Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain
BMC Biology volume 16, Article number: 24 (2018)
G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A1R or A2AR) can form A1R-A2AR heteromers (A1-A2AHet), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A1R and A2AR, allowing the heteromeric receptor to detect its concentration by integrating the downstream Gi- and Gs-dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A2AR impedes signaling via A1R.
We examined the mechanism by which A1-A2AHet integrates Gi- and Gs-dependent signals. A1R blockade by A2AR in the A1-A2AHet is not observed in the absence of A2AR activation by agonists, in the absence of the C-terminal domain of A2AR, or in the presence of synthetic peptides that disrupt the heteromer interface of A1-A2AHet, indicating that signaling mediated by A1R and A2AR is controlled by both Gi and Gs proteins.
We identified a new mechanism of signal transduction that implies a cross-communication between Gi and Gs proteins guided by the C-terminal tail of the A2AR. This mechanism provides the molecular basis for the operation of the A1-A2AHet as an adenosine concentration-sensing device that modulates the signals originating at both A1R and A2AR.
Adenosine is a purine nucleoside whose relevance in the central nervous system is mainly due to its role in regulating neurotransmitter release . The effects of adenosine are mediated by specific G-protein-coupled receptors (GPCRs) that are coupled to either Gs or Gi heterotrimeric Gαβγ proteins. The endogenous adenosine acts on four receptor subtypes – A1R, A2AR, A2BR, and A3R. Convergent and compelling evidence shows that GPCRs may form complexes constituted by a number of equal (homo) or different (hetero) receptor protomers . As agreed in the field, a GPCR heteromer displays characteristics that are different from those of the constituting protomers, thus giving rise to novel functional entities . Adenosine receptors have been used as a paradigm in the study of receptor homo- and heteromerization. For instance, A1R, which is Gi coupled, and A2AR, which is Gs coupled, form a functional heteromer .
The A1R-A2AR heteromer (A1-A2AHet) is found presynaptically in, inter alia, cortical glutamatergic terminals innervating the striatum and functions as a switch that differentially senses high and low concentrations of adenosine in the inter-synaptic space. Since adenosine has higher affinity for A1R than for A2AR, low concentrations predominantly activate A1R, engaging a Gi-mediated signaling, whereas higher adenosine concentrations also activate A2AR, engaging a Gs-mediated signaling . The physiological role of such a concentration-sensing device is remarkable as it allows adenosine to fine-tune modulate the release of neurotransmitters from presynaptic terminals. However, the mechanism by which A1-A2AHet integrates both Gi- and Gs-dependent signals is not yet understood. We have recently shown, using a combination of single-particle tracking experiments, bioluminescence resonance energy transfer (BRET) assays, and computer modeling, that the (minimal) functional A1-A2AHet/G protein unit is composed by a compact rhombus-shaped heterotetramer (with A1R and A2AR homodimers) bound to two different interacting heterotrimeric G proteins (Gs and Gi) . In the present study, we aim to understand the molecular intricacies underlying the signaling mediated by A1-A2AHet, in which (1) both receptors constituting the heteromer are activated by the same endogenous agonist and (2) is coupled to two different G proteins with opposite effects, i.e., one mediating the inhibition of the adenylate cyclase (Gi) and another mediating the activation of the enzyme (Gs). Our data identifies a new mechanism of signal transduction and provides the molecular basis to understand the unique properties of this heteromer, in which the C-terminal tail of the A2AR influences the Gi-mediated signaling of the partner A1R receptor.
Homodimerization of A1R and A2AR occurs through the transmembrane (TM) 4/5 interface and heterodimerization via the TM5/6 interface in the A1-A2AHet
Our recently published BRET-aided computational model of the A1-A2AHet predicted the TM interfaces involved in homo- (TM4/5) and heterodimerization (TM5/6) . To further confirm this arrangement, we used synthetic peptides with the sequence of TM domains of the A2AR (abbreviated TM1 to TM7) and the A1R (abbreviated TM5 to TM7), fused to the cell-penetrating HIV transactivator of transcription (TAT) peptide , to alter inter-protomer interactions in the A1-A2AHet. These peptides were first tested in bimolecular fluorescence complementation (BiFC) assays in HEK-293 T cells expressing receptors fused to two complementary halves of YFP (cYFP and nYFP) (see Methods). We detected fluorescence in HEK-293 T cells transfected with cDNAs for A2AR-nYFP, A2AR-cYFP, and non-fused A1R (broken lines in Fig. 1a), indicating the formation of the A2AR-A2AR homodimer. Notably, in the presence of interference peptides, we observed a fluorescence decrease only with TM4 and TM5 of A2AR (Fig. 1a), but not with A1R TM peptides (Fig. 1a) or with peptides derived from the orexin receptor (Additional file 1: Figure S1A) used as negative controls. Further negative controls show that A2AR peptides do not alter fluorescence in HEK-293 T cells expressing A1R-nYFP and A1R-cYFP (Additional file 1: Figure S1B). These results therefore confirmed the TM4/5 interface for A2AR homodimerization in the heteromer. Similarly, we detected fluorescence in cells expressing A1R-nYFP and A2AR-cYFP (broken lines in Fig. 1b), indicating formation of the A1-A2AHet. This fluorescence was only reduced in the presence of TM4, TM5, and TM6 peptides of A2AR (Fig. 1b). The involvement of TM5/6 in the heteromer interface was also confirmed by the fact that TM5 and TM6, but not TM7, of A1R reduced fluorescence in cells expressing A1R-nYFP and A2AR-cYFP (Fig. 1b). These results reinforce our previously proposed compact rhombus-shaped arrangement of protomers in which heteromerization of A1-A2AHet occurs via the TM5/6 interface (Fig. 1f). The fluorescence decrease induced by TM4 A2AR peptide indicates that the correct homomerization is a requisite for A1-A2AHet formation and/or that the TM4 peptide interferes with interactions of the TM4 of the external protomer of the A2AR homodimer with the internal protomer of the A1R homodimer (Fig. 1f) . Next, we evaluated whether receptor activation, by the A1R-selective agonist N6-cyclopentyladenosine (CPA), the A2AR-selective agonist 4-[2-[[6-Amino-9-(N-ethyl-β-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid (CGS-21680), or both, modify the heteromer TM interface. As clearly shown in Figs. 1c–e, none of the agonists, used either individually (Figs. 1c, d) or in combination (Fig. 1e), modified the effect of the TM peptides relative to the ligand-free experiments. Therefore, no rearrangements of the TM interface in the A1-A2AHet occurred upon receptor activation.
