Author response: Effector membrane translocation biosensors reveal G protein and βarrestin coupling profiles of 100 therapeutically relevant GPCRs (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and βarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology. Editor's evaluation The authors describe a novel set of biosensors to assess the coupling specificity of 100 therapeutically relevant G proteins-coupled receptors (GPCRs) to various G proteins. The utility of the assay system is well-supported by the data. These tools are likely to be useful for many specific studies of individual receptors, including efforts to discover ligands that display functional selectivity (bias) between G protein pathways or between G proteins and arrestins. The work provides a rich repository of data informing on the possible effector coupling of 100 GPCRs and a set of analytical tools that could guide the development of new drugs, including efforts to discover ligands that display functional selectivity (bias) between G protein pathways or between G proteins and arrestins. https://doi.org/10.7554/eLife.74101.sa0 Decision letter Reviews on Sciety eLife's review process Introduction G protein-coupled receptors (GPCRs) play crucial roles in the regulation of a wide variety of physiological processes and represent one-third of clinically prescribed drugs (Hauser et al., 2017). Classically, GPCR-mediated signal transduction was believed to rely on linear signaling pathways whereby a given GPCR selectively activates a single G protein family, defined by the nature of its Gα subunit (Oldham and Hamm, 2008). Gα proteins are divided into four major families (Gs, Gi/o, Gq/11, and G12/13) encoded by 16 human genes. Once activated, these proteins each trigger different downstream effectors yielding different biological outcomes. It has now become evident that many GPCRs can couple to more than one G protein family and that ligands can selectively promote the activation of different subsets of these pathways (Namkung et al., 2018; Quoyer et al., 2013). These observations extended the concept of ligand-biased signaling, which was first established for ligand-directed selectivity between βarrestin and G protein (Azzi et al., 2003; Wei et al., 2003), to functional selectivity between G proteins. Ligand-directed functional selectivity represents a promising avenue for GPCRs drug discovery since it offers the opportunity of activating pathways important for therapeutic efficacy while minimizing activation of pathways responsible for undesirable side effects (Galandrin et al., 2007; Kenakin, 2019). To fully explore the potential of functional selectivity, it is essential to have an exhaustive description of the signaling partners that can be activated by a given receptor, providing receptor- and ligand-specific signaling signatures. Currently, few assays allow for an exhaustive pathway-specific analysis of GPCR signaling; these include BRET-based G protein activation sensors platforms (Galés et al., 2005; Masuho et al., 2015; Maziarz et al., 2020; Mende et al., 2018; Olsen et al., 2020) and the TGF-α shedding assay (Inoue et al., 2019). However, several of these platforms require modification of G protein subunits that may create functional distortions. Moreover, these assays may detect non-productive conformational rearrangements of the G protein heterotrimer as was recently reported for G12 (Okashah et al., 2020). Here, we describe unique sensors that do not require modification of receptors or G proteins (except for Gs) for interrogating the signaling profiles of GPCRs. The platform includes 15 pathway-selective enhanced bystander bioluminescence resonance energy transfer (ebBRET) biosensors monitoring the translocation of downstream effectors to the plasma membrane for Gi/o, Gq/11, and G12/13, the dissociation of the Gα subunit from the plasma membrane for Gs and the recruitment of βarrestin to the plasma membrane. Overall, the new ebBRET-based Effector Membrane Translocation Assays, named EMTA, provide a readily accessible large scale and comprehensive platform to study constitutive and ligand-directed GPCR signaling. The signaling signatures of 100 GPCRs