or TM12 of AC5 did not change receptor signaling but also blocked the canonical interaction, while TM10 was ineffective (Fig. 6e). These results confirm the involvement of the AC5 subtype in striatal cultures and indicate that these AC5 TM peptides are not able to destabilize the interactions between AC5 and the receptors but induce an alteration of the quaternary structure of the complex that impedes the simultaneous binding of Gαs and Gαi subunit to AC5, the canonical interaction. Thus, the correct intermolecular interaction between AC5 and the A 2A R–D 2 R heterotetramer is also a necessary condition for the presence of the canonical Gs–Gi interaction at AC5.