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Agonist-induced formation of unproductive receptor-G12 complexes

Significance G protein-coupled receptors (GPCRs) are targeted by a large fraction of approved drugs and regulate many important cellular processes. Association of GPCRs with heterotrimeric G proteins in response to agonist activation is thought to invariably lead to G protein activation. We find instead that G12 heterotrimers can associate with agonist-bound receptors in a manner that does not lead to activation. These unproductive agonist–receptor-G protein ternary complexes sequester G12 heterotrimers and thus inhibit rather than support G12 signaling. These findings reveal a mechanism whereby agonist activation of GPCRs can inhibit as well as promote G protein signaling.

G proteins are activated when they associate with G proteincoupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V 2 receptors (V 2 R) associate with both G s and G 12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V 2 R-G s complexes, V 2 R-G 12 complexes are not destabilized by guanine nucleotides and do not promote G 12 activation. Activating V 2 R does not lead to signaling responses downstream of G 12 activation, but instead inhibits basal G 12 -mediated signaling, presumably by sequestering G 12 heterotrimers. Overexpressing G 12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V 2 R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G 12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G 12 complexes are not unique to V 2 R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.
GPCR | ternary complex | G protein-coupled receptor | arrestin G protein-coupled receptors (GPCRs) mediate important physiological activities and exert most of their effects through activation of G proteins. In the conventional model of coupling, unliganded receptors are poor recruiters and activators of G proteins, whereas agonist-bound GPCRs take on more active conformations that effectively recruit G protein heterotrimers (1,2). Productive receptor-G protein association promotes GDP release by stabilizing the nucleotide-free state of the Gα subunit, which in turn allows GTP binding, G protein activation, and downstream signaling (3,4). According to this model, agonist-dependent GPCR-G protein complex formation is essentially synonymous with G protein activation. Four families of G proteins (G s , G i/o , G q/11 , and G 12/13 ) can be activated, and each leads to a distinct set of downstream signaling outcomes. It is generally thought that selection of G protein subtypes by GPCRs occurs at the receptor-G protein association step, such that receptors interact with and activate cognate G protein subtypes and do not interact with noncognate G protein subtypes. Here we find that agonist-dependent GPCR-G protein association can occur without promoting subsequent G protein activation, thus, whether a G protein subtype is activated can be determined after initial receptor-G protein engagement. Moreover, noncognate G proteins can impede downstream events, perhaps by competing with other intracellular transducers for access to activated receptors. These findings revise the standard model of G protein coupling by indicating that agonist-induced GPCR-G protein association does not always promote G protein activation and may in some circumstances inhibit downstream signaling.
Results V 2 R Interacts with G 12 Heterotrimers. Conventional GPCR-G protein coupling is understood as an allosteric interaction where an agonist-bound active receptor mediates GDP release by stabilizing the nucleotide-free state of an associated Gα subunit (3)(4)(5).
Receptor complexes with nucleotide-free G proteins are quite transient when guanine nucleotides are present at concentrations similar to those found in cells, but are stabilized when guanine nucleotides are absent. In order to observe allosteric coupling we monitored receptor-G protein association under conditions that allowed us to control both ligand binding to the receptor and nucleotide binding to the G protein. We used bioluminescence resonance energy transfer (BRET) between GPCRs fused to Renilla luciferase (Rluc8) and Gβ 1 and Gγ 2 subunits fused to complementary fragments of Venus fluorescent protein (6)(7)(8) to monitor receptor-G protein association. These components and unlabeled Gα subunits were transfected into HEK 293 cells in which most of the endogenous G proteins had been deleted using CRISPR/Cas9-mediated gene editing (9,10). In order to control nucleotide binding to G proteins, cells were permeabilized and either supplemented with nucleotides or treated with apyrase to remove residual nucleotides.
