Specific pharmacological and Gi/o protein responses of some native GPCRs in neurons

Xu, Chanjuan; Zhou, Yiwei; Liu, Yuxuan; Lin, Li; Liu, Peng; Wang, Xiaomei; Xu, Zhengyuan; Pin, Jean-Philippe; Rondard, Philippe; Liu, Jianfeng

doi:10.1038/s41467-024-46177-z

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Published: 05 March 2024

Specific pharmacological and G_i/o protein responses of some native GPCRs in neurons

Nature Communications volume 15, Article number: 1990 (2024) Cite this article

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Abstract

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and are important drug targets. The discovery of drugs targeting these receptors and their G protein signaling properties are based on assays mainly performed with modified receptors expressed in heterologous cells. However, GPCR responses may differ in their native environment. Here, by using highly sensitive G_i/o sensors, we reveal specific properties of G_i/o protein-mediated responses triggered by GABA_B, α₂ adrenergic and cannabinoid CB1 receptors in primary neurons, different from those in heterologous cells. These include different profiles in the G_i/o protein subtypes-mediated responses, and differences in the potencies of some ligands even at similar receptor expression levels. Altogether, our results show the importance of using biosensors compatible with primary cells for evaluating the activities of endogenous GPCRs in their native environment.

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Introduction

G protein-coupled receptors (GPCRs) are ubiquitously expressed in every cell type, and constitute the largest family of membrane proteins¹. They participate in the regulation of a large variety of physiological functions². Their dysregulation or malfunction can be the cause of numerous diseases³. Accordingly, controlling their activity with selective drugs can have multiple therapeutic effects, as illustrated by the large proportion of the clinical drugs targeting GPCRs⁴.

Most of the physiological functions of GPCRs are mediated through the coupling of G proteins². Eventually, GPCRs are able to activate several G protein subtypes, such as G_i/o, G_s, G_q, and G_12/13 family^5,6,7,8, but this may vary depending on the use of ligands, a phenomenon called ligand biased effect^9,10, or/and the cellular environment known as system bias¹¹. This concept of functional selectivity¹¹, that combined effect of ligand and system bias, has been largely used in the characterization of ligands^12,13,14, but the signalling properties and identification of potential drug candidates targeting GPCRs are performed in heterologous cells. Since the cellular environment is critical, the analysis of G protein response profile of endogenous receptors in their native environment is therefore of much interest to validate drug candidates. Indeed, it becomes clear that many interacting proteins, post-translational modifications, localization in specific compartments, lipid membrane and ion environments largely influence GPCR signalling properties^15,16. It is thus essential to examine the effects of drugs on the signalling properties of GPCRs in their native environments.

Functional analysis of endogenous GPCRs in their native environment is usually performed through the measurement of second messengers, such as cAMP, inositol phosphate or Ca²⁺¹⁷, or ion channel regulation^2,18. Due to receptor reserves and signal amplification, such assays are not precise for estimating drug activity, potency and efficacy⁹. G proteins are first effectors of GPCRs, such that measuring their activation is less prone to signal amplification¹⁸. GTPγS binding assay can detect G protein activation in native cells or tissues^19,20. However it requires the preparation of membranes and these assays are not specific for one G protein subtype, unless an immunoprecipitation of the G protein is added²¹. Nowadays, much hope in studying native GPCRs is based on the development of specific optical biosensors that must be sensitive enough to detect the activity of native receptors²² usually expressed at very low levels, but most of them are still not compatible with these endogenously expressed receptors^{13,23,24,25,26}.

In the central nervous system, GPCRs are essential regulators of synaptic transmission, acting at both pre- and post-synaptic levels, as well as in glial cells²⁷. More than half of neuronal GPCRs can couple to one or more of the various G_i/o protein subtypes⁵, but their expression profiles vary in different native tissues or cell types and specific cellular function depends on distinct G_i/o protein subtypes^12,28,29. It is therefore essential to determine which G protein can be activated by a GPCR upon stimulation with various agonists in its native environment.

In the present study, we describe BRET-based sensors for each G_i/o protein subtypes that can be used to study endogenous GPCRs in living neurons. Our study focuses on three types of GPCRs that play important roles in modulating synaptic activity, the GABA_B³⁰, α₂ adrenergic^31,32 and cannabinoid CB1 receptors³³. We could detect the activation of G_i/o proteins from a small number of neurons and monitor both the kinetics and dose-response of the effect mediated by various compounds including agonists, antagonists and positive allosteric modulators, in different types of neurons. Our data reveal differences in the profile of G_i/o protein subtypes mediated responses and agonist potencies between these recombinant and native receptors in HEK293 cells and neurons, respectively. In addition, our results show a major difference in G_i1 versus G_oA protein activation induced by a CB1 receptor agonist in neurons, but not in HEK293 cells. Finally, different composition of Gγ subunits in the neurons can also lead to specific G_i/o protein responses. Altogether, our results reveal the importance of evaluating GPCR activities in their native environment and highlight the need for sensitive biosensors compatible with native receptors in their natural environment.

Results

G_i/o protein sensors for endogenous GPCRs in neurons

To measure the rearrangement or dissociation of the G_i/o proteins upon receptor activation in live cells and neurons, we used a series of BRET-based biosensors. They are based on the use of a luciferase as an energy donor inserted in the Gα subunit and the fluorescent protein Venus as an energy acceptor attached to the Gγ subunit, as reported for previous BRET- and FRET-based G protein sensors^23,24,25. To monitor these sensors in conditions close to the physiological ones, in our approach, the cells were cotransfected only with the luciferase-tagged Gα subunit and Venus-tagged Gγ subunit (^VenusGγ), while the endogenous Gβ subunits were used (Fig. 1a). In these experiments, the amount of Gβγ complexes that can produce a BRET signal with Gα is expected to be limited by the endogenous level of Gβ subunits (Supplementary Fig. 1a) that are able to form a complex with the ^VenusGγ. We used mostly the mouse cerebellum granule neurons (CGNs) that constitute the largest homogenous neuronal population of the mammalian brain³⁴.

