All receptor constructs are human unless otherwise specified. AT₂-Rluc8 (rat) and B₂-Rluc8 cDNA constructs were generated from plasmids containing the respective receptor cDNA tagged with Rluc. The Rluc coding region was replaced with Rluc8 cDNA from pcDNA3.1-Rluc8 kindly provided by Andreas Loening and Sanjiv Gambhir (Stanford University, CA) (33), as described previously for other constructs (34). AT₂-Rluc (rat), AT₂-Venus (rat) (5) and HA-AT₂ (rat; referred to as AT₂ in the BRET¹ and eBRET assays) were kindly provided by Walter Thomas (University of Queensland). B₂ and HA-B₂ (referred to from here-on-in as B₂) and EP₃ cDNA was obtained from the Missouri S&T cDNA Resource Center (www.cdna.org). B₂-Rluc was previously produced by PCR amplification of B₂ cDNA to remove the stop codon and ligation into pcDNA₃ containing Rluc. B₂-Venus was generated by replacing the Rluc8 coding region from B₂-Rluc8 with Venus cDNA. NES–Venus–mGsq was kindly provided by Nevin Lambert (Augusta University, Augusta, Georgia). Gα_q-Rluc8, Gα_i3-Rluc8 and Gβ₃ were from the TRUPATH kit, which was a gift from Bryan Roth (Addgene kit #1000000163), with Venus-Gγ₉ being generated from GFP2-Gγ₉, also from the TRUPATH kit (35). Gα_z-Rluc8 was kindly provided by Martina Kocan (The Florey Institute of Neuroscience and Mental Health). GRK2-Rluc8 was synthesized by GeneArt (ThermoFisher Scientific, Regensburg, Germany). The β-arrestin2-Venus cDNA construct was prepared previously from pCS2-Venus kindly provided by Atsushi Miyawaki (RIKEN Brain Science Institute, Wako-city, Japan) (34). Signal peptide and flag-tagged AT₂ (referred to as AT₂ in the NanoBRET ligand binding assays) and Nluc-AT₂ were generated previously (27). Nluc-B₂ was generated by replacing the AT₂ coding region from Nluc-AT₂ with B₂ cDNA. Ligands used were AngII, BK and PGE₂ (Sigma), icatibant and PD 123319 (Tocris Bioscience) and TAMRA-AngII (AnaSpec).

HEK293FT cells were maintained at 37°C, 5% CO₂ in complete medium (Dulbecco’s modified Eagle’s medium (DMEM) containing 0.3 mg/ml glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin) supplemented with 10% fetal calf serum (FCS) (GIBCO BRL, Carlsbad, CA). Transient transfections were carried using either GeneJuice (Merck, Kilsyth, Australia) or FuGENE (Promega) according to manufacturer’s instructions. All assays were carried out 48 hours post transfection.

Receptor-HIT is an assay that enables the identification and pharmacological profiling of receptor heteromers in live cell systems. The assay uses a proximity-based reporter system such as BRET to enable detection of heteromers through their ligand-dependent interaction with proteins or ligands (5, 25, 27, 29–32). The Receptor-HIT assay comprises three elements (Figure 1), which in these studies on the BRET platform are a BRET-tagged receptor, an untagged receptor, and a BRET-tagged interacting protein (Figure 1A) or a BRET-tagged interacting ligand [Figure 1B (27)]. If a change in BRET signal occurs upon addition of a ligand that is selective for the untagged receptor, this indicates proximity between the tagged receptor and the tagged interacting biomolecule. This Receptor-HIT signal signifies the close proximity of the two receptors, and is indicative of receptor heteromerization.