Significance G protein-coupled receptors (GPCRs) are targeted by a large fraction of approved drugs and regulate many important cellular processes. Association of GPCRs with heterotrimeric G proteins in response to agonist activation is thought to invariably lead to G protein activation. We find instead that G 12 heterotrimers can associate with agonist-bound receptors in a manner that does not lead to activation. These unproductive agonist-receptor-G protein ternary complexes sequester G 12 heterotrimers and thus inhibit rather than support G 12 signaling. These findings reveal a mechanism whereby agonist activation of GPCRs can inhibit as well as promote G protein signaling.
Because receptor-G protein complexes are transient it can be difficult to detect agonist-induced BRET signals between receptors and G proteins when guanine nucleotides are present (6). For example, arginine vasopressin (AVP) did not detectably increase BRET between vasopressin V 2 receptors (V 2 R) and G s heterotrimers in the presence of GDP (Fig. 1A). In contrast, AVP produced large BRET increases in the absence of nucleotides (Fig. 1A). Stabilization of agonist-receptor-G protein complexes when Gα subunits are nucleotide-free indicates conventional "productive" allosteric coupling and predicts that agonist-bound receptors will promote GDP release and G protein activation under physiological conditions. Nucleotide-free conditions also enhanced AVP-induced BRET between V 2 R and G i1 or G q heterotrimers (SI Appendix, Fig. S1). These results are consistent with cognate V 2 R activation of G s and G q heterotrimers (11,12) and predict some ability to activate G i1 heterotrimers. In contrast, we observed surprisingly robust agonist-induced BRET between V 2 R and G 12 heterotrimers in the presence of GDP that was not enhanced by nucleotide depletion (Fig. 1A). These results suggest that AVP-bound V 2 R can form complexes with GDP-bound G 12 heterotrimers that do not progress to the nucleotide-free state and therefore are not stabilized when GDP is removed (Fig. 1B). As an index of allosteric coupling we divide the increase in BRET produced by agonist in the presence of GDP (ΔBRET ag ) by the increase in BRET produced by the combined effect of agonist and nucleotide depletion (ΔBRET ag+apy ), and refer to this index as the GDP-resistance ratio, or R GDP . R GDP values that are less than 1 indicate conventional productive coupling, whereas an R GDP value of 1 indicates nucleotide-resistant or unproductive coupling. R GDP for V 2 R and G s was 0.08 ± 0.12 (mean ± SD; n = 6), whereas R GDP for V 2 R and G 12 was 1.01 ± 0.06 (n = 6). In contrast to V 2 R, we observed more conventional productive coupling of both endothelin A (ET A ) and thromboxane A 2 (TP) receptors with G 12 heterotrimers, with R GDP values of 0.66 ± 0.09 (n = 3) and 0.55 ± 0.09 (n = 6), respectively. Both of these receptors also coupled productively with G q heterotrimers, with R GDP values of 0.29 ± 0.08 (n = 3) and 0.11 ± 0.07 (n = 6), respectively (SI Appendix, Fig. S2).
Receptors that couple to one member of a Gα subunit family can usually couple to other members of the same family. Therefore, we examined V 2 R coupling to G 13 heterotrimers, the other member of the G 12/13 family (13). We found that stimulation with AVP increased BRET between V 2 R and G 13 heterotrimers in the presence of GDP (SI Appendix, Fig. S3). However, unlike what we observed with G 12 , these responses were enhanced by nucleotide depletion (R GDP = 0.67 ± 0.08; n = 4), indicating productive coupling, consistent with weak V 2 R-mediated activation of G 13 (14). Similar results were obtained with G 13 heterotrimers and ET A (R GDP = 0.21 ± 0.03; n = 4) and TP (R GDP = 0.18 ± 0.01; n = 4) receptors (SI Appendix, Fig. S3).