**Fig. 1: Design and validation of the G_i/o protein sensors in neurons.**

Our sensors rely on the use of a small luciferase, the Oplophorus nanoluciferase (Nluc; 19 kDa) that produces a more intense and sustained luminescence signal than other commonly used luciferases from Renilla luciferase (Rluc and Rluc8; 36 kDa)³⁵. Nluc was inserted in the helical domain of different Gα_i/o subunits similarly to previously reported G protein BRET sensors with Rluc^23,24 and Nluc³⁶, and the constructs were named Gα^Nluc (Fig. 1b). The Nluc was inserted after residue 91 (for Gα_i1, Gα_i2, Gα_i3, Gα_oA and Gα_oB), or 113 (for Gα_z) (Fig. 1c). Of note, Gα_oA has 94% sequence identity with Gα_oB and around 70% with Gα_i1, Gα_i2 and Gα_i3³⁷. To facilitate the insertion of Nluc, a short and flexible linker sequence (SGGGGS) was added at the N- and C-terminal ends of Nluc. For BRET signal measurement, Venus was fused to the N-terminus of Gγ₂ subunit with the amino acid sequence KLGT serving as a linker, and the construct is named ^VenusGγ₂ (Fig. 1b). To show the advantage of using Nluc over Rluc8 in measuring agonist-induced BRET change in neurons, Rluc8 was inserted at the same position as Nluc in Gα_i1 (named Gα_i1^Rluc8) and Gα_oA (named Gα_oA^Rluc8). These Gα_i1^Rluc8 and Gα_oA^Rluc8 constructs are highly similar to the constructs reported for the Gα protein of TRUPATH BRET sensors (Supplementary Fig. 1b). The Gα_oA^Nluc construct showed much higher luminescence intensity at emission 480 nm compared with the subunits Gα^Rluc8 in CGNs, for the same amount of cDNA co-transfected with ^VenusGγ₂ (Supplementary Fig. 1c). In addition, the basal BRET ratio of the constructs Gα_i1^Nluc and Gα_oA^Nluc co-transfected with ^VenusGγ₂ was also more stable over time, compared to the sensors that used subunits Gα^Rluc8 (Fig. 1d and Supplementary Fig. 1d). We also verified that the co-expression of the constructs Gα^Nluc + ^VenusGγ₂ for the different Gα subunits did not change the expression level of endogenous Gβ subunits (Supplementary Fig. 1e). Altogether, the G protein Nluc-biosensors are expected to produce a much improved signal-to-noise ratio for BRET measurement in neurons.

We then validated our different G_i/o protein sensors with the GABA_B receptor as a prototype G_i/o-coupled receptor, which is abundantly expressed in many types of neurons³⁸, including in CGNs³⁹. Interestingly, despite a low percentage of transfected neurons compared with HEK293 cells (Fig. 1e), the GABA_B receptor specific agonist baclofen largely decreased the BRET signal with the sensor Gα_i1^Nluc + ^VenusGγ₂ (Fig. 1d, Supplementary Fig. 2a) and Gα_oA^Nluc + ^VenusGγ₂ (Supplementary Fig. 1d, 2b), while the change was barely detectable with Gα_i1^Rluc8 + ^VenusGγ₂ (Fig. 1d) and Gα_oA^Rluc8 + ^VenusGγ₂ (Supplementary Fig. 1d). In kinetics experiments, baclofen induced a rapid and strong decrease of BRET signal in CGNs, that was reversed by the GABA_B receptor competitive antagonist CGP64213 for both the G_i1 (Fig. 1f) and G_oA (Supplementary Fig. 2c) sensors. As a control, no change of BRET signal was measured when Gα_i1^Nluc was expressed in the absence of ^VenusGγ₂ or with Venus (Supplementary Fig. 2d). Similarly, no change of BRET signal was measured with the dominant negative mutants Gα_i1^Nluc-S47A⁴⁰ or Gα_i1^Nluc-G202T⁴¹ (Supplementary Fig. 2e) when they are co-expressed with ^VenusGγ₂. This is consistent with the unability of these mutants to exchange their GDP for GTP. We also verified that the co-expression of Gβ with the Gα and Gγ₂ was not required for a large BRET change signal induced by baclofen (Supplementary Fig. 2f). Altogether, these results validated the use of the Gα_i1^Nluc and Gα_oA^Nluc -based BRET sensors in neurons.

These G_i/o protein Nluc sensors were highly sensitive even though only about 2% of CGNs expressed the sensor as measured by the fluorescence of the transfected ^VenusG_γ2. This low level of transfection is consistent with previous data reported with CGNs⁴², even though any transfected cell is expected to overexpress the sensors thanks to the strong promotor. The transfected neurons displayed normal morphology with dendrites and branches (Fig. 1e). It indicates that the Gα_i/o protein Nluc-sensors were sensitive enough to monitor endogenous GPCR activation in a small number of neurons. In addition, they are compatible with measurements in 96-well plates. The amount of cDNA to be transfected in CGNs was optimized for each sensor (Supplementary Fig. 2g). Finally, our sensors are also compatible with the measurement of the activation of endogenous GPCRs expressed in the HEK293 cells such as the lysophosphatidic acid receptor (Supplementary Fig. 3a). In these experiments, the G_i2 showed a weak response than those generated by the other G_i/o sensors (Supplementary Fig. 3a), and this is unrelated to differences in expression levels of these sensors (Supplementary Fig. 3b).

We then compared the agonist-induced BRET signals obtained with the same Gα_i/o sensor in the two different cell types, CGNs and HEK293 cells (Fig. 2a). Conditions were optimized such that the GABA_B receptor expression level in HEK293 cells was similar to that in CGNs (Fig. 2b). Since the basal BRET signal in the absence of agonist can be different between the G protein sensors (Supplementary Fig. 3c), the signal was expressed as the agonist-induced change in BRET ratio expressed as percentage of the basal signal. The results showed that in CGNs, the BRET changes induced by baclofen are significantly different from those measured in HEK293 cells except for the two G_o sensors (Fig. 2a). Indeed, a larger BRET signal is measured in CGNs with G_i1 and G_i3, and a large signal is observed with Gz in HEK293 cells while absent in CGNs. The differences observed are related neither to the expression levels of the Gα^Nluc sensor components (Fig. 2c) nor to the endogenous Gα_i/o mRNA expression levels (Fig. 2d). Though lower signal was observed in G_i2 in CGNs, the difference of baclofen response can be detected in dose-dependent manner (Supplementary Fig. 3d). Similar pEC₅₀ values were measured in three G_i subtype sensors and two G_o subtype sensors in CGNs upon baclofen activation (Supplementary Fig. 3d). Altogether, the data showed different abilities of the GABA_B receptor to generate responses by the different G_i/o subtypes in transfected HEK293 cells and in CGNs, then revealing the importance of studying GPCRs in their native environment.