Next, we investigated whether interference TM peptides, which are able to alter the quaternary structure of the A1-A2AHet as demonstrated by BiFC experiments, are also able to disrupt the heteromer. To do this, proximity ligation assays (PLA) were performed in HEK-293 T cells expressing A1R and A2AR. The PLA assay is a powerful technique to detect protein-protein interactions by assessing proximity between GPCR protomers with high resolution (< 40 nm). A1-A2AHet was observed as red punctate staining (Fig. 2), whereas pretreatment of cells with TM4, TM5, TM6, and TM7 of A2AR did not decrease PLA staining (Fig. 2), indicating that interference peptides can alter the quaternary structure of the heteromer but cannot disrupt heteromerization.
The complex formed by Gs, Gi, and the A1-A2AHet as a signal transduction unit
In order to test the ability of Gs and Gi proteins to interact with the A1-A2AHet, we used BRET assays . Cells were transfected with cDNAs of A1R-nYFP and A2AR-cYFP, which only upon complementation can act as a BRET acceptor (YFP), and Renilla luciferase (Rluc) as a BRET donor fused to either Gi (Gi-Rluc) or Gs (Gs-Rluc). We observed significant energy transfer (Additional file 1: Figure S1C), indicating that Gi and Gs are bound to their respective receptors in the A1-A2AHet.
Next, we tested whether the A1-A2AHet can signal through Gs- and Gi-dependent pathways by measuring cAMP levels in cells expressing both A1R and A2AR. The A1R-selective agonist CPA (100 nM, a concentration producing maximal effect), which was unable to modify cAMP levels in the absence of forskolin (Additional file 1: Figure S2A), decreased forskolin-induced cAMP due to its Gi coupling, and the A2AR-selective agonist CGS21680 (100 nM, a concentration producing maximal effect) increased cAMP due to a Gs coupling (Fig. 3a, control), indicating that both receptors signal via their cognate G protein. We performed the same experiments in cells treated with pertussis (PTX) or cholera (CTX) toxins, which impair Gi- and Gs-mediated signaling, respectively, and in cells transfected with minigenes that encode for peptides blocking the interaction of the receptor with the α subunits of Gi or Gs . As expected, we observed blockade of CPA-induced cAMP decrease by either PTX (Fig. 3a) or the Gi-specific minigene (Fig. 3b), and blockade of CGS21680-induced cAMP increase by CTX (Fig. 3a) or the Gs-specific minigene (Fig. 3b). Strikingly, PTX or Gi-specific minigene (blocking Gi-receptor interaction) also blocked the CGS21680-induced cAMP increase (Fig. 3a, b). Moreover, CTX or the Gs-specific minigene (blocking Gs-receptor interaction) also blocked the CPA-induced cAMP decrease (Fig. 3a, b). Control experiments using these agonists in cells expressing only A1R or A2AR did not show any crossover effect with either toxins or minigenes (Additional file 1: Figures S2B, C, E, F). These results demonstrate that both A1R- and A2AR-mediated signaling in the A1-A2AHet are dependent on the functional integrity of both Gi and Gs proteins. According to this, we observed by BRET experiments that the A2AR agonist-induced interaction between A1-A2AHet and Gs protein diminished in cells pre-treated with PTX (Additional file 1: Figure S1D). We hypothesize that this cross-communication could depend on the ability of α subunits of Gi and Gs coupled to the A1-A2AHet to establish mutual interactions (see below).
To further test for a cross-communication between G proteins in the Gs-Gi-heterotetramer signaling unit, we resolved the real-time signaling signature by using a label-free method, based on optical detection of dynamic changes in cellular density following receptor activation . The magnitude of the signaling by CPA or by CGS 21680 significantly decreased when cells co-expressing both receptors were pre-treated with either PTX or CTX (Fig. 3d). This phenomenon was not observed in cells expressing only A1R (Additional file 1: Figure S2G) or A2AR (Additional file 1: Figure S2H). Again, these results indicate the simultaneous coupling of interacting Gs and Gi proteins within the A1-A2AHet.
Simultaneous activation of both A1R and A2AR with CPA and CGS21680 increased cAMP to similar levels to those obtained with CGS21680 alone and the signal of co-activated receptors was inhibited by both PTX and CTX (Fig. 3c). Therefore, A1R agonist was able to decrease forskolin-induced cAMP (Fig. 3a, b) and yet was unable to decrease A2AR-mediated increases of cAMP (Fig. 3c). Consequently, when both receptors are co-activated in the heterotetramer, only the A2AR-mediated, but not the A1R-mediated signaling occurs. This finding was confirmed in label-free experiments, showing that receptor co-activation with CPA and CGS 21680 did not increase the time-response curve with respect to the activation with CGS 21680 alone (Fig. 3d green and yellow lines, respectively).