Because nucleotide-resistant V 2 R-G 12 association was unexpected we performed additional experiments to rule out the possibility that our standard BRET assay was simply detecting an agonist-induced change in V 2 R-Rluc8 conformation. We reasoned that if both ET A and V 2 R receptors were able to associate with G 12 the two receptors should compete for a common pool of heterotrimers. Indeed, we found that stimulation of unlabeled ET A receptors inhibited AVP-induced BRET between V 2 R-Rluc8 and G 12 heterotrimers in intact cells (SI Appendix, Fig. S4A). Conversely, stimulation of unlabeled V 2 R receptors inhibited endothelin-1-induced BRET between ET A -Rluc8 and G 12 heterotrimers (SI Appendix, Fig. S4B). Second, we found that stimulation of unlabeled V 2 R receptors increased BRET between Gα 12 -Rluc8 and Gβγ-Venus in intact cells (SI Appendix, Fig. S5 A and B). This increase persisted in permeabilized cells that were treated with apyrase and supplemented with GDPβS to prevent the possibility of heterotrimer activation by residual GTP (SI Appendix, Fig.  S5B). This suggests that active V 2 R receptors may impose a conformational change in G 12 heterotrimers that does not require GTP binding or G 12 activation. In contrast, stimulation of unlabeled ET A and TP receptors decreased BRET between Gα 12 -Rluc8 and Gβγ-Venus, and these decreases were largely blocked in permeabilized cells when only GDPβS was present (SI Appendix, Fig. S5C). Finally, we found that AVP increased luciferase complementation when a small fragment (SmBit) of Nanoluc was fused to V 2 R, and a large fragment of Nanoluc was fused to Gγ 2 , and these proteins were coexpressed with unlabeled Gα 12 and Gβ 1 (SI Appendix, Fig. S6). These results are consistent with AVPinduced association of V 2 R receptors and G 12 heterotrimers.
Additional experiments revealed that V 2 R recruited G s and G 12 heterotrimers at similar rates (Fig. 1C) and that agonistinduced V 2 R-G 12 complexes were equally stable in the presence of GDP or GTP (Fig. 1D). Stimulation of V 2 R with the agonist oxytocin produced similar responses to AVP, indicating BRET between V 2 R-Rluc8 and Gαβγ-Venus in the presence or absence of AVP (1 μM), and the presence or absence of GDP. When GDP was absent, apyrase (apy) was added to remove residual nucleotides. AVPinduced BRET to G s (Left) but not G 12 (Right) heterotrimers was enhanced when GDP was absent; **P < 0.005; n.s., not significant (P = 0.58); one-way ANOVA (Sidak's test) compared to GDP+AVP; n = 6. (B) Cartoon representation of two steps of V 2 R-G protein coupling: agonist-induced formation of receptor-G protein complexes, and GDP release. (C) Time course of BRET between V 2 R-Rluc8 and Gαβγ-Venus in response to injection of 1 μM AVP, followed by injection of 100 μM GTP in permeabilized cells treated with apyrase (mean ± SEM; n = 4-6). (D) BRET between V 2 R-Rluc8 and Gαβγ-Venus as a function of AVP concentration in permeabilized cells expressing either G s (Left) or G 12 (Right) heterotrimers treated with apyrase, GDP (100 μM), or GTP (100 μM). The logEC 50 for association with G s was −6.8 ± 0.5 in apyrasetreated cells, and the logEC 50 s for association with G 12 were −7.5 ± 0.3, −7.4 ± 0.4, and −7.5 ± 0.5 in the presence of apyrase, GDP, and GTP, respectively. Data points represent the change in BRET (ΔBRET) in response to AVP (mean ± SEM; n = 3). that nucleotide-insensitive V 2 R-G 12 interactions are not restricted to AVP (SI Appendix, Fig. S7A), and AVP-induced responses were inhibited by the antagonist mozavaptan (SI Appendix, Fig. S7B). V 2 R Does Not Activate G 12 Heterotrimers. The above results suggested that AVP-stimulated V 2 R should not activate G 12 heterotrimers. To test this prediction we turned to sensitive assays that monitor signaling downstream of G 12 activation. We first monitored translocation of full-length p115-RhoGEF and a fragment (amino acids 281-483) of PDZ-RhoGEF from the cytosol to the