**Fig. 2: Activity of endogenous GABA_B receptor detected by the G_i/o protein sensors.**

Lower agonist potencies for the endogenous GABA_B receptor in neurons

We have further validated these G protein sensors by measuring the potency of different orthosteric GABA_B receptor ligands (Fig. 3a) in both CGNs and HEK293 cells. Similar potencies were obtained for the G_i1 and G_oA for baclofen and other agonists, and for the antagonist CGP64213 in CGNs (Fig. 3b–f, Supplementary Table 1). The GABA_B receptor agonists APPA and SKF 97541 were more potent than GABA and baclofen in both CGNs (Fig. 3c–f) and HEK293 cells (Supplementary Fig. 4a), which was consistent with previous findings using the GTPγS assay in brain tissues²¹. pEC₅₀s obtained in CGNs were correlated with those measured in HEK293 cells (Fig. 3e, f), even though they were more than 10 times higher in HEK293 cells, except for baclofen where the difference is lower (Supplementary Table 1, Fig. 3e, f). Similar results were obtained in the hippocampal neurons and cortical neurons (Supplementary Fig. 4b–d and Supplementary Table 1). These differences in baclofen potencies observed between transfected HEK293 cells and native neurons were not due to the difference in receptor expression. Indeed, when the expression level GABA_B receptors was similar to that in CGNs (Fig. 2b), the baclofen pEC₅₀ was similar to that observed with higher expression levels, and still significantly different from that measured in CGNs (Fig. 3g).

**Fig. 3: Lower agonist potencies of the endogenous GABA_B receptor in CGNs.**

We have also validated our sensors for the analysis of positive allosteric modulators (PAMs). PAMs can synergistically enhance the activity of native neurotransmitter GPCRs in neurons⁴³ and have a greater potential for drug development with less side effect⁴⁴, including for the GABA_B receptor⁴⁵. It was recently demonstrated that these PAMs bind at the interface of the transmembrane domains of this dimeric receptor^46,47,48 (Fig. 3a). Here we showed that Rac BHFF increased strongly the potency of baclofen-induced G_i1 (Fig. 3h) and G_oA protein (Supplementary Fig. 5a) response in CGNs. However, in contrast to what is observed in HEK293 cells^48,49 (Fig. 3h and Supplementary Fig. 5a–b), Rac BHFF alone (10 μM) did not increase the net BRET signal in CGNs (Fig. 3h and Supplementary Fig. 5a). It is nicely illustrated by concentration responses curves of the Rac BHFF obtained with transfected HEK293 cells but not so efficiently with neurons (Supplementary Fig. 5c). It suggests that the Rac BHFF agonist activity observed with the recombinant GABA_B receptor is much higher than that measured for the receptor in its native environment. This slight agonist effect of Rac BHFF in CGNs is probably not due to the lower GABA_B receptor expression in neurons compared to HEK293 cells. Indeed, the agonist activity of Rac BHFF can still be observed in HEK293 cells that expressed GABA_B receptor at a similar level to that found in CGNs (Figs. 2b, 3h, Supplementary Fig. 5a).

Finally, we took advantage of our sensors to measure the constitutive activity⁵⁰, in the absence of agonist, of the GABA_B receptor that is usually observed in recombinant systems^{47,49,51,52,53,54,55}. This constitutive activity was reversed by the competitive antagonists known for its inverse agonist activity^49,51,55,56. Accordingly, the competitive antagonist CGP54626 increased the basal BRET signal between Gα and Gβγ for both the G_i1 and G_oA sensors in the HEK293 cells (Supplementary Fig. 5d), as previously reported⁴⁹. It is consistent with the properties of this inverse agonist to favour the Gαβγ heterotrimer by stabilizing the inactive conformation of the receptor. Interestingly, this increase of BRET signal was not observed in the CGNs (Supplementary Fig. 5d), indicating the native GABA_B receptor has no or a very low constitutive activity that cannot be observed with our sensor. It is consistent with the weak intrinsic agonist activity of Rac BHFF in neurons.

Altogether, our results show that GABA_B receptor ligands have different potencies and efficacies in the cell lines compared to neurons, which cannot be explained by a difference in GABA_B receptor expression levels between the two cell types.

Various efficacies of endogenous G_i/o-coupled GPCRs in neurons

We then examined the activities of other endogenous G_i/o-coupled GPCRs in the CGNs. First, the agonist CP 55940 that actives both the cannabinoid receptor type 1 (CB1) and 2 (CB2) induced a strong change in both G_i1 and G_oA BRET signal as observed for the GABA_B receptor (Fig. 4a). It was expected that it is due to the activation of CB1 since this receptor is highly expressed in cerebellum³³ compared to CB2 that is mainly expressed at the periphery⁵⁷. When testing other GPCR agonists, a strong or significant change of BRET signal was measured with different ligands used at saturation concentrations for their expected receptors: brimonidine (also named UK-14304), pramipexole, eletriptan and carbachol known to activate α₂ adrenoceptor⁵⁸, D_2/4 receptor⁵⁹, 5-hydroxytryptamine receptor 1B/1D⁶⁰ and the M_2/4 muscarinic receptors⁶¹, respectively. In contrast, agonists of the platelet-activating factor (PAF) receptor and δ/μ opioid receptor DAMGO failed to induce a BRET signal change (Fig. 4a). Dose-response of brimonidine, CP 55940, pramipexole and carbachol further confirmed the specific activation of these endogenous G_i/o-coupled receptors (Fig. 4b). Altogether, our data correspond to the receptors for which higher amount of mRNA were detected in these cultured CGNs at the developmental stage investigated⁶².