It has been shown that the mechanism for receptor-catalyzed nucleotide exchange in G proteins involves a large-scale opening of the α-helical domain (αAH) of the α-subunit, from the Ras domain, allowing GDP to freely dissociate [10,11,12,13]. Notably, our proposed model of the A1-A2AHet positions the αiAH and αsAH domains facing each other (Fig. 3e). The fact that both Gs- and Gi-specific toxins and Gs- and Gi-specific minigenes affect both Gs- and Gi-mediated coupling in the A1-A2AHet suggests that the proposed large-scale conformational changes of αAH domains is mutually dependent. We used molecular dynamics (MD) simulations of the A1-A2AHet in complex with Gs and Gi to evaluate intermolecular distances between the αsAH and αiAH domains when αiAH is in the closed conformation and αsAH is either in the open (Fig. 3e) or in the closed conformation (Fig. 3ef). In a previous report, double electron–electron resonance (DEER) distance distributions between spin labels attached to Arg90 (αiAH domain) and Glu238 (Ras domain) of Gi (the distance between Cα atoms is termed d[Arg90αi-Glu238αi] in the manuscript) or Asn112 (αsAH) and Asn261 (Ras) of Gs (d[Asn112αs-Asn261αs]) permitted to faithfully monitor the equilibrium within the open (distance of ~40 Å) and closed (~20 Å) conformation of the αAH domain . Here, we measured the intermolecular distance between the αsAH and αiAH domains using Cα atoms of Arg90 of αi and Asn112 of αs (d[Arg90αi-Asn112αs]). This d[Arg90αi-Asn112αs] intermolecular distance between αiAH in the closed conformation (d[Arg90αi-Glu238αi]: 11 Å, yellow line in Fig. 3e) and αsAH in the closed conformation (d[Asn112αs-Asn261αs]: 14 Å, green line in Fig. 3e) has an average value of 108 Å for inactive A1-A2AHet (Fig. 3e, dark red line). Activation of A2AR would trigger the opening of αsAH (d[Asn112αs-Asn261αs]: 52 Å; Fig. 3f, green line), necessary for GDP/GTP exchange, decreasing the d[Arg90αi-Asn112αs] distance between αiAH and αsAH to 60 Å (Fig. 3f, dark red line). Although the results are based on a single trajectory, it is unlikely that additional replicates would change, in a significant manner, the distances reported from the simulations. Moreover, the differences between the distances are so substantial that results from more simulations would not have a significant impact. We hypothesize that a similar change occurs with activation of A1R. This indicates that both receptors can signal via their cognate G protein by opening their αAH domain. However, in the compact rhombus-shaped A1-A2AHet model, simultaneous opening of both αAH domains (co-activation with CPA and CGS 21680) would not be possible due to a steric clash in such open conformations (Fig. 3g). Due to this steric clash, MD simulations of this open αiAH-open αsAH conformation in the absence of interference peptides (see below) cannot be performed.
Altering the heteromer interface of A1-A2AHet enables simultaneous Gi and Gs signaling
Next, we investigated whether the correct formation of the A1-A2AHet is a necessary condition for the crosstalk between the Gs- and Gi-signaling units using the interference peptides (TM4, TM5 and TM6 of A2AR, which alter receptor heterodimerization, and TM7 as a negative control). Remarkably, pretreatment of cells expressing A1-A2AHet with the interference peptides did not change receptor signaling when only one receptor is activated (Fig. 4a). Interestingly, in the presence of TM4, TM5 and TM6 peptides, simultaneous activation of both A1R and A2AR with CPA and CGS21680, respectively, allows CPA to decrease CGS21680-stimulated cAMP (Fig. 4a), in contrast to experiments in the absence of either interference peptides (Fig. 4a, control) or TM7 used as a negative control (Fig. 4a). Moreover, this decrease in cAMP accumulation in the CPA/CGS co-stimulated condition is mediated by activation of the A1R/Gi pathway as, in the presence of TM peptides, a selective A1R antagonist or the treatment with PTX blocks the CPA-induced effect (Additional file 1: Figure S2D). Thus, modification of the quaternary structure of the A1-A2AHet with peptides that penetrate within the heteromer interface abolishes inhibition of A1R by A2AR in the Gs-Gi-heterotetramer signaling unit. These experimental results suggest that synthetic peptides inserted between A1R and A2AR protomers, which are not able to disrupt the heteromer as seen by PLA (Fig. 2), increase the distance between Gi and Gs. This would allow the simultaneous opening of αiAH and αsAH domains for GDP dissociation. In order to verify this hypothesis, we modeled the A1-A2AHet with the TAT-fused peptide TM6 altering the heteromer interface between A1R and A2AR, in complex with Gs (open αsAH, d[Asn112αs-Asn261αs]: 56 Å; Fig. 4b, green line) and Gi (open αiAH, d[Arg90αi-Glu238αi]: 52 Å; Fig. 4b, yellow line). Due to the insertion of TM6, the distance between the binding site of A1R and A2AR increases by 17 Å, from 14 Å in the absence of TM6 (Fig. 3e, f, black line) to 31 Å in the presence of TM6 (Fig. 4b, black line). This increase in the distance between heteromers also moves the intracellular αiAH and αsAH domains apart, thus permitting their simultaneous opening (d[Arg90αi-Asn112αs]: 10 Å; Fig. 4b, dark red line) for GDP/GTP exchange.
A1-A2AHet as an adenosine concentration-sensing device
In order to illustrate the molecular device allowing adenosine to signal by one or the other receptor , we measured cAMP levels at increasing concentrations of adenosine in cells expressing the A1-A2AHet (Fig. 5a). Due to the higher affinity for the hormone, adenosine at a low concentration (30 nM) binds predominantly to A1R and engages a Gi-mediated signaling, which significantly decreases forskolin-induced cAMP accumulation. At higher concentrations, adenosine progressively binds to A2AR, which engages a Gs-mediated signaling. At high adenosine concentrations, full occupancy of both A1R and A2AR leads to marked increases in cAMP levels compatible with Gs activation and blockade of Gi, as depicted in the schemes of Fig. 5a. In these conditions, full active A2AR can increase cAMP over the forskolin-induced levels whilst the progressive blockade of A1R by A2AR cannot reduce cAMP accumulations. To demonstrate such blockade of A1R actions by A2AR, we performed the experiments in the presence of a peptide (A2AR TM6) that inserts into the heteromer interface (Fig. 5b). In the presence of the peptide, the device lost its concentration-sensing properties. In fact, high adenosine concentrations, in which both receptors are fully occupied and functional, led to a null response, i.e., the A2AR-mediated increase in forskolin-stimulated cAMP is counteracted by a similar Gi-mediated decrease of cAMP. Upon heteromer structure alteration by TM6, the A2AR becomes unable to block A1R-mediated signaling.