using the EMTA platform also provides a rich source of information to explore the principles underlying receptor/G protein/βarrestin coupling selectivity relationships. It thus provides a unique set of tools that is complementary to previously described platforms and existing datasets, and offers a map of the coupling potentials for individual GPCR that will stimulate future studies investigating the relevance of these couplings in different physiological systems. Results ebBRET-based G protein effector membrane translocation assay (EMTA) allows detection of each Gα protein subunit activation To detect the activation of Gα subtypes, we created an EMTA biosensor platform based on ebBRET (Namkung et al., 2016; Figure 1A). The biosensors at the heart of EMTA consist of sub-domains of the G protein-effector proteins p63-RhoGEF, Rap1GAP and PDZ-RhoGEF that selectively interact with activated Gq/11, Gi/o, or G12/13, respectively. These domains were fused at their C-terminus to Renilla luciferase (RlucII) and co-expressed with different unmodified receptor and Gα protein subtypes. Upon GPCR activation, the energy donor-fused effectors translocate to the plasma membrane to bind activated Gα proteins, bringing RlucII in close proximity to the energy acceptor, Renilla green fluorescent protein, targeted to the plasma membrane through a CAAX motif (rGFP-CAAX), thus leading to an increase in ebBRET. The same plasma membrane translocation principle is used to measure βarrestin recruitment (Namkung et al., 2016; Figure 1B, top). Because no selective soluble downstream effector of Gs exists, the assay was modified taking advantage of Gαs dissociation from the plasma membrane following its activation (Wedegaertner et al., 1996). In this configuration, the RlucII is directly fused to Gαs (Carr et al., 2014). Its activation upon GPCR stimulation leads to its dissociation from the plasma membrane (Martin and Lambert, 2016), resulting in a reduction in ebBRET (Figure 1B, bottom). Figure 1 Download asset Open asset EMTA ebBRET platform to monitor G protein activation and βarrestin recruitment. (A) Schematic of the G protein Effector Membrane Translocation Assay (GEMTA) to monitor Gα protein activation. Upon receptor activation, RlucII-tagged effector proteins (Effector-RlucII) translocate towards and interact with active Gα subunits from each G protein family, leading to increased ebBRET. (B) Principle of the Effector Membrane Translocation Assay (EMTA) monitoring βarrestin recruitment to the plasma membrane (top) and Gαs activation (bottom). Top; upon receptor activation, RlucII-tagged βarrestins (βarrestin-RlucII) translocate to the plasma membrane, thus increasing ebBRET with rGFP-CAAX. Bottom; Internalization of activated RlucII-tagged Gαs (Gαs-RlucII) following receptor stimulation decreases ebBRET with the membrane-anchored rGFP-CAAX. The sensitivity and selectivity of the newly created G protein EMTA biosensors, were validated using prototypical GPCRs known to activate specific Gα subtypes. The responses were monitored upon heterologous expression of specific Gα subunits belonging to Gi/o, Gq/11, or G12/13 families in the absence or presence of pharmacological inhibitors and using engineered cells lacking selected Gα subtypes. The dopamine D2 receptor was used to validate the ability of the Gi/o binding domain of Rap1GAP (Jordan et al., 1999; Meng et al., 1999) to selectively detect Gi/o activation. The dopamine-promoted increase in ebBRET between Rap1GAP-RlucII and rGFP-CAAX in the presence of Gαi/o subunits was not affected by the Gq/11-selective inhibitor UBO-QIC (a.k.a., FR900359; Schrage et al., 2015; Figure 2A, left), whereas the Gαi/o family inhibitor, pertussis toxin (PTX), completely blocked the response for all members of Gαi/o family except for Gαz, known to be insensitive to PTX (Casey et al., 1990; Figure 2A, right). Gonadotropin-releasing hormone (GnRH) stimulation of the GnRH receptor (GnRHR), used as a prototypical Gq/11-coupled receptor, promoted ebBRET between the RlucII-fused Gq/11 binding domain of p63-RhoGEF (p63-RhoGEF-RlucII; Lutz et al., 2007; Rojas et al., 2007) and rGFP-CAAX. The ebBRET increase observed in the presence of