**Fig. 4: G_i/o protein sensors report the activity of other endogenous G_i/o-coupled GPCRs in CGNs.**

Next, using α₂ adrenoceptor we show that our G protein sensors can determine ligand efficacies, by evaluating the full agonist brimonidine and several reported partial agonists (clonidine, oxymetazoline, tizanidine and xylazine)⁶³. In contrast, brimonidine exhibited the highest response of G_i1 and G_oA protein in CGNs, while others showed less response (Fig. 5a). However, the discrimination between brimonidine and other agonists was not significantly observed in HEK293 cells transfected with α_2A adrenoceptor (α_2AAR) (Fig. 5b). It was not due to the high level of expression of α_2AAR, since no discrimination between brimonidine and other agonists was observed in HEK293 cells that expressed α_2AAR at a similar level to that found in CGNs (Fig. 5c). In these conditions (15 ng of α_2AAR cDNA/well), the brimonidine pEC₅₀ was similar to that observed with higher expression levels (Supplementary Fig. 6a), and still significantly different from that measured in CGNs (Fig. 5d), as observed previously for the GABA_B receptor (Fig. 3g). Finally, the data showed a significant difference in the G protein responses mediated by the different G_i/o subtypes triggered by the recombinant α_2AAR in HEK293 cells and the endogenous one in CGNs (Fig. 5e and Supplementary Fig. 6b). Of note, similarly to experiments above with GABA_B receptor (Fig. 2c), the expression of the different Gα^Nluc proteins was within a factor of three in each type of cell (Supplementary Fig. 6c).

**Fig. 5: Activity of endogenous α₂AR detected by the G_i/o protein sensors.**

Our results demonstrated that G_i/o protein Nluc-biosensors can be widely applied to different endogenous G_i/o-coupled GPCRs and evaluate the ligand efficacies and G protein coupling profile in neurons. They also further revealed the importance of studying a GPCR in its native environment.

Difference between G_i and G_o proteins revealed for the endogenous CB1 receptor

Finally, we took advantage of the major change in BRET signal observed for the cannabinoid receptor to investigate the G protein response in CGNs. While level of expression of the CB1 receptor in the two systems was similar in western blotting experiments (Fig. 6a), a higher CB1 receptor-mediated G protein response was observed with G_i1 and G_i3 only, with the native receptor in CGNs compared to the recombinant one in HEK293 cells (Fig. 6b, Supplementary Fig. 7a). Similar responses were observed with the other G_i/o subtype sensors between two types of cells. Again, as with the GABA_B receptor (Fig. 2b) and α_2AAR (Supplementary Fig. 6c), the expression of the different Gα^Nluc proteins was within a factor of three in each type of cell (Supplementary Fig. 7b). And a small overexpression of the CB1 receptor by transfection of CGNs with the recombinant CB1 receptor gave similar results (Supplementary Fig. 7c, d). Interestingly, our G protein sensors revealed a higher potency of CP 55940, Win 55,212-2 and Bay 59-3074 in CGNs compared with transfected HEK293 cells for both G_i1 and G_oA response (Fig. 6c, d and Supplementary Table 2). It is different from GABA_B and α₂AR, which showed higher agonist potencies in transfected HEK293 cells. Of note, when taking Win 55,212-2 as a reference ligand, Bay 59-3074 showed G_oA bias in both CGNs and HEK293 cells with a bias factor of 1.6 and 1.3 respectively. CP 55940 showed a G_i1 bias in CGNs and a G_oA bias in HEK293 cells with a bias factor of 1.6 and 1.4 respectively, indicating a difference between G_i1 and G_oA proteins revealed by CP 55940 (Fig. 6c, d and Supplementary Tables 2 and 3).

**Fig. 6: Difference of G_i1 and G_oA responses by cannabinoid receptor CB1 agonists.**

Major influence of the Gγ subunit on the G protein coupling in CGNs

The influence of Gβγ subunit composition on G protein coupling^64,65 was largely reported even though the underlying molecular mechanism is largely unknown. Therefore, we have compared sensors composed by Gα^Nluc and different Gγ including ^VenusGγ₂, ^VenusGγ₈ or ^VenusGγ₉, which are well-used in the previously reported G protein BRET sensors^25,36. ^VenusGγ₈ or ^VenusGγ₉ belong to two different groups of Gγ subunits (Supplementary Fig. 8). Gγ₈, like Gγ₂, is well expressed in some regions of the brain, in contrast to Gγ₉ that seems restricted to retina⁶⁶. Interestingly, a major difference in G protein response profile of the GABA_B receptor and α₂ adrenoceptor was measured depending on the Gγ subunits used (Fig. 7a–c and Supplementary Fig. 9a–d), that clearly shows the impact of the Gγ subunit in these GPCR-mediated responses.

**Fig. 7: Gγ subunit influences G protein responses in CGNs.**

Discussion

Our study compares the pharmacological and G protein responses of native, versus recombinant G_i/o-coupled receptors in primary neurons and in heterologous cells, respectively. Such an analysis was made possible thanks to the sensitive BRET-based G_i/o protein sensors. These sensors allow a simple analysis of different types of GPCR ligands on each G_i/o protein subtypes in primary neurons in a medium throughput format (in 96-well plate). For the same G protein sensor, our study reveals important differences in agonist potencies between neurons and heterologous HEK293 cells (Fig. 8a). GABA_B and α₂ adrenergic receptor agonists displayed higher potencies in cell lines versus neurons even at similar receptor expression levels, in contrast to CB1 agonist. And when comparing the profile of response of different G protein sensors in the same cellular system, major differences of potencies can be observed, such as for a CB1 agonist (CP 55940) that shows high potency in mediating G_i1 response compared to G_oA in neurons. These data highlight the importance of evaluating the activities of endogenous GPCRs in their native environment, and of developing biosensors allowing such analyses.

**Fig. 8: Major differences in agonist potencies and the G protein response profile for some GPCRs between heterologous and native cells.**

In order to be able to compare the properties of recombinant and native G_i/o-coupled GPCRs, we needed sensors for the various G_i/o proteins compatible with measurements of their activity in primary neurons. Here we showed that Nluc-based BRET sensors, could do so, most likely due to the high emission of Nluc, compared to Rluc8 used previously^24,25, since the efficacy of transfection of these constructs in primary neurons remains very low (around 2%). The principle of our optical biosensors is not novel, since the rearrangement between Gα and Gβγ upon receptor activation using resonance energy transfer techniques was previously reported by FRET²³, BRET^24,25 including with the use of Nluc³⁶. However, these biosensors have never been tested in primary neurons. Interestingly, other kind of biosensors compatible with the detection of endogenous G protein activity were recently reported^22,36. Among them, the unimolecular “BERKY” sensors were used in live primary cells but they required the use of a viral vector. In addition, these sensors were not able to discriminate between different G protein subtypes, such as between G_i1, G_i2, G_i3, G_oA, G_oB and G_z since it is a unimolecular sensor using a peptide recognizing the GTP form of these different G proteins. Accordingly, they cannot be used to analyse the influence of the different Gβγ subunits as our sensors and the previous BRET sensors^24,25,36 do. Instead, our sensors nicely revealed specific responses of each G_i/o protein triggered by some native GPCRs in primary neurons, which are different from what can be observed in heterologous cells. They work well for the most abundant neuronal GPCRs, but would likely need to be further improved for receptors expressed at lower levels.