plasma membrane using bystander BRET assays (15,16). These proteins bind to activated Gα 12 subunits at the plasma membrane to regulate Rho GTPase activity and actin fiber formation (17,18). ET A and TP receptors robustly recruited p115-RhoGEF to the plasma membrane in a G 12 -dependent manner ( Fig. 2A). In contrast, stimulation of V 2 R receptors failed to recruit p115-RhoGEF and instead decreased the baseline abundance of this reporter at the plasma membrane ( Fig. 2A). Similar results were obtained with V 2 R and TP receptors and PDZ-RhoGEF recruitment to the plasma membrane ( Fig. 2B) and direct recruitment of the RGS homology domain (amino acids 1-246) of p115-RhoGEF, p115-RGS-GFP, to Gα 12 -RlucII (Fig. 2C). The AVP-induced decrease in p115-and PDZ-RhoGEF at the plasma membrane suggests that active V 2 R may sequester G 12 heterotrimers, preventing activation by endogenous receptors. Consistent with this suggestion, we found that activation of V 2 R could significantly reduce p115-RhoGEF recruitment mediated by activation of ET A receptors (Fig. 2D). A second sensitive assay of G 12 activity is gene transcription driven by activation of the serum response element (SRE) (19). Stimulation of V 2 R receptors failed to activate SRE-dependent gene transcription, whereas stimulation of both ET A and TP receptors could activate SRE in a G 12dependent manner (Fig. 2E). A similar trend was observed with transcription driven by nuclear factor kappa-light-chain enhancer of activated B cells (NFκB; Fig. 2F). These results demonstrate that active V 2 R receptors do not detectably activate G 12 heterotrimers, even though the two proteins interact in an agonist-dependent manner. V 2 R Activates G 12 Chimeras and Mutants. Canonical GPCR-mediated activation of G proteins involves extension of the Gα subunit C terminus (helix 5; H5) into the active receptor core (20,21). This region of Gα is necessary for productive coupling and is also a key determinant of receptor-G protein selectivity. Therefore, we hypothesized that exchanging the Gα 12 C terminus with C-terminal peptides from other Gα subunits might allow productive coupling with V 2 R. Indeed, we found that Gα 12 chimeras bearing the last 10 amino acids of either Gα s or Gα q (Fig. 3A) interacted with AVP-activated V 2 R in a GDP-sensitive manner; R GDP values were significantly less than 1 for G 12s and G 12q heterotrimers and were similar to R GDP for G s and G q heterotrimers (Fig. 3B). G 12s and G 12q chimeras also supported V 2 R-mediated translocation of p115-RhoGEF to the plasma membrane, consistent with productive coupling to these heterotrimers and activation of G 12 signaling pathways (Fig. 3C). Conversely, we found that replacing the C-terminal peptides of either Gα s or Gα q with that of Gα 12 (Fig. 3A) dramatically increased R GDP , indicating much less productive coupling to G s12 and unproductive coupling to G q12 (Fig. 3B). We next made point mutations in the Gα 12 C terminus to introduce residues with properties shared by the corresponding residues in Gα s and Gα q (Fig. 3A). We found that Gα 12 mutants with a hydrophobic residue in the −1 position (Q381L and Q381V) still coupled unproductively with V 2 R ( Fig. 3 D and E). In contrast, Gα 12 mutants with a tyrosine in the −4 position (I378Y) coupled productively with V 2 R; R GDP was less than 1 (Fig. 3D), and I378Y supported V 2 R-mediated translocation of p115-RhoGEF (Fig. 3E). Similar weak but productive coupling to V 2 R was observed when Gα 12 was simply truncated by a single amino acid (Δ1; Fig. 3 D and E). These results indicate that the Gα 12 C terminus is required for unproductive coupling to active V 2 R. Together with the observation that subtle modifications of the Gα 12 C terminus overcome the barrier to productive coupling, this result suggests that G 12 heterotrimers are likely to interact with active V 2 R in a manner that is structurally similar to canonical GPCR-G protein complexes.