different Gαq/11 subunits was not significantly (p = 0.077, 0.0636 and 0.073 for Gq, G11, and G14, respectively) affected by PTX (Figure 2B, right), whereas UBO-QIC completely blocked the response for all members of Gαq/11 family except for Gα15, known to be insensitive to UBO-QIC (Schrage et al., 2015; Figure 2B, left). These two G protein-specific EMTA were sensitive enough to detect responses elicited by endogenous G proteins since deletion of Gi/o (ΔGi/o) or Gq/11 (ΔGq/11) subtypes completely abolished the responses induced by D2 or GnRHR activation in the absence of heterologously expressed G proteins (Figure 2—figure supplement 1I). It should however be noted that relying on endogenous proteins does not allow the identification of specific members of Gi/o (i.e.: Gi1, Gi2, Gi3, GoA, GoB, or Gz) or Gq/11 (i.e.: Gq, G11, G14, or G15) families. Figure 2 with 6 supplements see all Download asset Open asset Validation of EMTA ebBRET-based platform to monitor Gα protein activation. (A) Pharmacological validation of the Gαi/o activation sensor. HEK293 cells were transfected with the D2 receptor and the Gαi/o family-specific sensor, along with each Gαi/o subunit. Concentration-response curve using the Gαi/o activation sensor, in the presence or absence of UBO-QIC (left) or PTX (right) inhibitors. Insets; Emax values determined from concentration-response curves of inhibitor-pretreated cells. (B) Pharmacological validation of the Gαq/11 activation sensor. HEK293 cells were transfected with the GnRH receptor and the Gαq/11 family-specific sensor, along with each Gαq/11 subunit. Concentration-response curve using Gαq/11 activation sensor, in the presence or absence of UBO-QIC (left) or PTX (right) inhibitors. Insets; Emax values determined from dose-response curves of inhibitor-pretreated cells. (C) Validation of the Gα12/13 activation sensor. Cells were transfected with the CB1 receptor and one of the Gα12/13 activation sensors, along with the Gα12 or Gα13 subunits. Concentration-response curves of HEK293 cells (top) or the parental and devoid of G12/13 (ΔG12/13) HEK293 cells (bottom) using the PDZ-RhoGEF-RlucII/rGFP-CAAX (top and bottom left) or PKN-RBD-RlucII/rGFP-CAAX (bottom right) sensors, pretreated or not with UBO-QIC or PTX (top). (D) Pharmacological validation of the Gαs activation sensor. HEK293 cells were transfected with the GPBA receptor and the Gαs activation (left and central) or the EPAC (right) sensors. Left: Concentration-response curves using the Gαs activation sensor in the presence or absence of UBO-QIC or PTX, inhibitors of Gαq or Gαi/o, respectively. Central: Concentration-response activation of the Gαs sensor using CTX, a Gαs activator. Right: Concentration-response curve using the EPAC sensor. Inset; Emax values determined from dose-response curves of inhibitors-pretreated cells. Data are expressed as BRET ratio for the concentration-response curves or expressed in % of respective control cells (Emax graphs) and are the mean ± SEM of 3 (A–C) or 4 (D) independent experiments performed in one replicate. Unpaired t-test (A–D): *p < 0.05 and ***p < 0.001 compared to control cells. Figure 2—source data 1 Raw data of Figure 2. https://cdn.elifesciences.org/articles/74101/elife-74101-fig2-data1-v2.xlsx Download elife-74101-fig2-data1-v2.xlsx The selectivity of the G12/13 binding domain of PDZ-RhoGEF (Fukuhara et al., 2001) was confirmed using the cannabinoid receptor type 1 (CB1). The ebBRET between PDZ-RhoGEF-RlucII and rGFP-CAAX in the presence of Gα12 or Gα13 promoted by the cannabinoid agonist WIN-55,212–2 was not affected by UBO-QIC (Figure 2C, top left), nor PTX (Figure 2C, top right). Given the lack of selective G12/13 pharmacological inhibitor, we used HEK293 cells genetically deleted for Gα12 and Gα13 proteins (ΔG12/13) to further confirm the response selectivity. As expected, PDZ-RhoGEF-RlucII/rGFP-CAAX ebBRET was observed only following reintroduction of either Gα12 (ΔG12/13_+G12) or Gα13 (ΔG12/13_+G13) (Figure 2C, bottom left). The G12/13 coupling of CB1 was further confirmed by monitoring the recruitment of PKN to the plasma membrane (Figure 2C, bottom right) in agreement with previous reports (Inoue et al., 