HEK293FT cells were transfected with cDNA as described in figure legends. BRET¹ and eBRET assays used rat AT₂ constructs. For all BRET¹ assays (with the exception of Figures 6E, F), 5 µM coelenterazine h (Promega) was added and basal BRET was measured for 10-20 mins before adding agonist or vehicle and then continuing to measure BRET. Antagonist assays had a pretreatment of antagonist or vehicle (30 min) prior to addition of coelenterazine h. For the BRET¹ assays in Figures 6E, F, cells were pretreated for 30 min with agonist or vehicle, with cells in Figure 6F having an additional pretreatment of antagonist or vehicle (30 min) prior to treatment with agonist. Following pretreatment, coelenterazine h was added to a final concentration of 5 µM and BRET was measured immediately. For eBRET assays, cells were incubated at 37°C, 5% CO₂ for 2 hours with 30 µM EnduRen (Promega) to ensure substrate equilibrium was reached. Basal BRET was measured for 10-20 mins before adding agonist or vehicle and then continuing to measure BRET. Antagonist assays had a pretreatment of antagonist or vehicle (30 min) prior to addition of coelenterazine h. All BRET¹ and eBRET measurements were taken at 37°C using either a LUMIstar Omega plate reader (BMG Labtech, Mornington, Victoria, Australia) with 460–490 nm and 520–550 nm filters; a CLARIOstar plate reader (BMG Labtech) with 420–480 nm and 520–620 nm filters; or a VICTOR Light plate reader (Perkin Elmer) with 400–475 nm and 520–540 nm filters. The ligand-induced BRET signal was calculated by subtracting the ratio of the long wavelength emission over the short wavelength emission for a vehicle-treated cell sample from the same ratio for a second aliquot of the same cells treated with agonist, as described previously (34, 36). In this calculation, the vehicle-treated cell sample represents the background, eliminating the requirement for measuring a donor-only control sample (34, 36). For BRET kinetic assays, the final pretreatment reading is presented at the zero time point (time of agonist/vehicle addition).

HEK293FT cells were transfected with cDNA as described in figure legends. NanoBRET assays used human AT₂ constructs. For NanoBRET assays, cells were pretreated for 30 min with PD 123319 and then TAMRA-AngII was added (final concentration of 1 μM). Following another 30 min incubation, furimazine was added and BRET measured immediately at 37°C using a PHERAstar FS plate reader (BMG Labtech) with 420–500 nm and 610–LP filters or a LUMIstar Omega plate reader (BMG Labtech) with 410–490 nm and 610–LP filters. The BRET signal was calculated by subtracting the ratio of the long wavelength emission over the short wavelength emission and the data were normalized as percentage of TAMRA-AngII binding.

Measurement of IP₁ accumulation was performed using the IP-One Tb kit (Cisbio Bioassays) according to manufacturer’s instructions. Cells were treated for 30 minutes at 37°C with agonists or vehicle. Antagonist assays had an additional pre-treatment with antagonist or vehicle for 30 mins at 37°C, which was removed prior to treatment with agonist. The cells were then lysed by adding the supplied assay reagents, and the assay was incubated for 1 hour at room temperature. Fluorescence was measured at 620 nm and 665 nm 50 µs after excitation at 340 nm using the EnVision 2102 multilabel plate reader (PerkinElmer).

Data were presented and analyzed using Prism 9 software (GraphPad). Competition binding data and concentration-response data were fitted using logarithmic nonlinear regression (three parameter). Unpaired t-tests, one-way ANOVAs and two-way ANOVAs were used to determine statistical significance where appropriate (*p < 0.05).

Following activation by an agonist, GPCRs typically interact with and activate heterotrimeric G proteins to initiate intracellular signaling cascades. The B₂ receptor primarily couples to the Gα_q class of G proteins (37) while the AT₂ receptor is an unusual GPCR in that it does not readily couple to any G proteins (38). To investigate Gα_q coupling by the receptors, we used a Venus-tagged mini G (mG) protein construct that comprises an engineered GTPase domain of the Gα_s protein that has been modified to confer Gα_q specificity (NES-Venus-mG_sq). As expected, no ligand-induced recruitment of NES-Venus-mG_sq to the Rluc8-tagged AT₂ receptor (AT₂-Rluc8) was observed (Figure 2A). In contrast, and also as expected, coexpression of NES-Venus-mG_sq with the B₂ receptor tagged with Rluc8 (B₂-Rluc8) resulted in a BK-induced BRET signal (Figure 2B) indicative of recruitment of Gα_q to the receptor.