The V 2 R-G 12 Interaction Interferes with Other Transducers. The robust agonist-induced BRET signal between V 2 R receptors and G 12 heterotrimers in the presence of nucleotides suggested that this interaction might be stable enough to interfere with recruitment of other intracellular transducer molecules to V 2 R. As V 2 R receptors canonically activate G s heterotrimers (11), we first asked how overexpressing G 12 would influence activation of adenylyl cyclase and cAMP accumulation. We found that overexpressing G 12 resulted in modest inhibition of G s activation, as (A) Activation of V 2 R decreases bystander BRET between p115RhoGEF-Rluc8 and the plasma membrane marker Venus-Kras when G 12 is expressed, whereas activation of ET A and TP receptors increases this signal, indicating association of p115RhoGEF-Rluc8 and active Gα 12 at the plasma membrane; *P < 0.05; paired t test compared to mock-transfected control (−); n = 3-4. (B) V 2 R activation decreases BRET between PDZ-RhoGEF-RlucII and the plasma membrane marker rGFP-CAAX, whereas TP activation increases this signal (mean ± SEM; n = 3).
(C) TP activation increases BRET between two different Gα 12 -RlucII constructs and p115-RGS-GFP, whereas V 2 R activation has no effect on this signal; ***P < 0.0005; n.s., not significant; one-sample t test compared to zero; n = 6. RlucII was fused to Gα 12 after amino acids 84 and 136 in the two different probes. Data points in A-C represent the change in BRET (ΔBRET) in response to agonist, and the broken gray line represents zero. (D) Activation of V 2 R reduces p115-RhoGEF recruitment mediated by activation of ET A . Activation of ET A (ET1; 100 nM) increases BRET between p115RhoGEF-Rluc8 and Venus-Kras, and this response is significantly inhibited when V 2 R receptors are coexpressed and activated (AVP; 1 μM); n.s., not significant (P = 0.43); **P < 0.005; one-way ANOVA (Sidak's test); n = 4. Data points represent the change in bystander BRET (ΔBRET) in response to ET1, and the broken gray line represents zero. (E) Activation of V 2 R fails to activate the SRE when G 12 is expressed, whereas activation of ET A and TP receptors increases SRE-driven gene expression; *P < 0.05; n.s., not significant; paired t test compared to (−); n = 5. (F) V 2 R receptors fail to activate NFκB-driven gene expression when G 12 is expressed; n.s., not significant; paired t test compared to (−); n = 3. Data points in E and F represent luminescence normalized to vehicletreated controls, and the broken gray line represents one (no change). Agonists were AVP (1 μM), ET1 (100 nM), and U-46619 (10 μM).
indicated by a G s biosensor (SI Appendix, Fig. S8A). Surprisingly, this did not lead to detectable inhibition of V 2 R-mediated cAMP accumulation, as indicated by two different cAMP sensors (SI Appendix, Fig. S8 B and C). Active V 2 R receptors are phosphorylated by G protein receptor kinases (GRKs), and phosphorylated V 2 R bind tightly to β-arrestins (22). Remarkably, we found that overexpressing G 12 significantly reduced AVP-induced BRET between V 2 R-Rluc8 and β-arrestin2-Venus (Fig. 4 A and  B). A much smaller but still significant reduction was observed after overexpressing G s heterotrimers (Fig. 4 A and B). In contrast, overexpressing G 12 did not significantly reduce β-arrestin2 recruitment to ET A , β 2 -adrenergic, or angiotensin AT 1 receptors (SI Appendix, Fig. S9). Because V 2 R-β-arrestin interactions are very stable and because phosphorylated V 2 R can accommodate G protein and arrestin binding simultaneously (23), we suspected that G 12 overexpression was acting upstream of arrestin binding to inhibit V 2 R interactions with GRKs. Consistent with this hypothesis, we found that G 12 overexpression greatly reduced the AVP-induced interaction of V 2 R and GRK2 (SI Appendix, Fig. S10). Because arrestin binding is critical for agonist-dependent V 2 R internalization (22) we then asked if G 12 overexpression would inhibit receptor endocytosis. Indeed, overexpression of G 12 but not G s heterotrimers inhibited V 2 R trafficking from the plasma membrane to the endosomal compartment as assessed by enhanced bystander BRET (ebBRET; Fig. 4 C and D). Conversely, there was a small but significant enhancement of V 2 R internalization in cells lacking Gα 12 and Gα 13 subunits (SI Appendix, Fig. S11).