2019). To further assess the selectivity of each EMTA biosensor, we took advantage of the fact that the endothelin-1 receptor (ETA) can activate Gq/11, Gi/o, and G12/13 family members. As shown in Figure 2—figure supplement 2, only over-expression of the Gα family members corresponding to their selective effectors (Rap1GAP for Gi/o, p63-RhoGEF for Gq/11, and PDZ-RhoGEF for G12/13) significantly increased the recruitment of the effector-RlucII to the plasma membrane. A recent study (Chandan et al., 2021) showed that Gi/o can also activate full length PDZ-RhoGEF. Although the domain of PDZ-RhoGEF required for this activation has not been identified yet, the selectivity of our PDZ-RhoGEF sensor for G12/13 vs. all other G protein families most likely results from the fact that we used a truncated version of PDZ-RhoGEF that only contains the G12/13 binding domain and lacks the PDZ domain involved in protein-protein interaction, the actin-binding domain and the DH/PH domains involved in GEF activity and RhoA activation (Aittaleb et al., 2010). It should be noted that in the heterologous expression configuration, competition with endogenous G proteins did not occur to a significant extent since the potencies of the responses to a given G protein subtype were not affected by genetic deletion of the different G protein family members (Figure 2—figure supplement 1 and Supplementary file 1A). Similarly, overexpression of G proteins, GPCRs or effectors-RlucII did not affect the potencies of the responses observed (Figure 2—figure supplement 3 and Supplementary file 1B-D), indicating that, in our experimental conditions, overexpression of the different components of EMTA sensors must likely not bias the coupling response. In addition to spectrometric assessment of coupling selectivity (above) and activation kinetics (Figure 2—figure supplement 4), EMTA allows to image the real-time recruitment of the G protein effectors to the plasma membrane (Videos 1–3) thus providing spatiotemporal resolution for the imaging detection of Gαi/o, Gαq/11, and Gα12/13 activation. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg BRET-based imagery of p63-RhoGEF-RlucII recruitment to the plasma membrane upon AT1 activation. HEK293 cells expressing the p63-RhoGEF-RlucII/rGFP-CAAX sensors with Gαq and AT1 were stimulated with Angiotensin II. BRET levels (the ratio of the acceptor photon count to the total photon count) are expressed as a color code (lowest being black and purple, and highest being red and white). Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg BRET-based imagery of Rap1GAP-RlucII recruitment to the plasma membrane upon D2 activation. HEK293 cells expressing the Rap1GAP-RlucII/rGFP-CAAX sensors with Gαi2 and D2 were stimulated with dopamine. BRET levels (the ratio of the acceptor photon count to the total photon count) are expressed as a color code (lowest being black and purple, and highest being red and white). Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg BRET-based imagery of PDZ-RhoGEF-RlucII recruitment to the plasma membrane upon TPαR activation. HEK293 cells expressing the PDZ-RhoGEF-RlucII/rGFP-CAAX + Gα13 and TPαR were stimulated with U46619. BRET levels (the ratio of the acceptor photon count to the total photon count) are expressed as a color code (lowest being black and purple, and highest being red and white). The sensitivity of the EMTA platform is illustrated by a direct side-by-side comparison of the signals detected with EMTA vs. BRET assays based on Gαβγ dissociation (Gαβγ) (Galés et al., 2005; Galés et al., 2006; Olsen et al., 2020), that reveals a significantly larger assay windows for EMTA for the 6 Gα subunits tested for eight selected receptors, (Figure 2—figure supplement 5). For the Gαs translocation biosensor, the bile acid receptor (GPBA) was chosen for validation (Kawamata et al., 2003). As expected, lithocholic acid stimulation resulted in a concentration-dependent decrease in ebBRET between Gαs-RlucII and rGFP-CAAX (Figure 2D, left). Cholera toxin (CTX), which directly activates Gαs (De Haan and Hirst, 2004), led to a decrease in ebBRET (Figure 2D, center), confirming that loss of Gαs plasma membrane