We have to be cautious when comparing the profile of response of different G protein sensors in the same cellular system (Figs. 7 and 8b). Indeed, each G protein BRET sensor might have its own properties, with its specific range of signal, even though they were constructed similarly, and Gα_i/o subtypes have a high sequence identity³⁷. G protein sensor response can result either from a complete dissociation between Gα and Gβγ but also from a repositioning of Gβγ relative to Gα without a real dissociation²³. Within the G_i/o protein family, using similar FRET-based G protein sensors between Gα and Gβγ, Frank et al. have proposed that receptor-activated G_i1, G_i2, G_i3 and G_z undergo subunit rearrangement rather than subunit dissociation, whereas G_o proteins either rearrange with a very distinct pattern or dissociate during activation⁶⁷. But even though there is good evidence that the activated Gα and Gβγ subunits actually loose affinity to each other and exchange faster depending of the G proteins, there is no evidence that the subunits separate completely in intact cells⁶⁸.

When analysing the same G protein sensor, our study reveals major differences in G protein responses and agonist potencies between neurons and heterologous HEK293 cells. The difference in the profiles of G protein response using different Gγ subunits (Gγ₂, Gγ₈ or Gγ₉) (Fig. 7) shows clearly the impact of Gγ in CGNs. It shows that not only the Gα subunit is important, but also the Gβ and Gγ, consistent with other recent studies^65,66. Therefore, it brings to our attention that the G protein-mediated response also depends detected on the biosensor components used, which is the limitation of biosensors that only detect the response of a well-defined G protein. Meanwhile, the difference can be due to the intrinsic properties of the system under study. For example, the association of these receptors with specific protein partners that may influence their coupling to some Gα_i subtypes, present in neurons but not in HEK293 cells. Indeed, specific proteins interacting with the GABA_B receptor were identified in brain tissues^15,69, while they are not expressed in HEK293 cells. For example, the soluble form of APP, AJAP-1 and PIANP bind to the extracellular sushi domain of GABA_B^70,71, TRPV1 channels interact in the membrane⁷², and the intracellular KCTDs and 14-3-3 proteins⁷³ modulate GABA_B receptor downstream signalling. The KCTD proteins are constitutively associated with GABA_B and they control its kinetics of activation⁷³.

Our sensors showed a difference between Gi and Go proteins revealed by a CB1 agonist CP 55940, which has a higher potency in generating G_i1 than G_oA responses in neurons, relative to the other agonists tested. Indeed, such biased signalling between G_i and G_o proteins have already been reported upon agonist stimulation^12,13,74, by allosteric modulators⁴⁹ or as a result of genetic variation⁷⁵. In our study, the molecular bases of this difference in agonist effect is unclear. CP 55940 and Win 55212-2 (K_i 1.1 nM and 62.3 nM for CB1 receptor, respectively)^76,77 have different scaffolds. Their differential effects might be explained by the ligand-binding kinetics⁷⁸, by specific active conformation stabilized by the agonist⁷⁹, or due to specific components associated to the receptor in these neurons, better stabilizing the CP 55940/CB1/Gi complex.

Our sensors also revealed larger differences in some agonist potencies in neurons compared to transfected HEK293 cells. This is nicely illustrated for the CB1 and GABA_B agonists CP 55940 and APPA, respectively, that show a higher potency than the other agonists in neurons (Fig. 8a). In addition and unexpectedly, no intrinsic agonist activity of the PAM Rac BHFF was detected with the native GABA_B receptor in contrast to the recombinant one in HEK293 cells. Finally, the important differences in GABA_B receptor agonist potencies that are higher in transfected HEK293 cells compared to neurons are most probably not due to the higher expression in the transfected cells in our study. Indeed, a 10-times higher affinity for GABA in neurons compared to HEK cells⁸⁰ and similar agonist potencies in GTPγS binding experiments in the two systems⁸¹ were previously reported. Possible explanations might come from the environment of the receptor in neurons, such as its compartmentalization and its interaction with extracellular and intracellular proteins^15,50, including other endogenous receptors.

Our sensors also revealed specific ligand-independent G protein response in neurons compared to HEK293 cells. Indeed, no detectable constitutive activity of the GABA_B receptor was observed in CGNs in contrast to transfected cell lines^49,51,52. As discussed above, this lack of constitutive activity might be explained by the environment of the receptor in neurons. The importance of the intracellular protein partners for the constitutive activity of a GPCR was nicely illustrated for the postsynaptic metabotropic glutamate receptor mGlu5. Its constitutive activity was maintained low in neurons by the long isoform of Homer, while it is revealed when the short isoform of Homer is expressed⁸².

In conclusion, our study reveals the importance of evaluating the pharmacological and G protein response properties of native GPCRs in primary cells to better understand their signalling, identify and characterize ligands expected to have therapeutic effects. It also reveals the powerfulness of using G protein sensors compatible with native cells, though biosensors compatible with single cell analysis and imaging will certainly help revealing the potential cell-to-cell or sub-compartment heterogeneity in GPCR and G protein coupling^83,84. In the future, methods measuring the activation of a specific endogenous G protein in live cells should be envisioned. Regardless of the criticism of our approach, our study clearly reveals that data generated in recombinant systems should be taken with caution before going further into pre-clinical and clinical development of drugs candidates only characterized this way.