Recruitment of β-arrestin-2 by the A1-A2AHet
We used BRET assays to detect the interaction between a protomer and β-arrestin-2. Thus, cells were transfected with cDNAs of β-arrestin-2 fused to Rluc (Arr-Rluc) as the BRET donor and A1R or A2AR fused to YFP (A1R-YFP, A2AR-YFP) as the BRET acceptor. Control experiments in cells expressing only A1R-YFP or A2AR-YFP and Arr-Rluc show the ability of both receptors to recruit β-arrestin-2 (Additional file 1: Figure S3A) and the selectivity of each agonist (Additional file 1: Figure S3B). Similar experiments in cells additionally expressing non-fused A2AR (Arr-Rluc/A1R-YFP + A2AR) or non-fused A1R (Arr-Rluc/A2AR-YFP + A1R) were performed (Additional file 1: Figure S3B). Interestingly, in cells expressing Arr-Rluc, A2AR-YFP and non-fused A1R (control in Fig. 6a and Additional file 1: Figure S3B) or Arr-Rluc, A1R-YFP and non-fused A2AR (control in Fig. 6b and Additional file 1: Figure S3B), a similar degree of BRET was induced by CGS-21680 (white bars) or by CGS-21680 plus CPA (striped bars). This suggests that agonist binding to A2AR inhibits the CPA ability to stimulate β-arrestin-2 recruitment to A1R. In order to rationalize these results, we have used the recent crystal structure of rhodopsin bound to visual arrestin-1  to model the A1-A2AHet in complex with β-arrestin-2. The finger loop of arrestin, which adopts a short α-helix, is inserted into the intracellular cavity of the external protomer, whereas the C-domain of arrestin points towards the internal protomer of the homodimer. Figure 6c shows key intermolecular distances between the center of mass of the N- and C-domains of two arrestin molecules bound to A1R and A2AR obtained from MD simulations. These data suggest that the A1-A2AHet quaternary structure permits the binding of two arrestin molecules to the external protomers of both A1R and A2AR, similarly to the simultaneous binding of Gi and Gs to the heterotetramer. Moreover, similar simulations of A1-A2AHet in complex with Gi and β-arrestin-2 (Fig. 6d) show no steric clashes between Gi (bound to A1R) and arrestin (bound to A2AR). These results suggest that sustained activation of Gs (Gβγ moving away from Gαs to facilitate the interaction of Gαs with the catalytic domain of adenylate cyclase) by agonist binding to A2AR enables β-arrestin-2 recruitment to A2AR. As stated above, within the A1-A2AHet, CPA cannot activate Gi in the presence of the A2AR agonist CGS-21680 (Fig. 3) and, consequently, CPA does not trigger additional β-arrestin-2 recruitment to A1R (control in Figs. 6a, b and Additional file 1: Figure S3B).
Using the TAT-fused synthetic peptides we investigated whether the quaternary structure of the A1-A2AHet determines its putative selective A2AR-dependent β-arrestin-2 recruitment. As a negative control, we first corroborated that TM4, TM5, and TM6 peptides of A2AR do not interfere with A1R-mediated signaling (Additional file 1: Figure S3C). Pretreatment of cells expressing Arr-Rluc, A2AR-YFP and non-fused A1R (Fig. 6a), or Arr-Rluc, A1R-YFP and non-fused A2AR (Fig. 6b) with TM4, TM5, and TM6 peptides, but not in the absence of peptides (control) or with the TM7 peptide (negative control), allowed the detection of positive BRET (recruitment of β-arrestin-2) not only when cells were treated with the A2AR-selective agonist CGS-21680 (white bars), but also when treated with the A1R-selective agonist CPA (black bars) (Figs. 6a, b). Importantly, when cells expressing Arr-Rluc, A2AR-YFP, and non-fused A1R were co-activated by CPA and CGS-21680 (striped bars), BRET measurement in the presence of TM4, TM5, or TM6 peptides, but neither in the absence of peptides nor in the presence of TM7 peptide, significantly increased relative to the values obtained by the action of a single agonist (Fig. 6a). The trend is similar in cells expressing Arr-Rluc, A1R-YFP, and non-fused A2AR, but not statistically significant (Fig. 6b). These results indicate that alteration of the A1R-A2AR heteromer interface within the A1-A2AHet allows simultaneous recruitment of β-arrestin-2 to A1R and A2AR when both receptors are activated. Interference peptides abolish cross-communication of G proteins, permitting CPA to activate Gi (Gβγ moving away from Gαi) and recruitment of β-arrestin-2 to A1R, as well as Gs activation by CGS-21680 (Gβγ moving away from Gαs) and simultaneous recruitment of β-arrestin-2 to A2AR.