Although neither of these two receptors is known to activate G 12 we directly assessed activation of downstream G 12 signaling pathways by FPR2. As was the case with V 2 R, activation of FPR2 failed to recruit p115-RhoGEF to the plasma membrane and failed to activate SRE-dependent gene transcription (SI Appendix, Fig. S12). Formyl peptide 1 receptors (FPR1) are highly homologous with FPR2 (68% identical), and we found that FPR1 also coupled productively with G i heterotrimers (R GDP = 0.26 ± 0.01; n = 3), but unproductively with G 12 heterotrimers (R GDP = 0.96 ± 0.02; n = 3). When we examined association of GPCRs with G proteins from all four Gα subtype families, we found that highly GDP-resistant interactions (R GDP > 0.5) were restricted to G 12 heterotrimers (Fig. 6). For 19 of the 20 interactions that were studied with G s , G i , and G q heterotrimers, R GDP was <0.3, whereas this was the case for only two of the nine interactions we studied with G 12 heterotrimers. These results suggest that receptor-G 12 complexes may generally be more stable than other receptor-G protein complexes when G proteins are bound to GDP. Boxes indicate residues whose properties are shared between Gα s and Gα q but not Gα 12 . (B) GDP resistance (R GDP ) of interactions between V 2 R-Rluc8 and heterotrimers incorporating the indicated Gα subunits; n.s., not significant; all other groups significantly different from one; P < 0.05; one-sample t test; n = 4-10. (C) Activation of V 2 R decreases bystander BRET between p115RhoGEF-Rluc8 and Venus-Kras when G 12 is expressed, but increases bystander BRET when G 12s or G 12q are expressed, indicating receptor-mediated activation of these chimeras; all groups were significantly different from mock-transfected controls (−); P < 0.05; one-way ANOVA (Dunnett's test); n = 7. (D) GDP resistance of interactions between V 2 R-Rluc8 and G 12 heterotrimers bearing the indicated point mutations; n.s., not significant; all other groups significantly different from one; P < 0.05; onesample t test; n = 3-14. (E) Activation of V 2 R decreases p115RhoGEF-Rluc8 translocation when G 12 wt, Q381L, or Q381V are expressed, but increases translocation when G 12 I378Y or Δ1 are expressed; all groups were significantly different from mock-transfected controls (−); P < 0.05; one-way ANOVA (Dunnett's test); n = 3-13.

Discussion
Taken together, our results suggest that several GPCRs bind to G 12 heterotrimers in an activation-dependent manner, but the resulting GPCR-G 12 complexes are insensitive to guanine nucleotides. These interactions do not activate G 12 signaling, but may instead have a negative effect on RhoGEF recruitment and signaling by sequestering G 12 , thus preventing activation by other receptors. These interactions may also interfere with recruitment of other intracellular transducers and thus change signaling or trafficking of receptors that recruit but fail to activate G 12 heterotrimers. Whether or not these inhibitory effects occur under physiological conditions will depend on several factors, most notably the local abundance of G 12 heterotrimers and the stoichiometry of receptors and intracellular transducers. The normal physiological role of the V 2 R is to enhance water reabsorption in the kidney by stimulating G s , which ultimately leads to incorporation of aquaporin-2 water channels to the luminal surface of collecting duct cells (25). An inhibitory effect of V 2 R activation on G 12 signaling could conceivably contribute to the physiological activity of this receptor, as Rho activity has been reported to act as an inhibitor of aquaporin transport (26). An inhibitory effect of the V 2 R-G 12 interaction on arrestin recruitment could also play a regulatory role to limit receptor internalization. Although we found that G 12 overexpression weakly inhibited V 2 R-mediated G s activation when assessed using a direct G s activation assay, we were surprised to find that this did not lead to a detectable decrease in cAMP accumulation. It is possible that GRK and arrestin recruitment are more sensitive to competition with G 12 than cAMP accumulation because cAMP signals are amplified downstream of G s . Similar observations have been made after expression of some intrabodies that