Methods

Materials

GABA (Cat. A2129), CP 55940 (Cat. C1112) and carbachol (PHR1511) were purchased from Sigma-Aldrich (Shanghai, China). R-baclofen (Cat. 0796), SKF 97581 (Cat. 0379) and CGP54626 (Cat. 1088) were obtained from Tocris Biosciences (Shanghai, China). 3-APPA (Cat. ab120329) was purchased from Abcam (Shanghai, China). Win 55,212-2 (T4458), Bay 59-3074 (T3699), DAMGO (T4351), eletriptan HBr (T0216), pramipexole 2HCl monohydrate (T6951), brimonidine tartrate (T6422), tizanidine hydrochloride (T0290), oxymetazoline hydrochloride (T0252), xylazine (T7046), clonidine hydrochloride (T1247), and 1-oleoyl lysophosphatidic acid sodium (T21654) were purchased from TargetMol (Shanghai, China). PAF C-16 (74389-68-7) was purchased from Santa Cruz Biotechnology (Shanghai, China). CGP64213 was a gift from Prof. Fajun Nan (Shanghai Institute of Materials Medicine, China). Coelenterazine h (Cat. S2001) and furimazine (Cat. N1120) were purchased from Promega (Madison, WI, USA).

Plasmids

The coding sequences of Nluc and Rluc8 were PCR-amplified and inserted into the coding sequence of human Gα_i1, Gα_i2, Gα_i3, Gα_oA and Gα_oB (between residues 91 and 92) and Gα_z (between residues 113 and 114) in pcDNA3.1, using the flexible linkers SGGGGS and SGGGEF, before and after the sequence of Nluc, as shown in Supplementary Figs. 10c, d and 12. The pRK5 plasmid encoding the rat GABA_B subunits (HA-tagged GABA_B1 (GB1) and Flag-tagged GABA_B2 (GB2)) and pcDNA3.1 encoding the human Gβ₁ and human ^VenusGγ₂ were provided by the Institut de Génomique Fonctionnelle (Montpellier, France). The cDNA of mouse α_2AAR, mouse CB1, human Gγ₈ and human Gγ₉ were bought from Miaoling Bio (Wuhan, China) and inserted in pcDNA3.1. Venus was inserted at the N-terminus of Gγ₂, Gγ₈ and Gγ₉ between the HindIII and KpnI restriction enzymatic sites and the linker KLGT between Venus and the Gγ subunits was used. The schematic representation of the constructs (Supplementary Fig. 10) and their protein sequences (Supplementary Figs. 11–13) were shown.

HEK293 cell culture and transfection

HEK293 cells (ATCC, CRL-1573, lot: 3449904) were cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified incubator containing 5% CO₂. For transfections, cells were suspended and transfected using Lipofectamine 2000 with the appropriate expression constructs as previously described⁴⁹. The cDNA amounts used per well in 96-well plate were as following unless indicated in figure legends: rat GABA_B receptor, GB1 (3 ng, 6 ng, 9 ng, 30 ng) and GB2 (4 ng, 8 ng, 12 ng, 40 ng), mouse α_2AAR (10 ng, 15 ng, 20 ng, 50 ng), mouse CB1 receptor (80 ng), Gα_i1^Nluc (1 ng), Gα_i2^Nluc (3 ng), Gα_i3^Nluc (3 ng), Gα_oA^Nluc (1 ng), Gα_oB^Nluc (1 ng), Gα_z^Nluc (1.5 ng), Gβ₁ (10 ng) and ^VenusGγ₂ (10 ng). The ratio of DNA to Lipofectamine 2000 ratio was 1:2. After a 24 h culture in 96-well plates, the cells were ready for the bioluminescence resonance energy transfer (BRET) experiments.

Primary neuron culture and transfection

For the primary culture of neurons, all experiments were specifically designed to minimize the number of animals used and were approved by the Animal Experimentation Ethics Committee of the College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. Kunming mice were obtained from the Center for Disease Control and Prevention of Hubei Province. The mice were raised in a specific pathogen free (SPF) environment with an ambient temperature of 18–22 °C, a humidity of 50%-60%, and a 12 h light-dark cycle.

Primary cerebellar granule neuronal cultures were prepared from one-week-old newborn Kunming mice as previously described³⁹. The dissected tissue was gently triturated after Versene (15040066; Gibco, Shanghai, China) treatment for 5 min at 37 °C, and the homogenate was centrifuged at 170 g for 5 min. In the meantime, mixtures of the DNA and Lipofectamine 2000 (Ref 11668019; Thermo Fisher Scientific, Shanghai, China) in a 1:3 ratio in an Opti-minimal essential medium (Opti-MEM) (Ref 31985070; Thermo Fisher Scientific, Shanghai, China) were prepared following the manufacturer’s protocols and as previously described⁴². The pellet was re-suspended in culture medium DMEM-F12 (Ref 11320-033; Gibco) supplemented with 2 mM glutamine, 30 mM KCl, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% foetal bovine serum (FBS) and seeded into 96-well plates (100 μL/well) previously coated with poly-L-ornithine (Sigma-Aldrich). Then, the mixtures of DNA and Lipofectamine 2000 (50 μL/well) were added to the wells. BRET experiments were performed three or four days after transfection. BRET experiments were performed at DIV3 or DIV4. The cDNA amounts used per well in 96-well plate were as following: Gα_i1^Nluc (25 ng), Gα_i2^Nluc (75 ng), Gα_i3^Nluc (75 ng), Gα_oA^Nluc (25 ng), Gα_oB^Nluc (25 ng), Gα_z^Nluc (25 ng), ^VenusGγ₂ (50 ng), ^VenusGγ₈ (50 ng) and ^VenusGγ₉ (50 ng).

Primary cortical and hippocampal neuronal cultures were prepared from embryonic day 17.5 mice as previously described⁸⁵. The cortex or hippocampi were digested with trypsin, and cells were seeded in neurobasal medium (Ref 21103049; Gibco) supplemented with 2% B27 (Ref 17504-044; Gibco), 4 mM GlutaMAX, 25 μM glutamic acid, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% FBS in 96-well plates. After three days in culture (DIV3), the culture medium was supplemented with 5 μM cytosine β-D-arabinofuranoside hydrochloride (C6645; Sigma-Aldrich) and incubated overnight. Then, 75% of the medium was replaced by neurobasal medium supplemented with B27, GlutaMAX and antibiotics. Neurons were then transfected with Gα_i1^Nluc or Gα_oA^Nluc (50 ng/well), together with ^VenusGγ₂ (50 ng/well) using Lipofectamine 2000 on DIV7. The ratio of DNA to Lipofectamine 2000 ratio was 1:2. BRET experiments were performed on DIV10 or DIV11.