The C-terminal domain of A2AR is responsible for the dominant A2AR-mediated signaling
Despite the apparent structural symmetry of the GPCR/G protein macromolecular complex, a major difference is the length of the intracellular C-terminal domain of adenosine receptors (16 amino acids in A1R versus 102 in A2AR). The short C-terminal tail of the A1R does not have any known specific function, while the C-terminus of A2AR, albeit dispensable for ligand binding , dimerization , and agonist induced cAMP signaling , influences constitutive signaling . Due to the shorter C-terminus of A1R and the proposed orientation of the C-tail of A2AR toward αsAH (see Additional file 1: Figure S4a for details), as well as the proposed role of the C-terminal tail in downstream signaling cascade activation , we speculated that the C-terminus of A2AR could modulate the prevailing Gs-mediated signaling upon A1R and A2AR co-activation. To test this hypothesis, we engineered two A2AR mutants, one lacking most of the C-terminal end (A2AΔCTR) and another lacking the last 40 amino acids (A2AΔ40R). First, we tested whether these truncated versions of A2AR could form heteromers with A1R. We observed similar BRET saturation curves in HEK-293 T cells expressing a constant amount of A1R-Rluc cDNA and increasing amounts of either A2AR-YFP, A2AΔ40R-YFP, or A2AΔCTR-YFP, indicating that A2AΔ40R and A2AΔCTR form heteromers with A1R (Fig. 7a; BRETmax in mU: 91 ± 3 A2AR, 99 ± 3 A2AΔ40R, and 90 ± 8 A2AΔCTR). Heteromers were also detected by BiFC assays in HEK-293 T cells transfected with cDNAs for A1R-nYFP and A2AΔCTR-cYFP (Fig. 7b, dashed line). In these cells, fluorescence was reduced in the presence of TM4, TM5, and TM6 peptides of A2AR (Fig. 7b). Thus, heteromerization of A2AΔCTR with A1R occurs via the TM5/6 interface, similarly to the interaction of A2AR with A1R.
We measured cAMP production in cells expressing A1R and wild-type or truncated A2AR receptors (Fig. 7c). Truncated A2AR were able to signal as wild-type receptors. Interestingly, the dominant Gs-mediated signaling when A1R and A2AR were co-activated decreased progressively with the shortening of the A2AR C-tail (Fig. 7c, striped bars). In fact, CPA inhibited CGS-21680-induced cAMP accumulation when truncated receptors were expressed, showing that, in these heteromers, A1R were functional (Additional file 1: Figure S5). Figure 7e shows a detailed view of the orientation of the C-tail (102 amino acids, Gln311-Ser412) of both A2AR protomers in the A1-A2AHet, which was modeled as suggested for the OXER , together with the structure of β-arrestin-2 in complex with V2 vasopressin receptor . It is important to note that the exact conformation of the A2AR C-tail cannot unambiguously be determined, thus, we only predict its orientation as explained in detail in Additional file 1: Figure S4. The fact that the C-tail of the αs-unbound A2AR protomer points toward the αsAH domain suggests that this C-tail is influencing the conformational changes required to open the αsAH, and thus controlling the balance between Gs and Gi activation. Next, we measured β-arrestin-2 recruitment by BRET assays in cells expressing A1R and wild-type or truncated A2AR receptors. In cells expressing non-fused A1R, Arr-Rluc and A2AR-YFP, A2AΔ40R-YFP, or A2AΔCTR-YFP, the A1R agonist CPA could increase BRET values only when the heteromer is formed with A2AR-truncated receptors. In these conditions, co-activation with CPA and CGS-21860 induced a BRET increase higher than the one obtained with CGS-21680 alone (Fig. 7d). These results indicate that the selective A2AR-dependent β-arrestin-2 recruitment in the A1-A2AHet decreases progressively with the shortening of the A2AR C-tail (Fig. 7d).
As previously reviewed [2, 3, 22], the intercommunication between protomers of a GPCR heteromer can be observed at the level of agonist binding, ligand-induced cross-conformational changes between receptor protomers, and the binding of GPCR-associated proteins, including heterotrimeric G proteins and β-arrestins. The intercommunication between protomers is a consequence of a defined quaternary structure that is responsible for the specific functional characteristics of the heteromer. For GPCR heteromers, such as A1-A2AHet, constituted by receptors sensing the same hormone but producing opposite signaling effects, it is not obvious how a defined quaternary structure achieves this dual behavior. A1-A2AHet acts as a concentration-sensing device that allows adenosine to signal by one or the other coupled G protein (Gs or Gi) to fine-tune modulate the release of neurotransmitters from presynaptic terminals. In the present study, we solved this question by discovering a new mechanism of signal transduction, a cross-communication between Gi and Gs in the A1-A2AHet guided by the A2AR C-terminal domain.
We have shown that cross-communication between Gi and Gs proteins involves the formation of a GPCR heterotetramer (i.e., one homodimer of A1R and one of A2AR) that has a 2:2:1:1 (A2AR:A1R:Gs:Gi) stoichiometry. From our data, it is deduced that the cross-talk between Gi and Gs resides on the structural constraints surrounding the mechanism for GDP/GTP exchange, which involves the opening of the αAH domain of the α-subunit of any given G protein. We propose that cross-communication in the Gs-Gi-heterotetramer signaling unit is a property associated with a specific quaternary structure, the compact rhombus-shaped A1-A2AHet (the TM4/5 interface for homodimerization and the TM5/6 interface for heterodimerization), which positions the αiAH and αsAH domains in close proximity, making their conformational changes mutually dependent in a way that simultaneous opening of both αAH domains would not be possible due to a steric clash in such open conformations. Alterations of this quaternary structure of the A1-A2AHet by insertion of synthetic peptides between A1R and A2AR blocks this cross-communication without disrupting the heteromer and permits simultaneous activation of Gi and Gs in the heteromer. Since the cross-talk between Gi and Gs resides on the structural constraints imposed by defined TM interfaces in the heteromer, it is important to note that other heterotetramers, mainly those sensing different hormones and with a different quaternary structure, might not display this cross-communication among G proteins. Moreover, although, from a structural point of view, the A1-A2AHet is capable to recruit not only two G proteins but also two β-arrestins, the cross-talk between Gi and Gs, in which Gs activation inhibits the simultaneous activation of Gi, blocks A1R agonist-promoted arrestin recruitment. Alteration of the A1-A2AHet by insertion of synthetic peptides between A1R and A2AR facilitates simultaneous activation of Gi and Gs and the corresponding binding of two β-arrestins to A1R and A2AR. Our finding that Gi is dependent on Gs-mediated signaling strengthens the conclusion that cross-talk across G proteins is a potentially important functional property of GPCR heteromers. Remarkably, when both receptors are co-activated in this heterotetramer, only A2AR-mediated, but not A1R-mediated signaling occurs. We show that the ability of blunting A1R-mediated signaling when Gs is engaged is dependent of the long C-terminus of the A2AR. In the absence of A2AR activation by agonists, or in the absence of the C-terminal domain of A2AR, the A1R-mediated signaling via Gi is totally functional. The most straightforward hypothesis is that the opening of αsAH parallels a movement of the C-tail to block the opening of αiAH.