We then investigated Gα_q coupling using the Receptor-HIT assay, again using NES-Venus-mG_sq. Receptor-HIT uses a proximity-based reporter system such as BRET to enable detection and characterization of heteromers through their ligand-dependent interactions with labelled proteins or ligands (5, 25, 29–32) (Figure 1). Upon coexpression of the unlabeled B₂ receptor in cells expressing AT₂-Rluc8 and NES-Venus-mG_sq we now observed a BK-induced BRET signal (Figure 2C), indicating recruitment of NES-Venus-mG_sq proximal to AT₂-Rluc8. This Receptor-HIT signal indicates the close proximity of the AT₂ receptor and the B₂ receptor, and suggests their interaction within a heteromeric complex. Coexpression of untagged AT₂ receptor to cells expressing B₂-Rluc8 and NES-Venus-mG_sq did not alter the BRET signal (Figure 2D) from that seen without AT₂ expression (Figure 2B).

We investigated the mG_sq Receptor-HIT signal further by conducting concentration-response analysis. Figure 2E shows that there is no change in potency of mG_sq coupling to AT₂-B₂ heteromers compared to B₂ receptors (pEC₅₀ ± SEM = 7.94 ± 0.29 vs. 7.73 ± 0.19, respectively; p > 0.05, unpaired t-test). When we conducted the mG_sq Receptor-HIT assay in the presence of selective antagonists (Figure 2F), we saw that the AT₂ receptor antagonist PD 123319 did not inhibit coupling of mG_sq to AT₂-B₂ heteromers. In contrast, the putative B₂ receptor antagonist icatibant was able to significantly reduce the level of mG_sq recruitment, indicating the requirement of B₂ receptor activation for Gα_q coupling.

Finally, we investigated the specificity of the Receptor-HIT signal by conducting a similar experiment but instead using a GPCR not known to heteromerize with the AT₂ receptor, the prostaglandin E receptor 3 (EP₃ receptor). Here we found that only coexpression and activation of the B₂ receptor resulted in a Receptor-HIT signal between AT₂-Rluc8 and NES-Venus-mG_sq (Figure 2G). No signal was observed when EP₃ was coexpressed with AT₂-Rluc8 and NES-Venus-mG_sq and treated with PGE₂ (Figure 2G), despite both B₂ and EP₃ being expressed within the cells, as shown by their activation of G protein (Figure 2H; Gα_q for B₂, and Gα_i3 for EP₃).

Gα_q activation initiates a signaling cascade that leads to inositol phosphate signaling, which can be monitored by measuring the accumulation of the metabolite IP₁. Using an IP₁ assay and aliquots of transfected cells also used in the β-arrestin2 assays described below, we next investigated downstream Gα_q signaling mediated by the receptors. As expected, we found that AngII did not induce IP₁ production in cells expressing AT₂/Rluc8 (Figure 3A). However, coexpression of the B₂ receptor resulted in robust BK-induced IP₁ production (Figure 3A). When we conducted concentration-response analysis, we found that there was no significant difference in the potency of IP₁ production between cells expressing just the B₂ receptor and cells expressing the B₂ receptor and the AT₂ receptor (Figure 3B; pEC₅₀ ± SEM = 8.55 ± 0.20 vs. 8.45 ± 0.04, respectively; p > 0.05, unpaired t-test), just as we saw no difference in potency of mG_sq recruitment in the BRET assay. When we conducted these IP₁ assays with an antagonist pretreatment, we found that 10 μM of the AT₂ receptor antagonist PD 123319 had no inhibitory effect on 0.1 μM BK-induced IP₁ production (Figure 3C). Interestingly, in this assay 10 μM of the putative B₂ selective antagonist icatibant was also unable to inhibit 0.1 μM BK-induced IP₁ production. Indeed, it acted as a partial agonist in this assay, as can be seen by the substantial IP₁ production in cells treated only with icatibant and no BK. Further analysis illustrated the concentration-dependent effect of IP₁ production mediated by icatibant (Figure 3D). This concentration-response analysis also showed that high concentrations of icatibant were in fact able to inhibit BK-induced IP₁ production. However, the potency of this effect was shifted substantially to the right of its inhibitory actions on BK-induced β-arrestin2 recruitment. These findings support reports of the partial agonism of icatibant, which has previously been observed mediating IP₁ production through the B₂ receptor (39).