recognize the active state of β 2 -adrenergic receptors (27). Further studies with native systems will be required to determine if unproductive GPCR-G 12 association has physiological significance. At present, our findings significantly change the current model of GPCR coupling by demonstrating robust agonist-induced receptor-G protein interactions that do not lead to nucleotide exchange and G protein activation. GPCRs are thought to have access to all G protein subtypes expressed in a given cell, but possible interactions with noncognate heterotrimers (defined as G proteins that cannot be activated by a given GPCR) have, with a few exceptions (28), been overlooked. It is commonly assumed that stable agonist-induced GPCR-G protein interactions are restricted to cognate G proteins and are associated with G protein activation. One implication of this idea is that the conventional selection process whereby receptors reject noncognate G proteins occurs at an early stage of receptor-G protein association, such that complexes with noncognate G proteins do not progress past weak and transient encounter complexes. This seems to be true in the majority of cases, as several previous studies using sensitive methods have shown that interactions between GPCRs and noncognate G proteins are usually undetectable (7,29). In contrast, our results suggest that some receptors functionally reject G 12 heterotrimers despite forming relatively stable GPCR-G 12 complexes. It is thought that GPCR-G protein complexes evolve through multiple intermediate conformations prior to receptor-stimulated nucleotide release (30)(31)(32)(33)(34). It is possible that receptors such as V 2 R and FPR2 form similar intermediate complexes with G 12 heterotrimers that are unusually stable (Fig. 1B) and are unable to promote the changes in G 12 that lead to GDP release. Spontaneous GDP release from  G 12 heterotrimers is particularly slow (35), and it may be that receptor-mediated GDP release requires relatively stable complexes with G 12 -GDP, even for receptors that do activate G 12 . G protein chimeras and mutants revealed that the Gα 12 C terminus is necessary for unproductive complexes with V 2 R, implying that these complexes share some structural features with conventional productive ternary complexes. Our results with intramolecular G 12 BRET sensors suggest that V 2 R and FPR2 may promote conformational changes in G 12 that do not lead to activation. However, the resolution of such probes is insufficient to determine if the heterotrimer itself changes conformation or, alternatively, if only the attached BRET donor and acceptor labels are rearranged. In either case, these results suggest that changes in G protein conformation reported by sensors similar to those used here do not necessarily indicate G protein activation. In summary, our results reveal a mode of GPCR-G protein interaction wherein agonist-activated receptors bind to G 12 heterotrimers but do not promote nucleotide exchange and activation. These findings show that receptors can inhibit as well as activate G proteins, adding to the complexity of GPCR-mediated signaling.
Cell Culture and Transfection. HEK 293 cells (ATCC) were propagated in plastic flasks and on 6-well plates according to the supplier's protocol. HEK 293 cells with targeted deletion of GNAS and GNAL (G s knockouts; GSKO), targeted deletion of GNAS, GNAL, GNAQ, GNA11, GNA12, and GNA13 (G protein three family knockouts; 3GKO), and HEK 293 cells with additional targeted deletions to the 3GKO cells of GNAI1, GNAI2, GNAI3, GNAT1, GNAT2, GNAZ, and GNAO1 (G protein four family knockouts; 4GKO) were derived, authenticated, and propagated as previously described (9,10). HEK 293 cells with additional targeted deletion of ARRB1 and ARRB2 (beta-arrestin knockouts; ARRBKO) were derived, authenticated, and propagated as previously described (37,38). Cells were transfected in growth medium using linear PEI MAX (MW 40,000) at a nitrogen/phosphate ratio of 20 and were used for experiments 12-48 h later. Up to 3.0 μg of plasmid DNA was transfected in each well of a 6-well plate. For ebBRET experiments, up to 1.0 μg of plasmid DNA was transfected in suspension to a cell density of 350,000 cells/mL in white 96-well plates.