Adenosinergic signaling in mammalians is important for energy and temperature homeostasis and for neuroregulation. Multiplicity of adenosine actions is due to a balance between the expression of specific receptors and producing/degrading enzymes and to the biological diversity due to a membrane network established by the interaction among purinergic receptors . Ciruela et al.  first identified the occurrence of heteromers formed by A1R-Gi- and A2A-Gs-coupled adenosine receptors that participate in the regulation of glutamate release by neurons projecting from the cortex to the striatum. The same A1-A2AHet can be found in astrocytes modulating the transport of γ-amino butyric acid (GABA) . Differently from the modulation of neuronal glutamate release, the A1R-Gi-coupled receptor activates and the A2AR-Gs-coupled receptor inhibits the modulation of GABA transport. Under conditions of high extracellular adenosine concentrations, such as hypoxic conditions , the nucleoside will bind to both the high (A1R) and the low (A2AR) affinity receptors in the heteromer, and the predominant A2AR-mediated signaling via Gs will result in counteraction of astrocytic GABA transport. Our results show that the asymmetric signaling is possible because the long C-terminus of A2AR blunts Gi-mediated signaling. We have therefore elucidated the mechanism by which the A1-A2AHet functions as an adenosine concentration-sensing device that can promote even opposite signaling responses depending on the extracellular concentration of adenosine. The molecular mechanism involves the C-terminal domain of the activated Gs-coupled A2AR, which hinders the activation of A1R coupled to Gi.
Using a convergent approach including biochemical, biophysical, cell biology, and molecular biology techniques, together with in silico molecular models, we here provide the mode of action of a membrane receptor complex that responds depending on the concentration of adenosine, a hormone and a neuroregulatory molecule. The concentration sensor is a heteromer composed of four adenosine receptors (two A1 and two A2A) and two G proteins (Gi and Gs). Despite Gi sits underneath the A1 receptor dimer and Gs sits underneath the A2A receptor dimer, both G proteins do interact and are able to convey allosteric regulation depending on how the functional unit is activated. At low adenosine concentrations Gi is engaged via A1 activation without affecting/engaging Gs signaling. At higher concentrations Gs is engaged via A2A activation, and this engagement blocks Gi-mediated signaling. The reason why a rhombus-shaped apparently symmetric structure results in asymmetric signaling is due to the long C-terminal tail of the A2A receptor. In fact, both deletion of the C-terminal end or treatment with interfering peptides derived from the sequence of TM segments of the receptors impair allosteric cross-interaction between receptors and G proteins within the macromolecule, and the device loses its concentration sensing properties.
Cell culture and transient transfection
HEK-293 T cells were grown at 37 °C in in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 2 mM L-glutamine, 100 U/mL penicillin/streptomycin, and 5% (v/v) heat inactivated fetal bovine serum (all supplements were from Invitrogen, Paisley, Scotland, UK). Cells were transiently transfected with cDNA corresponding to receptors, fusion proteins, A2AR mutant constructs, or minigene vectors using polyethylenimine (Sigma-Aldrich, Cerdanyola del Vallés, Spain) as described elsewhere .
Expression vectors, A2AR mutants and minigenes
Sequences encoding amino acid residues 1–155 or 155–238 of YFP-Venus protein, were subcloned in pcDNA3.1 to obtain the YFP Venus hemi-truncated proteins (nYFP and cYFP). The human cDNAs for A2AR, mutant A2AR, A1R, and Gi or Gs proteins cloned into pcDNA3.1, were amplified without their stop codons using sense and antisense primers harboring unique EcoRI and BamHI sites to subclone receptors in pcDNA3.1RLuc vector (pRLuc-N1 PerkinElmer, Wellesley, MA, USA) and EcoRI and KpnI to subclone receptors in pEYFP-N1 (enhanced yellow variant of GFP; Clontech, Heidelberg, Germany), pcDNA3.1-nVenus, or pcDNA3.1-cVenus vectors. The amplified fragments were subcloned to be in-frame with restriction sites of the corresponding vectors to give the plasmids that express receptors fused to RLuc, YFP, nYFP or cYFP on the C-terminal end (A1R-Rluc, A2AR-Rluc, Gi-RLuc, Gs-RLuc, A1R-YFP, A2AR-YFP, A2AΔ40R-YFP, A2AΔCTR-YFP, A1R-nYFP, A2A-nYFP, and A2A-cYFP). Expression of constructs was tested by confocal microscopy and the receptor-fusion protein functionality by second messengers, ERK1/2 phosphorylation and cAMP production as described previously [4, 26,27,28]. Mutants with a deletion of aa 372 to aa 412 (A2AΔ40R) or aa 321 to aa 412 (A2AΔCTR) on the C-terminal domain of A2AR were generated as previously described . “Minigene” plasmid vectors are constructs designed to express relatively short polypeptide sequences following their transfection into mammalian cells. Here, we used minigene constructs encoding 11 amino acid residues from the C-terminus sequence of α subunit of Gi1/2 or Gs. The peptide coded by every minigene inhibits the coupling of the G (Gi1/2 or Gs) protein to the receptor and, consequently, it inhibits the G-protein-mediated cellular response, as previously described . The cDNA encoding the last 11 amino acids of human Gα subunit corresponding to Gi1/2 (IKNNLKDCGLF) or Gs (QRMHLRQYELL), inserted in a pcDNA 3.1 plasmid vector, was generously provided by Dr. Heidi Hamm.