We next investigated Gα_z protein recruitment to the receptors, using Gα_z tagged with Rluc8 (Gα_z-Rluc8). As Gα_z is not a known signaling partner for either the AT₂ receptor or the B₂ receptor, we did not expect to observe any recruitment, and this was confirmed in our BRET assay expressing either Venus-tagged receptor and Gα_z-Rluc8 (Figures 4A, B). Coexpression of the untagged B₂ receptor did not alter the BRET signal between AT₂-Venus and Gα_z-Rluc8 (Figure 4C), however, coexpression of the untagged AT₂ receptor interestingly resulted in a marked decrease in the BRET signal between B₂-Venus and Gα_z-Rluc8 upon treatment with BK (Figure 4D). A decrease in the BRET signal suggests that there is a preformed complex between B₂-Venus and Gα_z-Rluc8, which either disassociates or undergoes conformational rearrangement that increases the distance between the two BRET tags (40–42). In either case, this BRET signal provides further evidence in support of the existence of a functional AT₂-B₂ heteromer, and illustrates completely novel pharmacology it has adopted.

We also investigated the concentration-dependence of the Gα_z BRET signal (Figure 4E) and found a similar potency of BK-induced concentration-dependence as observed for mG_sq coupling (pEC₅₀ ± SEM = 7.94 ± 0.29, unpaired t-test). Likewise, when we conducted the assay in the presence of selective antagonists, we again found that the Gα_z BRET signal could be blocked by B₂ receptor inhibition (icatibant), but not AT₂ inhibition (PD 123319) (Figure 4F).

Following agonist stimulation, GPCR kinases (GRKs) are rapidly recruited to GPCRs, where they phosphorylate the receptor’s C terminal tail. This initiates receptor desensitization and interaction with β-arrestin proteins. We investigated GRK recruitment using BRET with Rluc8-tagged GRK2 (GRK2-Rluc8) and Venus-tagged receptors. There was no ligand-induced recruitment of GRK2-Rluc8 to the Venus-tagged AT₂ receptor (AT₂-Venus; Figure 5A). This lack of GRK2 recruitment is expected, as it is well known that the AT₂ receptor does not recruit β-arrestin or internalize upon stimulation with AngII (43–45), and therefore it is unlikely it would recruit GRKs. In contrast, but also as expected, when cells expressing GRK2-Rluc8 and Venus-tagged B₂ receptor (B₂-Venus) were treated with BK (but not AngII) we saw an immediate increase in the BRET signal, indicating rapid recruitment of GRK2 to the B₂ receptor (Figure 5B).

When we coexpressed untagged B₂ receptor in cells expressing AT₂-Venus and GRK2-Rluc8, we saw BK-induced recruitment of GRK2 proximal to the AT₂ receptor (Figure 5C). Interestingly, this BRET signal had a much more sustained signal than that observed between GRK2-Rluc8 and B₂-Venus (Figure 5B), which declined steadily over time. As with the mG_sq Receptor-HIT assay, this Receptor-HIT signal indicates the close proximity of the AT₂ receptor and the B₂ receptor, and suggests their interaction within a heteromeric complex. Coexpression of untagged AT₂ receptor did not alter the BRET signal between B₂-Venus and GRK2-Rluc8 (Figure 5D) from that seen without AT₂ expression (Figure 5B).

We also investigated the GRK2 Receptor-HIT signal further by conducting concentration-response analysis. Figure 5E shows that there is a significant leftward shift in the potency of GRK2 recruitment to AT₂-B₂ heteromers compared to B₂ receptors (pEC₅₀ ± SEM = 8.07 ± 008 vs. 7.35 ± 0.06, respectively; p < 0.05, unpaired t-test). When we conducted the GRK2 Receptor-HIT assay in the presence of selective antagonists (Figure 5F) we saw, as in the mG_sq and Gα_z Receptor-HIT assays, that the AT₂ receptor antagonism did not inhibit the recruitment of GRK2 to AT₂-B₂ heteromers, while B₂ receptor antagonism significantly reduced the level of GRK2 recruitment, indicating the specificity of the BRET signals.

