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

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.

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

a Scheme of the BRET-based sensors to detect the activity of the endogenous GPCR in neurons. b Scheme of the Gα_i/o^Nluc and ^VenusGγ₂ constructs co-transfected in the neurons. Sequence of the linker (L) before and after Nluc was indicated. Gγ was fused with Venus in the N-terminal. c Composition of G protein sensors made of the different Gα_i/o^Nluc and ^VenusGγ₂. d Kinetics of the BRET signal between the indicated Gα constructs (Gα_i1^Nluc or Gα_i1^Rluc8) and ^VenusGγ₂ in CGNs. Buffer or baclofen (100 μM) were injected at the indicated time (arrow). The data are representative of BRET ratios from five independent experiments. e Representative fluorescent images of Venus and DAPI in CGNs transfected with Gα_oA^Nluc and ^VenusGγ₂ or in HEK293 cells transfected with Gα_oA^Nluc, G_β1 and ^VenusGγ₂ from three independent experiments. Scale bar: 20 μm. f Kinetics of the BRET signal between Gα_i1^Nluc and ^VenusGγ₂ in CGNs. Buffer or baclofen (100 μM) or competitive antagonist CGP64213 (10 μM) were injected at the indicated time. The data are representative of the mean ± SEM of BRET ratios from three independent experiments. The raw data and p-values are available in source data provided as a Source Data file.

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

a Baclofen-induced change in BRET ratio between Gα^Nluc and ^VenusGγ₂ in CGNs or transfected HEK293 cells for the indicated G_i/o proteins, and expressed as percentage of the basal signal ((BRET_basal-BRET_agonist/BRET_basal) x 100). CGNs were co-transfected with ^VenusGγ₂ (50 ng) and the Nluc-tagged Gα_i1 (25 ng), Gα_i2 (75 ng), Gα_i3 (75 ng), Gα_oA (25 ng), Gα_oB (25 ng) or Gα_z (25 ng), while HEK293 cells were co-transfected with GB1 (9 ng), GB2 (12 ng), Gβ₁ (10 ng)_, ^VenusGγ₂ (10 ng) and the Nluc-tagged Gα_i1 (1 ng), Gα_i2 (3 ng), Gα_i3 (3 ng), Gα_oA (1 ng), Gα_oB (1 ng) or Gα_z (1.5 ng) per well in 96-well plate. b Expression of GB1 subunit of GABA_B receptor in the cell membrane of CGNs, mock HEK293 cells, and HEK293 cells transfected with indicated amount of GB1 and GB2, detected by western blotting. Values are mean ± SEM normalized as fold of GB1 expression in CGNs from four independent experiments. c Expression of the Nluc-tagged Gα transfected in CGNs or in HEK293 cells in (a), measured by the luminescent signal at 480 nm. The values of individual well are shown. Values are mean ± SEM from four biologically independent experiments each performed in triplicates or quadruplicate in (a) and (c). Data in (a) are analysed using unpaired t-test (two-tailed) to determine significance. Data in (c) are analysed using one-way analysis of variance (ANOVA) with a Dunnett’s post-hoc multiple comparison test to determine significance (compared with G_i1 group). ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05 and not significant (ns). d Expression of the genes encoding indicated Gα in CGNs (mouse species: Gnai1, Gnai2, Gnai3, Gnao-1, Gnao-2, Gnaz) and HEK293 cells (human species: GNAI1, GNAI2, GNAI3, GNAO-1, GNAO-2, GNAZ). Values were determined by qRT-PCR and mean ± SEM normalized to Rplp0 (mouse) or RPLP0 (humans) from three biologically independent experiments each performed in quadruplicate. The values of individual sample are shown. The raw data and p-values are available in source data provided as a Source Data file.

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

a Scheme of the GABA_B receptor, made of GB1 and GB2 subunits, in CGNs and its various specific ligands (agonist, red; antagonists, blue; PAM, black). b Change of BRET signal induced by the GABA_B receptor antagonist CGP64213 in presence of 20 μM baclofen (EC₈₀) or different doses of baclofen in CGNs measured by G_i1 or G_oA sensor. c, d Change of BRET ratio induced by various GABA_B receptor agonists measured by G_i1 (c) or G_oA (d) sensor. e, f Correlation of the agonist potencies (pEC₅₀) between HEK293 cells and CGNs determined by the G_i1 (e) or G_oA (f) BRET sensors. Dotted line is the correlation of pEC₅₀ determined with HEK293 cells. Red line is fit of pEC₅₀ in CGNs with the same slope as the dotted line. g The pEC₅₀ of baclofen in CGNs and transfected HEK293 cells with indicated amount of GB1 and GB2 measured by G_i1 or G_oA sensor. Data are mean ± SEM from at least three biologically independent experiments each performed in triplicate and analysed using one-way ANOVA with a Dunnett’s post-hoc multiple comparison test to determine significance. The n number of G_i1 and G_oA group for CGNs, and HEK293 cells transfected with the indicated amount of GB1 and GB2 are 10, 4, 3, and 4, 3, 3, respectively. ****p < 0.0001, **p < 0.01 and not significant (ns). h Change of BRET ratio induced by baclofen with or without indicated concentrations of the GABA_B PAM Rac BHFF in transfected HEK293 cells and CGNs measured by G_i1 sensor. Data are normalized to maximum of baclofen response and mean ± SEM from at least three biologically independent experiments each performed in triplicate in (b–d) and (h). b n = 3; c n = 6; d n = 4; h CGNs n = 3, HEK293 cells n = 4. The raw data and p-values are available in source data provided as a Source Data file.