Peptides with the sequence of the TM of A1R and A2AR fused to the HIV TAT peptide (YGRKKRRQRRR) were used as oligomer-disrupting molecules (synthesized by Genemad Synthesis Inc. San Antonio, TX, USA). The cell-penetrating TAT peptide allows intracellular delivery of fused peptides . The TAT-fused TM peptide can then be inserted effectively into the plasma membrane because of the penetration capacity of the TAT peptide and the hydrophobic property of the TM moiety . To obtain the right orientation of the inserted peptide, the HIV-TAT peptide was fused to the C-terminus or to the N-terminus as indicated:
MEYMVYFNFFVWVLPPLLLMVLIYLYGRKKRRQRRR for TM5 of A1R, RRRQRRKKRGYLALILFLFALSWLPLHILNCITLF for TM6 of A1R, ILTYIAIFLTHGNSAMNPIVYAFRIYGRKKRRQRRR for TM7 of A1R, VYITVELAIAVLAILGNVLVCWAVWYGRKKRRQRRR for TM1 of A2AR, YGRKKRRQRRRYFVVSLAAADIAVGVLAIPFAITI for TM2 of A2AR, LFIACFVLVLTQSSIFSLLAIAIYGRKKRRQRRR for TM3 of A2AR, YGRKKRRQRRRAKGIIAICWVLSFAIGLTPMLGW for TM4 of A2AR, MNYMVYFNFFACVLVPLLLMLGVYLYGRKKRRQRRR for TM5 of A2AR, YGRKKRRQRRRLAIIVGLFALCWLPLHIINCFTFF for TM6 of A2AR, LWLMYLAIVLSHTNSVVNPFIYAYYGRKKRRQRRR for TM7 of A2AR.
YGRKKRRQRRRILGIWAVSLAIMVPQAAVME for TM4 of OX1R, SSFFIVTYLAPLGLMAMAYFQIFYGRKKRRQRRR for TM5 of OX1R, YASFTFSHWLVYANSAANPIIYNFYGRKKRRQRRR for TM7 of OX1R
Bimolecular fluorescence complementation assay (BiFC)
HEK-293 T cells were transiently transfected with equal amounts of the cDNA for fusion proteins of the hemi-truncated Venus (1 μg of each cDNA). At 48 h after transfection, cells were treated for 4 h at 37° with medium or TAT peptides (4 μM) before plating 20 μg of protein in 96-well black microplates (Porvair, King’s lynn, UK). To quantify reconstituted YFP Venus expression, fluorescence at 530 nm was read in a Fluoro Star 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 fluorescence of the sample minus the fluorescence of cells not expressing the fusion proteins (basal). Cells expressing receptor-cVenus and nVenus or receptor-nVenus and cVenus showed similar fluorescence levels than untransfected cells.
Bioluminescence resonance energy transfer (BRET)
HEK-293 T cells were transiently transfected with a constant amount of cDNA for Rluc fusion proteins and increasing amounts of cDNA for YFP fusion proteins. At 48 h after transfection, 20 μg of cell suspension were plated in 96-well black microplates for fluorescence detection or in 96-well white microplates for BRET readings and Rluc quantification. YFP fluorescence at 530 nm was quantified in a Fluoro Star Optima Fluorimeter as described above. BRET signal was collected 1 min after addition of 5 μM coelenterazine H (Molecular Probes, Eugene, OR, USA) using a Mithras LB 940. The integration of the signals detected in the short-wavelength filter at 485 nm and the long-wavelength filter at 530 nm was recorded. To quantify protein-RLuc expression, luminescence readings were also performed after 10 minutes of adding 5 μM coelenterazine H. The net BRET is 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 milli-BRET units (net BRET × 1000). To calculate maximum BRET (BRETmax) from saturation curves, data were fitted to a nonlinear regression equation, assuming a single-phase saturation curve with GraphPad Prism software (San Diego, CA, USA).
Proximity ligation assay (PLA)
HEK293T cells were grown on glass coverslips and fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline containing 20 mM glycine, permeabilized with the same buffer containing 0.05% Triton X-100, and successively washed with tris-buffered saline. Heteromers were detected using the Duolink II in situ PLA detection Kit (OLink; Bioscience, Uppsala, Sweden) following supplier’s instructions. A mixture of the primary antibodies (mouse anti-A2AR antibody (1:100; 05-717, Millipore, Darmstadt, Germany; RRID:AB_309931) and rabbit anti-A1R antibody (1:100; ab82477, Abcam, Bristol, UK; RRID: AB_2049141)) was used to detect A1-A2AHet together with PLA probes detecting mouse or rabbit antibodies. Then, samples were processed for ligation and amplification with a Detection Reagent Red and were mounted using a DAPI-containing mounting medium. Samples were analyzed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with 405 nm and 561 nm laser lines. For each field of view, a stack of two channels (one per staining) and 4–6 Z-stacks with a step size of 1 μm were acquired. Images were opened and processed with Image J software (National Institutes of Health, Bethesda, MD, USA).
cAMP determination assays
HEK-293 T cells expressing adenosine receptors were incubated for 4 h in serum-free medium containing 50 μM zardeverine. Cells were plated in 384-well white microplates (1500 cells/well), pre-treated with toxins or the corresponding vehicle for the indicated time, stimulated with agonists for 15 min before adding medium or 0.5 μM forskolin, and incubated for an additional 15 min. cAMP production was quantified by a TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) methodology using the LANCE Ultra cAMP kit (PerkinElmer) and fluorescence at 665 nm was analyzed on a Pherastar Flagship Microplate Reader (BMG Labtech, Ortenberg, Germany).