Following GRK recruitment and subsequent receptor phosphorylation, GPCRs recruit the scaffold protein β-arrestin, which desensitizes the receptor from classical cell surface G protein signaling and initiates internalization (46). Individual GPCRs have different β-arrestin recruitment profiles resulting in unique desensitization and internalization characteristics. Upon treatment with BK, the B₂ receptor rapidly recruits β-arrestin leading to swift desensitization and extensive internalization (47, 48). In contrast and as already mentioned, the AT₂ receptor does not recruit β-arrestin or internalize upon stimulation with AngII (43–45).

As expected, there was no ligand-induced recruitment of β-arrestin2-Venus to AT₂-Rluc8 (Figure 6A), whereas when we coexpressed B₂-Rluc8 with β-arrestin2 tagged with Venus (β-arrestin2-Venus) we observed strong and rapid BK-induced recruitment of β-arrestin2-Venus to B₂-Rluc8 (Figure 6B). When AT₂-Rluc8 was co-expressed with the untagged B₂ receptor in the Receptor-HIT configuration, there was a marked increase in ligand-induced BRET when the cells were treated with BK but not AngII (Figure 6C), indicating BK-dependent translocation of β-arrestin2-Venus proximal to the B₂ receptor. This BRET signal between AT₂-Rluc8 and β-arrestin2-Venus confirms the close proximity of AT₂-Rluc8 and the B₂/β-arrestin2-Venus complex and is indicative of AT₂-B₂ heteromerization. Additionally, and similar to what was seen with GRK2, β-arrestin2-Venus recruitment to AT₂-B₂ heteromers had an altered kinetic profile to what was seen with B₂ monomers/homomers. When we conducted the Receptor-HIT assay in the reverse configuration by coexpressing the untagged AT₂ receptor with B₂-Rluc8 and β-arrestin2-Venus there was no change in BRET signal (Figure 6D) from that seen without AT₂ expression (Figure 6B).

To further investigate the β-arrestin2 Receptor-HIT signal we again conducted concentration-response analysis (Figure 6E). This showed that there was no significant difference in potency between BK-induced β-arrestin2-Venus recruitment to B₂ receptors and AT₂-B₂ heteromers (pEC₅₀ ± SEM = 7.64 ± 0.06 vs. 7.94 ± 0.19, respectively; p > 0.05, unpaired t-test). We then conducted the β-arrestin2 Receptor-HIT assay in the presence of selective antagonists. Similar to the previous Receptor-HIT assays, we saw that the BK-induced recruitment of β-arrestin2 to the AT₂-B₂ heteromer could be blocked by B₂ receptor inhibition but not AT₂ inhibition (Figure 6F), which demonstrates the importance of B₂ receptor coexpression and activation.

We lastly investigated the AT₂-B₂ heteromer using the NanoBRET ligand binding assay (28). In this assay, the NanoLuc (Nluc) luciferase (49) is fused to the N-terminus of a GPCR, and binding of fluorescent ligands can be detected with BRET. In our study, we fused Nluc to the N-terminus of the AT₂ receptor (Nluc-AT₂) and treated cells with an AngII analogue tagged with the TAMRA fluorophore (TAMRA-AngII) (Figures 7A, B). When we treated cells with increasing concentrations of the AT₂ receptor antagonist PD 123319 in a competition binding assay, we were able to see a reduction in the BRET signal, indicating displacement of TAMRA-AngII binding to Nluc-AT₂.

We then conducted the ligand binding assay in the Receptor-HIT configuration, as recently published (27). Here, Nluc was fused to the N-terminus of the B₂ receptor (Nluc-B₂) and was coexpressed with the untagged AT₂ receptor. A BRET signal upon addition of TAMRA-AngII would indicate both the binding of TAMRA-AngII to the untagged AT₂ receptor and also its close proximity to Nluc-B₂, and this specific binding of TAMRA-AngII to AT₂ receptors would be confirmed by displacement with PD 123319 (Figure 7C). When we conducted the assay, this was precisely what we observed, a TAMRA-AngII-induced BRET signal that could be displaced by increasing concentrations of PD 123319 (Figure 7D). When we compared the pIC₅₀ values between cells expressing Nluc-AT₂ and cells expressing Nluc-B₂ and AT₂ we found no significant differences (pIC₅₀ ± SEM = 6.33 ± 0.10 vs. 6.84 ± 0.20, respectively; p > 0.05, unpaired t-test).