localization results from its activation. The potency of lithocholic acid to promote Gs dissociation from the plasma membrane was well in line with its potency to increase cAMP production as assessed using a BRET²-based EPAC biosensor (Leduc et al., 2009; Figure 2D, right). The Gs-plasma membrane dissociation ebBRET signal was not affected by UBO-QIC or PTX (Figure 2D, left), confirming the selectivity of the biosensor. Signaling signatures of one hundred therapeutically relevant receptors reveals distinct G protein and βarrestin selectivity profiles We used EMTA to assess the signaling signature of a panel of 100 human GPCRs that are either already the target of clinically used drugs (74 receptors), considered for pre- or clinical drug development (6 receptors), or pathophysiologically relevant (Supplementary file 2A). To establish the coupling potentials for each receptor, we quantified its ability to activate 15 pathways: Gαs, Gαi1, Gαi2, GαoA, GαoB, Gαz, Gα12, Gα13, Gαq, Gα11, Gα14, Gα15 and βarrestin 2 as well as βarrestin 1 and 2 in the presence of GRK2 (Supplementary file 3). Emax and pEC50 values were determined (Supplementary file 2) and, based on the pre-determined threshold criteria (Emax ≥mean of vehicle-stimulated +2*SD; see Materials and methods), a ‘yes or no’ agonist-dependent activation was assigned to each signaling pathway and summarized using radial graph representations (Figure 3—figure supplement 1). To assess whether endogenous receptors could contribute to the observed responses, assays were also carried out in cells not transfected with the studied receptor (Figure 3—figure supplement 2). When an agonist-promoted response was observed in non-transfected parental HEK293 cells, this response was not considered as a receptor-specific response (see Materials and methods). To compare the signaling profiles across all receptors and pathways and to overcome differences in receptor expression levels and individual biosensor dynamic windows, we first min-max normalized Emax and pEC50 values (between 0 and 1) across receptors as a function of a reference receptor yielding the largest response for a given pathway (Figure 3A, left). Then, these values were again min-max normalized (between 0 and 1) for the same receptor across pathways, using the pathway with the largest response for this receptor as the reference (Figure 3A, right; see description in Materials and methods). Such double normalization allows direct comparison of the coupling efficiency to different G proteins for a given receptor and across receptors for a given G protein. This coupling efficiency is summarized as heatmaps (Figure 3B) that reveals a high diversity of signaling profiles. The selectivity toward the different G protein families varies considerably among GPCRs (Figure 4). In our dataset, which is the first using unmodified GPCRs and Gα proteins (except for Gs), 29% of the receptors coupled to only one family, whereas others displayed more promiscuity by coupling to 2, 3, or 4 families (36%, 25%, and 10%, respectively). Receptors coupling to a single G protein family favored the members of the Gi/o family. Indeed, 27% of the receptors coupling to Gi/o only activated this subtype family in comparison to 0, 2.4 and 9.1% for receptors activating G12/13, Gq/11, and Gs, respectively, thus displaying more promiscuous coupling. A detailed comparative analysis of the selectivity profiles that we observed using the EMTA sensors with that of the chimeric G protein-based assay developed by Inoue et al., 2019 and the IUPHAR/BPS Guide to Pharmacology database (GtP; https://www.guidetopharmacology.org/) is in the (Hauser et al., Supplementary file allows a direct comparison of the potency determined using EMTA for the new and the already known (i.e.: identified in As can be in the in many the potency for the novel couplings is this is not a since for receptors, the for the new couplings are G12 for for G12/13 for and Gi/o for hormone receptor 1 and G protein-coupled receptor or for for 3 and 3 G12 for 2 A receptor 6 and G12/13 for and than for the ones. in many the potency for the newly couplings are to for which is than for their G proteins, a with the of