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

a Net BRET signal of the G_i1 and G_oA sensors in CGNs induced by specific agonists of various G_i/o-coupled GPCRs using a saturating concentration of the indicated ligands (PAF, 10 μM; DAMGO, 100 μM; eletriptan, 100 μM; brimonidine, 10 μM; carbachol, 100 μM; pramipexole, 100 μM; CP 55940, 20 μM and baclofen, 100 μM). Values are mean ± SEM from at least biologically independent experiments each performed in triplicate. The n number of the indicated treatments in G_i1 and G_oA are 6, 4, 5, 4, 6, 4, 6, 7, 8, and 8, 6, 4, 6, 6, 6, 6, 6, 7, 8, respectively. Data are analysed using one-way ANOVA with a Dunnett’s post-hoc multiple comparison test to determine significance (compared with no treated group). ****p < 0.0001, *p < 0.05 and not significant (ns). b Dose-response of the agonists brimonidine, CP 55940, carbachol and pramipexole for the α₂ adrenoceptor, CB1, muscarinic M_2/4 and dopamine D_2/4 receptors, respectively, in CGNs co-transfected with Gα_i1^Nluc and ^VenusGγ₂ or Gα_oA^Nluc and ^VenusGγ₂. Data are normalized to maximum response of each compound and mean ± SEM from at least three biologically independent experiments each performed in triplicate. G_i1, brimonidine (n = 3), CP 55940 (n = 4), carbachol (n = 4) and pramipexole (n = 4); G_oA, brimonidine (n = 3), CP 55940 (n = 3), carbachol (n = 4) and pramipexole (n = 3). The raw data and p-values are available in source data provided as a Source Data file.

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

a Effect of the indicated α₂AR agonists on the net BRET of the G_i1 and G_oA sensors in CGNs co-transfected with Gα_i1^Nluc and ^VenusGγ₂ or Gα_oA^Nluc and ^VenusGγ₂ (amounts of cDNA as in Fig. 2a). b Effect of the indicated α₂AR agonists on the net BRET of the G_i1 and G_oA sensors in HEK293 cells co-transfected with the mouse α_2AAR (10 ng, 20 ng or 50 ng), and Gβ_1, ^VenusGγ₂ and Gα_i1^Nluc or Gα_oA^Nluc as in Fig. 2a. Saturating concentrations of brimonidine (10 μM), oxymetazoline (100 μM), xylazine (50 μM), clonidine (100 μM), tizanidine (100 μM) were used in (a, b). c Detection of α₂AR in cell membranes of CGNs, mock-transfected HEK293 cells and HEK293 cells transfected with indicated amount of α₂AR, by western blotting. Values are mean ± SEM normalized as fold of α₂AR expression in CGNs from four independent experiments. d The pEC₅₀ of brimonidine in CGNs (n = 3) and transfected HEK293 cells (n = 4) with indicated amount of α₂AR measured by G_i1 or G_oA sensor. e Percentage of change in BRET ratio between Gα^Nluc and ^VenusGγ₂ induced by brimonidine between Gα^Nluc and ^VenusGγ₂ in HEK293 cells (n = 3) (α₂AR: 15 ng/well per 96-well plate) or CGNs (n = 4) for the indicated Gα_i1, Gα_i2, Gα_i3, Gα_oA, Gα_oB or Gα_z sensors (amounts of cDNA as in Fig. 2a). Values are mean ± SEM from at least three biologically independent experiments each performed in triplicate or quadruplicate in (a, b, d, e). a, n = 4; b, n = 3; (d, e), CGNs, n = 3; HEK293 cells, n = 4. Data are analysed using one-way ANOVA with a Dunnett’s post-hoc multiple comparison test to determine significance (compared with brimonidine in a, b). Data are analysed using unpaired t-test (two-tailed) in (e). ****p < 0.0001, ***p < 0.001, **p < 0.01 and not significant (ns). The raw data and p-values are available in source data provided as a Source Data file.