incubation for 1 d, transfected cells were harvested with 0.5 mM EDTAcontaining DPBS, centrifuged and suspended in 4 mL of HBSS containing 0.01% BSA (fatty acid-free grade; SERVA) and 5 mM Hepes (pH 7.4) (assay buffer). The cell suspension was dispensed in a white 96-well plate at a volume of 80 μL per well and loaded with 20 μL of 50 μM coelenterazine (Carbosynth) diluted in the assay buffer. After 2 h incubation at room temperature, the plate was measured for baseline luminescence (Spectramax L, Molecular Devices), and 20 μL of titrated ligand (AVP) were manually added. The plate was immediately read at room temperature for the following 10 min at a measurement interval of 20 s with an accumulation time of 0.17 s per read. The luminescence counts over 5-10 min after ligand addition were averaged and normalized to the initial count. The foldchange values were further normalized to that of vehicle-treated samples. Translocation of p115RhoGEF. Cells were transfected with an untagged GPCR, Gα, Gγ 2 , Gβ 1 , p115RhoGEF-Rluc8, Venus-Kras, and PTX-S1 in a (2:12:4:4:1:6:2) ratio. Glosensor cAMP assay. GSKO cells were transfected with a GPCR, Gα subunit, Gγ 2 , Gβ 1 , Glosensor 22F, and either pcDNA3.1(+) or PTX-S1 in a (1:1:1:1:4:1) ratio. After a 24-h incubation, cells were washed twice with DPBS and treated with trypsin-EDTA (0.05%). Detached cells were harvested and centrifuged at 250 × g for 5 min, and the cell pellet was resuspended in equilibration buffer supplemented with 10% FBS by volume and 2 mM D-luciferin. Cells were incubated at room temperature for 1 h and then distributed to opaque white 96-well plates. Luminescence measurements were made from cells treated with vehicle, agonist, or 100 μM forskolin.
HiBiT-based V 2 R internalization. Parental HEK 293 cells, G 12/13 -deficient HEK 293 cells (39), or β-arrestin1/2-deficient HEK 293 cells (37) in growth phase were seeded in a 6-well culture plate at a concentration of 2 × 10 5 cells mL −1 . Cells were transfected with 100 ng of HiBiT-V 2 R, which contained an Interleukin 6-derived signal sequence followed by a HiBiT sequence and a linker at the N terminus (MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVSGWRLFKKISGGSGGGGSG; gene synthesized with codon optimization) and an unintended SmBiT tag at the C terminus. After 1 d, cells were harvested, suspended in 1 mL of assay buffer, dispensed in a white 96-well half-area plate at a volume of 25 μL per well, and mixed with 25 μL of 2× substrate buffer consisting of 1:200 of a LgBiT stock solution (Promega) and 20 μM furimazine in the assay buffer. After 40 min at room temperature, the plate was measured for baseline luminescence, and a titrated ligand (10 μL) diluted in the 1× substrate buffer was manually added. The plate was immediately read at room temperature for the following 30 min at a measurement interval of 30 s with an accumulation time of 0.4 s per read. The luminescence counts over 27-30 min after ligand addition were averaged and normalized to the initial count. BRET, luminescence measurements. Steady-state BRET and luminescence measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH). Kinetic BRET and luminescence time course measurements were made using a Polarstar Optima plate reader (BMG Labtech). Coelenterazine h (5 μM; Nanolight) or furimazine (NanoGlo; 1:1,000, Promega) were added to all wells immediately prior to making measurements with Rluc8 and Nluc, respectively. Raw BRET signals were calculated as the emission intensity at 520-545 nm divided by the emission intensity at 475-495 nm. Net BRET signals were calculated as the raw BRET signal minus the raw BRET signal measured from cells expressing only the Rluc8 donor.
Data Availability. All study data are included in the article and SI Appendix.