Dynamic mass redistribution (DMR) assays
The heteromer-induced cell signaling signature was determined using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) by a label-free technology. Refractive waveguide grating optical biosensors, integrated in 384-well microplates, allow extremely sensitive measurements of changes in local optical density in a detecting zone up to 150 nm above the surface of the sensor. Cellular mass movements induced upon receptor activation were detected by illuminating the underside of the biosensor with polychromatic light and measured as changes in wavelength of the reflected monochromatic light, which is a sensitive function of the index of refraction. The magnitude of this wavelength shift (in picometers) is directly proportional to the amount of DMR. Briefly, 24 h before the assay, cells were seeded at a density of 7500 cells per well in 384-well sensor microplates with 40 μL growth medium and cultured for 24 h (37 °C, 5% CO2) to obtain 70–80% confluent monolayers. Previous to the assay, cells were pre-treated with medium or toxins as indicated and incubated for 2 h in 40 μL per well of assay-buffer (HBSS with 20 mM HEPES, pH 7.15) in the reader at 24 °C. Thereafter, the sensor plate was scanned and a baseline optical signature was recorded prior to addition of 10 μL of receptor agonist dissolved in assay buffer containing 0.1% DMSO. DMR responses were monitored for at least 8000 s and data were analyzed using EnSpire Workstation Software v. 4.10.
The structural model of the A1-A2AHet bound to Gs (closed αsAH domain) and Gi (closed αiAH domain) was taken from our previous work . This previous structural model contains a A2AR-based homology model of A1R. The structure of the adenosine A1R has recently been revealed , showing a remarkably similar structure (Additional file 1: Figure S6A). This structure of A1R contains a TM4/5 dimer interface that is in close agreement with our model (Additional file 1: Figure S6B). An intermediate conformation (obtained using the g_morph tool of the GROMACS package ) between the closed αAH domain (PDB id 1AZT) and the conformation observed in the crystal structure of the β2-AR in complex with Gs (PDB id 3SN6) was used to model the open αAH domain (Additional file 1: Figure S6C). This conformation is supported by DEER spectroscopy, deuterium-exchange and electron microscopy data [11,12,13]. The active state of β-arrestin-2 was built using a multi-template alignment combining the structure of the active β-arrestin-1 (PDB id 4JQI)  and the structure of rhodopsin in complex with visual β-arrestin (PDB id 4ZWJ) . Structural models of the A1-A2AHet bound to β-arrestin-2 were modeled using the crystal structure of rhodopsin bound to β-arrestin (PDB id 4ZWJ) . The structure of TM6 of A2AR fused to the cell-penetrating TAT peptide was modeled from the structure of A2AR. Molecular models of the A1-A2AHet with the TAT-fused TM6 peptide, disrupting the heteromer interface between A1R and A2AR, in complex with Gs (open αsAH domain) and Gi (open αiAH domain), was built from the structure of A1-A2AHet. The conformation of the proximal C-tail of A2AR (Ser305-Ala317) was modeled based on squid rhodopsin . The remaining part of the C-tail (1Gly319–Ser412), cannot be unambiguously determined and it was modeled as suggested for the oxoeicosanoid receptor (OXER) , together with the structure derived from the human V2 vasopressin receptor in complex with β-arrestin-2  (see Additional file 1: Figure S4 for details). Additional file 2: Table S1 shows the template structures used in the protein models. Modeller 9.12 was used to build these models . The molecular models of A1-A2AHet in complex with Gs and Gi or β-arrestin, in the absence or presence of the TAT-fused TM6 peptide, were embedded in a pre-equilibrated box containing a lipid bilayer (~800 POPC molecules) with explicit solvent (~110,000 waters) and 0.15 M concentration of Na+ and Cl– (~1800 ions). These initial complexes were energy-minimized and subsequently subjected to a 21 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. Computer simulations were performed with the GROMACS 4.6.3 simulation package , using the AMBER99SB force field as implemented in GROMACS and Berger parameters for POPC lipids. This procedure has been previously validated .
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We would like to thank Jasmina Jiménez for technical help (University of Barcelona). RF, 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.
This study was supported by grants from the Spanish Ministerio de Economía y Competitividad (SAF2015-74627-JIN, BFU2015-64405-R and SAF2016-77830-R; they may contain FEDER funds) and by the intramural funds of the National Institute on Drug Abuse to SF. RF, PJM, and LP participate in the European COST Action CM1207 (GLISTEN).
Availability of data and materials
The crystal structures 4EIY, 2Z73, 3SN6, 4JQI, 1AZT, 1AGR, 4ZWJ, 4JQI, 2PSD, and 2RH7 used to build the presented computational models are available from PDB (http://www.rcsb.org). All other relevant data are within the paper and its Additional files 1 and 2.
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Figures S1–S6. Figure S1. Control experiments on the effect of interfering peptides on the A1-A2AHet structure and Gs and Gi coupling to A1-A2AHet. Figure S2. Receptor signaling through A1R and A2AR. Figure S3. Recruitment of β-arrestin-2 by the A1-A2AHet. Figure S4. Modeling the orientation of the C-tail of A2AR. Figure S5. The influence of the C-terminal domain of A2AR in the signaling properties of the A1-A2AHet in the presence of pertussis toxin. Figure S6. Modeling A1R homodimer and αsAH and αiAH in closed and open conformations. (DOCX 5727 kb)
Table S1. List of target sequences and template structures used to construct the computer models of A1-A2AHet in complex with Gi and Gs. (PDF 23 kb)
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Navarro, G., Cordomí, A., Brugarolas, M. et al. Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain. BMC Biol 16, 24 (2018). https://doi.org/10.1186/s12915-018-0491-x