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

a Detection of CB1 receptor in cell membranes of CGNs, mock-transfected HEK293 cells and HEK293 cells transfected with mouse CB1 cDNA (80 ng/well in 96-well plate), by western blotting. Values are mean ± SEM normalized as fold of CB1 expression in CGNs from three independent experiments. b Percentage of change in BRET ratio between Gα^Nluc and ^VenusGγ₂ induced by CP 55940 in HEK293 cells (CB1 cDNA; 80 ng/well per 96-well plate) or CGNs for the indicated Gα_i1, Gα_i2, Gα_i3, Gα_oA, Gα_oB or Gα_z sensors (amounts of cDNA as in Fig. 2a). Values are mean ± SEM from four biologically independent experiments each performed in triplicate. Data are analysed using unpaired t-test (two-tailed) to determine significance. ****p < 0.0001, *p < 0.05 and not significant (ns). c, d Change of BRET signal between Gα^Nluc and ^VenusGγ₂ induced by the indicated CB1 receptor agonists in HEK293 cells and CGNs measured by G_i1 and G_oA sensors. The transfection was the same as in (b). Inset, correlation of the agonist potencies (pEC₅₀) between HEK293 cells and CGNs determined by the G protein sensors. Dotted lines are the correlation of pEC₅₀ determined with HEK293 cells. Red lines are the fit of pEC₅₀ in CGNs with the same slope as the dotted lines. Data are normalized to maximum CP 55940 response and are mean ± SEM from at least three biologically independent experiments each performed in triplicate (HEK293 cells: G_i1, CP 55940 (n = 5), Win 55,212-2 (n = 5) and Bay 59-3074 (n = 4); G_oA, CP 55940 (n = 5), Win 55,212-2 (n = 4) and Bay 59-3074 (n = 4). CGNs: G_i1, CP 55940 (n = 4), Win 55,212-2 (n = 4) and Bay 59-3074 (n = 4); G_oA, CP 55940 (n = 4), Win 55,212-2 (n = 4) and Bay 59-3074 (n = 3)). The raw data and p-values are available in source data provided as a Source Data file.

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

a, b Percentage of change in BRET ratio between Gα^Nluc and ^VenusGγ₈ (a) or ^VenusGγ₉ (b) induced by baclofen, brimonidine and CP 55940 in CGNs for the indicated G_i/o proteins. CGNs were co-transfected with the indicated ^VenusGγ and Nluc-tagged Gα_i1, Gα_i2, Gα_i3, Gα_oA, Gα_oB or Gα_z. Values are mean ± SEM from biologically independent experiments (baclofen and brimonidine, n = 5; CP 55940, n = 3) each performed in triplicate. c Scheme illustrating the difference in G_i/o (G_i1, G_i2, G_i3, G_oA, G_oB and Gα_z) and indicated Gγ protein (Gγ₂, Gγ₈ and Gγ₉) responses in CGNs for the receptors GABA_B, CB1 and α₂AR in presence of the indicated agonists. For each receptor and G_i/o sensor, the size of the circle is the percentage of change in BRET ratio upon the agonist stimulation, for GABA_B (Figs. 2a and a, b), α₂AR (Figs. 5e and a, b) and CB1 (Figs. 6b and a, b) normalized to the G_i/o sensor that has the highest response: for Gγ₂, G_i1 for both the GABA_B and CB1, and Gα_z for α₂AR; for Gγ₈, G_oA for GABA_B and α₂AR and G_i1 for CB1; and for Gγ₉, G_i1 for GABA_B and CB1, and Gα_z for α₂AR. For Gγ8, the empty circle indicates an increase in the BRET ratio in contrast to other conditions where a decrease BRET ratio is measured. The raw data and p-values are available in source data provided as a Source Data file.

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

a Scheme illustrating the difference in agonist or PAM potencies for the GABA_B and CB1 receptors between HEK293 cells and CGNs for the G_i1 sensor. For each cell and receptor, the relative size of the triangles is according to the pEC₅₀ values of the indicated agonists. b Scheme illustrating the difference in G_i/o protein response (G_i1, G_i2, G_i3, G_oA, G_oB and G_z) between CGNs and HEK293 cells for the receptors GABA_B, CB1 and α₂AR in presence of the indicated agonists, when Gγ₂ is used for all these G_i/o sensors. For each cell and receptor, the size of the circle is the percentage of change in BRET ratio upon the agonist stimulation for GABA_B (Fig. 2a), α₂AR (Fig. 5e) and CB1 (Fig. 6b) normalized to the G_i/o sensor that has the highest response: in HEK293 cells, G_oA for GABA_B, α₂AR and G_i1 for CB1, and in CGNs, G_i1 for both the GABA_B and CB1, and G_z for α₂AR.