Fluorescent imaging

Cerebellar granule neurons (CGNs) and transfected HEK293 cells were fixed with 4% formaldehyde and blocked with 2% bovine serum albumin (BSA) and 0.1% Triton X-100 in phosphate-buffered saline (PBS). The CGNs were incubated with primary GFP antibody (1:200; ab1218, Abcam, Shanghai, China) at 4 °C overnight. After washing three times with PBS, the cells were incubated with secondary anti-mouse antibody Alexa Fluor® 488 AffiniPure Donkey Anti-Mouse IgG (H + L) (1:500, 715-545-150, Jackson ImmunoResearch, Shanghai, China) at 25 °C for 2 h. The CGNs were then stained with DAPI for 15 min. The cells were washed with PBS and mounted with FluorSave reagent (AR1109, Boster Biological Technology Co. Ltd., Wuhan, China). Images were obtained with an Olympus FV1000 laser scanning confocal microscope (60 x objective for HEK293 cells and 40x objective for CGNs, Olympus Corporation, Tokyo, Japan) equipped with appropriate fluorescence and filters (FITC: 488/530 nm; DAPI: 405/449 nm). The images were digitized and saved in TIFF format.

BRET measurement

CGNs were starved in HEPES-buffered saline (HBS) containing 10 mM HEPES pH 7.4, 140 mM NaCl, 4 mM KCl, 2 mM MgSO₄, and 1 mM KH₂PO₄. Cortical and hippocampal neurons were starved with artificial cerebrospinal fluid (aCSF) buffer containing 140 mM NaCl, 2 mM CaCl₂, 3 mM KCl, 10 mM HEPES, and 10 mM D-glucose, at 37 °C for 1 h before the BRET experiments. BRET measurements were performed as previously described using the Mithras LB 940 multimode microplate reader (Berthold Technologies, Bad Wildbad, Germany)⁴⁹ with the programme MikroWin (Version 4.41) or PHERAstar FS (BMG Labtech, USA)⁸⁶ with the programme PHERAstar control (Version 4.00 R4). The signals emitted by the donor (460–500 nm band-pass filter, Em 480 nm) and the acceptor (510–550 nm band-pass filter, Em 530 nm) were recorded by Mithras LB 940 after the addition of 10 μM furimazine. All measurements were performed at 37 °C. The BRET signal was determined by calculating the ratio between the emission of acceptor and donor (Em 530 nm / Em 480 nm). The basal BRET ratio (BRET_basal) of cells was recorded before the stimulation with drugs or buffer. The change in BRET ratio (net BRET) was obtained by subtracting the BRET ratio after agonist treatment from the basal BRET (BRET_basal-BRET_agonist). Agonist-induced change in BRET ratio for the different G_i/o sensors was expressed as percentage of the basal signal ((BRET_basal-BRET_agonist/BRET_basal) x100). To study the kinetics, the BRET was measured in real time with a counting time of 0.5 s, and the drugs were injected using the Mithras LB 940 injectors at the indicated time.

Quantitative reverse transcription PCR

Total RNA was extracted using standard methods (Trizol, Invitrogen) from CGNs and HEK293 cells. Quantitative reverse transcription PCR (qRT-PCR) was carried out with SYBR Green (Vazyme Biotechnology, Nanjing, China) according to the manufacturer’s protocol as reported⁸⁷. Rplp0 was used as an internal reference for normalization, and the ΔΔCt method was adopted to analyse quantitative PCR data. The primer sets used were referred to previous references^75,88. For mouse (CGNs), Gnai1 (NM_010305), Forward (Fw): 5’-AAGCTGACTCGCCTTCCCAG-3’, Reverse (Rv): 5’-GTAGTTTACAGTTCTCCACACG-3’; Gnai2 (NM_008138), Fw: 5’-TGCCTTGAGTGTGTCTGCGTG-3’, Rv: 5’-CTCAGTGACGTTGGCAGTTG-3’; Gnai3 (NM_010306), Fw: 5’-GTGCAGTCCGTGTACAAGAG-3’, Rv: 5’-GATGAATGGATCCGAGCCAC-3’; Gnao1 transcript isoform A (NM_010308) Fw: 5’-AGGAAGACGGACTCCAAGATG-3’, Rv: 5’-AGTCGAAGAGCATGAGAGAC-3’; Gnao1 transcript isoform B (NM_001113384) Fw: 5’-AGGAAGACGGACTCCAAGATG-3’, Rv: 5’-AGATGTGTCTGTGAACCACTTG-3’; Gnaz (NM_010311), Fw: 5’-CAGCCGTGCTTAGAAACATCG-3’, Rv: 5’-TCTAGTGACACTCCACCTCC-3’ and Rplp0 (NM_007475), Fw: 5’-CTCACTGAGATTCGGGATATG-3’, Rv: 5’-CTCCCACCTTGTCTCCAGTC-3’. For human (HEK293 cells), GNAI1 (NM_002069), Fw: 5’-CATCTCTGACCTTGTTTCAGC-3’, Rv: 5’-CTTCAACCCAGTGACAACACG-3’; GNAI2 (NM_002070), Fw: 5’-ACTCCGTGCCTTGAGTGTG-3’, Rv:5’-TTGTCTGGAACAGCCCTTGG-3’; GNAI3 (NM_010306), Fw: 5’-GGAAAGTTACGTTCACTTCAACC-3’, Rv: 5’-TTGGACCCCAAAAGGCACTG-3’; GNAO1 transcript isoform A (NM_020988) Fw: 5’-AGAAAGGCTGACGCCAAGAT-3’, Rv: 5’-AGTCGAAGAGCATGAGAGAC-3’; GNAO1 transcript isoform B (NM_138736) Fw: 5’-AGAAAGGCTGACGCCAAGAT-3’, Rv: 5’-TGGACGTGTCTGTGAACCAT-3’; GNAZ (NM_002073), Fw: 5’-CTACGAGGATAACCAGAC-3’, Rv: 5’-TACGTGTTCTGGCCCTTG-3’ and RPLP0 (NM_053275), Fw: 5’-ATGCAGCAGATCCGCATGT-3’, Rv: 5’-TTGCGCATCATGGTGTTCTT-3’.