βarrestins in signaling at the plasma membrane. The potency differences observed for the activation of different G protein subtypes by a given receptor may to activation of pathways This selectivity is likely to be by G protein subtype expression The physiological of selectivity to be Figure 3 with 3 supplements see all Download asset Open asset the diversity of receptor-specific signaling signatures detected with the EMTA ebBRET (A) values each pathway were normalized to the response observed across all receptors = left). These values were normalized across pathways for the same receptor, with the pathway as the reference = right). (B) of double normalized Emax (left) and pEC50 (right) data. cells no detected coupling. receptor are Figure 4 with 1 supplement see all Download asset Open asset The EMTA ebBRET platform has a unique ability to coupling selectivity between G protein families. (A) the of receptors coupled to each G protein family in the EMTA ebBRET biosensor (B) of receptors coupling of receptors that couple to members of 2, 3, or 4 G protein families. (C) of G protein subunit coupling of receptors that activate each Gα subunit. (D) of receptors of receptors that do not or that either or or βarrestin data are based on double normalized Emax values from Figure Figure data 1 Raw data of Figure Download When the of coupling for each Gα subunit family (Figure the Gi/o family members were the most activated, with of the tested receptors activating a Gi/o family In only and of the receptors activate Gs, G12/13, or Gq/11 family respectively. and with its reported promiscuous Gα15 was found to be activated by of the For receptors, we also observed coupling of distinct members a subtype family (Figure 3—figure supplement 1). For of receptors can couple to only a of the family (Figure supplement 1A). For the Gq/11 family, only activate all family members with activating only Gα15 and only two or members of the family. A expressing the % of receptors a specific Gα subtype that also activated is illustrated in Figure supplement When individual the Gi/o family was The were observed between and either or Gαz, and the between and A of coupling selectivity is the that activates only and and that selectively activates Similarly, the responses our threshold criteria (see Materials and methods), and receptors activation of and Gαz, whereas the and receptors activate GαoB, GαoA, and all members of a given family are found to be activated, receptors activate specific family members with potencies (Supplementary file When βarrestin our analysis that of receptors did not βarrestin 1 or 2, in the presence of GRK2 (Figure the receptors to only a selectively or most of βarrestins in the presence of GRK2 (Figure of GRK2 the recruitment of for of receptors the of GRK2 expression in βarrestin activation (Supplementary 3 and 2). with previous reveals and crucial differences We compared the signaling profiles with by Inoue et al., 2019 and the this comparison only the reported couplings that in the study were based on the criteria of coupling and coupling and is by the different and normalization used in the two A comparison of couplings using Emax normalization and of G proteins into families is in the (Hauser et al., As can be in Supplementary file among the receptors to couplings were detected in our study than reported in Inoue et for Gαs vs. vs. Gαq vs. and vs. In more receptors activating Gα12 vs. vs. Gα13 vs. vs. and Gα15 vs. were detected in our When with data in that reports couplings for G protein families (i.e.: Gs, Gi/o, Gq/11, or G12/13) and not at the single G protein subtype we detected couplings than was reported in for Gαs vs. more for Gαi/o vs. Gαq/11 vs. and Gα12/13 vs. among the receptors to (Supplementary file the comparative analysis reveals and of couplings between the EMTA and or datasets, respectively. unique couplings and couplings found in the other two The for these differences are to differences in the assays For for G12/13 and the with the most likely results from the fact that in most G12/13 or activation were determined their recent description Quoyer et al., Schrage et al., 2015; et al., Olsen et al., 2020), no readily assay