Cell membrane preparations

The cell membranes were prepared as reported⁸⁹. HEK293 cells or CGNs were washed three times with PBS, then scraped and collected by centrifugation at 170 g for 5 min. Cells were resuspended in 500 μl Tris buffer (50 mM Tris pH 7.4, 50 mM NaCl) with cOmplete protease inhibitor cocktail (Roche) and crushed through a 26 gauge 5/8 inch needle attached to a syringe for 30 passages. After centrifugation at 860 g at 4 °C for 5 min, liquid supernatants were transferred to high speed centrifuge tubes and centrifuged at 44000 g at 4 °C for 20 min by Optima MAX-TL ultracentrifuge (Beckman Coulter, Brea, CA, USA). The precipitated membranes were diluted gently in 50 μl Tris-NaCl buffer. The protein amount was further determined using BCA protein assay kit and 20 μg protein was loaded for western blotting detection.

Western blotting analysis

Cells were treated using RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin; Beyotime Bio., Cat. P0013C, China) and protein concentrations were determined using the BCA protein assay kit. Equal amounts of protein (20 μg) from total cell lysis or cell membranes were separated by SDS–polyacrylamide gel electrophoresis (PAGE) on 10 to 12% gels. Proteins were transferred to nitrocellulose membranes and washed in blocking buffer (5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20) for 2 h at 25 °C. The blots were incubated with the primary monoclonal antibodies anti-GB1 mAb (1:1000, ab55051, Abcam, Shanghai, China), anti-Gβ₂ rabbit (1:1000, A9643, ABclonal Technology, Wuhan, China), anti-β-actin (1:3000, KM9001T, Sungene Biotech., Tianjin, China), the rabbit polyclonal antibodies anti-CB1 (1:1000, A1447, ABclonal, Wuhan, China), anti-α_2AAR (1:1000, A2809, ABclonal, Wuhan, China), anti-Gβ₁ (1:1000, A1867, ABclonal, Wuhan, China), anti-Gβ₃ (1:1000, A1387, ABclonal, Wuhan, China) and anti-Gβ₅ (1:1000, A4447, ABclonal, Wuhan, China). The primary antibodies were at the relevant dilution overnight at 4 °C and then incubated with DyLight 800 4 X PEG conjugated secondary antibodies (1:20,000, anti-mouse IgG, #5257; 1:20,000, anti-rabbit IgG, #5151, Cell Signaling Technology, Shanghai, China) for 2 h at 25 °C. Membranes were imaged using an Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA) at 700 nm.

Statistical analysis

Results are presented as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using GraphPad Prism 9.5.1 software (GraphPad Software Inc., San Diego, CA, USA). Dose-response experiments were analysed using nonlinear curve fitting for the log (agonist) vs. response (three parameters) curves. Statistical analysis was performed using the Ordinary one-way ANOVA with a Dunnett’s post-hoc multiple comparison test or unpaired t-test (two-tailed) or paired t-test. P < 0.05 was considered to be statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data generated in this study are provided in the main text, Supplementary information and source data files. The raw data and p-values for all Figures and Supplementary Figs. are available in Source Data file accompanying this paper. Source data are provided with this paper.

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Acknowledgements

We thank Dr Julie Perroy (IGF) for providing us Nluc cDNA. This work was supported by grants from the National Natural Science Foundation of China (NSFC) (grant number 32271198 to C.X.), the National Key R&D Programme of China (grant number 2022YFA1302901 to C.X.), the National Natural Science Foundation of China (NSFC) (grant number 32330049 and 82320108021 to J.L.), National Key R&D Programme of China (grant number 2022YFE0116600 and 2021ZD0203302 to J.L.), interdisciplinary Research Programme of HUST (grant number 2023JCYJ006 to C.X.), the Agence Nationale de la Recherche (ANR 18-CE11-0004-01 to J.-P.P.), and the Fondation Recherche Médicale (DEQ 20170336747 to J.-P.P. and EQU202303016470 to P.R.). P.R. and J.-P.P. were supported by the Institut National de la Santé et de la Recherche Médicale (INSERM; International Research Programme «Brain Signal») and the Franco-Chinese Joint Scientific and Technological Commission (CoMix) from the French Embassy in China.

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Yiwei Zhou
Present address: Kindstar Global Precision Medicine Institute, Wuhan, China
These authors contributed equally: Chanjuan Xu, Yiwei Zhou, and Yuxuan Liu.

Authors and Affiliations

Cellular Signaling Laboratory, Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei, China
Chanjuan Xu, Yiwei Zhou, Yuxuan Liu, Li Lin, Peng Liu, Xiaomei Wang, Zhengyuan Xu & Jianfeng Liu
Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, 510005, Guangzhou, China
Chanjuan Xu & Jianfeng Liu
Institut de Génomique Fonctionnelle (IGF), Université de Montpellier, CNRS, INSERM, 34094, Montpellier, France
Jean-Philippe Pin & Philippe Rondard

Authors

Chanjuan Xu
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Yiwei Zhou
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Yuxuan Liu
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Li Lin
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Peng Liu
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Xiaomei Wang
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Zhengyuan Xu
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Jean-Philippe Pin
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Philippe Rondard
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Jianfeng Liu
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Contributions

C.X., J.L., P.R. and J.-P.P. designed the experiments. C.X. initiated the project, designed the sensors, set up the protocols and screened different agonists; C.X., Y.Z. and Y.L. detected the Gi/o protein response in HEK293 cells and CGNs; Y.L. optimized the transfection amount in HEK293 cells and prepared cell membranes; Y.L. and Y.Z. performed all the western blotting experiments; L.L. performed the dose-response in HEK293 cells treated with GABA_B receptor agonists; Y.L., L.L. and Z.X. produced constructs. P.L. and X.W. helped with the plasmid constructs and neuron preparations. C.X., Y.Z. and Y.L. collected and analysed the data. C.X., J.L., J.-P.P. and P.R. wrote the manuscript with input from all of the authors.

Corresponding authors

Correspondence to Jean-Philippe Pin, Philippe Rondard or Jianfeng Liu.

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Competing interests

P.R. and J.-P.P. are involved in a collaborative team between the CNRS and Cisbio Bioassays, Revvity group. The remaining authors declare no competing interests.

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Nature Communications thanks Moritz Bünemann, Laura Humphrys, Bryan Roth and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Xu, C., Zhou, Y., Liu, Y. et al. Specific pharmacological and G_i/o protein responses of some native GPCRs in neurons. Nat Commun 15, 1990 (2024). https://doi.org/10.1038/s41467-024-46177-z

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Received: 08 August 2022
Accepted: 15 February 2024
Published: 05 March 2024
DOI: https://doi.org/10.1038/s41467-024-46177-z

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