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. 2024 Feb;105(2):104–115. doi: 10.1124/molpharm.123.000795

Signaling Specificity and Kinetics of the Human Metabotropic Glutamate Receptors

Tyler W McCullock 1, Loren P Cardani 1, Paul J Kammermeier 1,✉
PMCID: PMC10794986  PMID: 38164584

Abstract

Metabotropic glutamate receptors (mGluRs) are obligate dimer G protein coupled receptors that can all function as homodimers. Here, each mGluR homodimer was examined for its G protein coupling profile using a bioluminescence resonance energy transfer-based assay that detects the interaction between a split YFP-tagged Gβ1γ2 and a Nanoluciferase tagged free Gβγ sensor, MAS-GRK3-ct- nanoluciferase with 14 specific Gα proteins heterologously expressed, representing each family. Canonically, the group II and III mGluRs (2 and 3 and 4, 6, 7, and 8, respectively) are thought to couple to Gi/o exclusively. In addition, the group I mGluRs (1 and 5) are known to couple to the Gq/11 family and generally thought to also couple to the pertussis toxin-sensitive Gi/o family some reports have suggested Gs coupling is possible as cAMP elevations have been noted. In this study, coupling was observed with all eight mGluRs through the Gi/o proteins and only mGluR1 and mGluR5 through Gq/11, and, perhaps surprisingly, not G14. None activated any Gs protein. Interestingly, coupling was seen with the group I and II but not the group III mGluRs to G16. Slow but significant coupling to Gz was also seen with the group II receptors.

SIGNIFICANCE STATEMENT

Metabotropic glutamate receptor (mGluR)-G protein coupling has not been thoroughly examined, and some controversy remains about whether some mGluRs can activate Gαs family members. Here we examine the ability of each mGluR to activate representative members of every Gα protein family. While all mGluRs can activate Gαi/o proteins, only the group I mGluRs couple to Gαq/11, and no members of the family can activate Gαs family members, including the group I receptors alone or with positive allosteric modulators.

Introduction

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors and consist of eight members (mGluR1–8), organized by sequence homology, signaling effectors, and general localization (Gregory, 2021). The group I mGluRs include mGluR1 and mGluR5, are dual-coupled through Gαq and Gαi/o (McCool et al., 1998; Kammermeier and Ikeda, 1999; Avet et al., 2020; Hauser et al., 2022), and exhibit post-synaptic expression (Senter et al., 2016) in the nervous system, and there have been reports of cAMP accumulation in response to mGluR1 and mGluR5 activation giving rise to speculation of Gs coupling by these receptors (Kubo and Tateyama, 2005; Tateyama and Kubo, 2007; Nasrallah et al., 2018; Sebastianutto et al., 2020). The group II mGluRs consist of mGluR2 and mGluR3 and are thought to couple exclusively to the Gαi/o pathway (Niswender and Conn, 2010). These receptors can be found at either the pre- or post-synapse participating in cAMP-based synaptic plasticity as well as acting as auto receptors via Gβγ to limit the amount of glutamate released during action potentials (Upreti et al., 2013). The group III mGluRs consist of mGluR4, mGluR6, mGluR7, and mGluR8. These receptors also believed to solely couple to Gi/o signaling pathways (Niswender and Conn, 2010). mGluRs 4, 7, and 8 are typically found acting as auto receptors on the presynaptic terminus (Mao et al., 2013) while mGluR6 expresses exclusively post-synaptically in retinal ON bipolar cells (Nakajima et al., 1993). Interestingly though, mGluR7 is only poorly responsive to millimolar concentrations of glutamate, and no other native agonist has been identified for it (Okamoto et al., 1994; Conn and Pin, 1997; Habrian et al., 2019). While the G protein coupling tendencies of the mGluRs are generally known, a comprehensive assessment of mGluR-G protein coupling has not been published, although some studies have examined the coupling of representative members of each group (Avet et al., 2022).

Due to their widespread expression in the nervous system, mGluRs participate in many neuronal physiologic processes and pathophysiological behaviors. For this reason, mGluRs have been considered potential therapeutic targets for a wide range of pathologies including addiction, epilepsy, schizophrenia, and Parkinson’s disease (Ritzen et al., 2005). Here we use optimized adaptations of state of the art bioluminescence resonance energy transfer (BRET) assays to assess the G protein signaling of each member of the mGluR family as homodimers in detail in HEK293T cells.

Our results indicate that all members of the mGluR family can activate members of the Gαi/o family, while only the group I receptors, mGluR1 and mGluR5, couple to Gq and G11. Interestingly, we observe coupling through G16 through mGluRs 1, 5, 2, and 3 only, although coupling with mGluR3 was quite weak. None of the mGluRs exhibited coupling to the G12/13 or GS families, even mGluR5 in the presence of the positive allosteric modulator VU0424465 (5-[2-(2-(3-Fluorophenyl)ethynyl]-N-[(1R)-2-hydroxy-1,2-dimethylpropyl]-2-pyridinecarboxamide), which had been reported to promote αs coupling (Nasrallah et al., 2018). In addition, the kinetics and potencies of each mGluR coupling to its corresponding Gα proteins were also examined. In general, the group II mGluRs appeared to be the most efficient activators of Gα proteins. Glutamate activated the group II and III receptors coupled to Go proteins with the highest potency and activated the group I receptors coupled to Gq with the highest potency.

Materials and Methods

Plasmids and Molecular Biology

Plasmids encoding Gi1, Gi2, Gi3, GoA, GsS, Ric8B, mGluR6, and lysophosphatidic acid receptor 2A were gifts from Dr. Cesare Orlandi (University of Rochester) (Masuho et al., 2015b). Plasmids encoding GoB, Gz, Gq, G11, G14, G13, GsL, Golf, Gβ1, Gγ2, masGRK3ct, EGFP-PTX-S1, and the D5 dopamine receptor (D5R) were gifts from Dr. Stephen Ikeda (National Institute on Alcohol Abuse and Alcoholism). The pmVenus-N1 plasmid was a gift Dr. Steven Vogel (National Institute on Alcohol Abuse and Alcoholism). The CMV-hEAAT3 plasmid was a gift from Susan Amara (National Institute of Mental Health; Addgene plasmid #32815). The pH1R-P2A-mCherry-N1 was a gift from Dorus Gadella (Addgene plasmid # 84330) (van Unen et al., 2016). A plasmid encoding for nanoluciferase (NLuc) was a gift from Dr. John Lueck (University of Rochester) (Ko et al., 2022). All plasmids were verified by full sanger sequencing before use.

The Gβγ-masGRK3ct sensor components mVenus(156-239)-Gβ1, mVenus(1-155)-Gγ2, and masGRK3ct-NL were assembled to be identical to those previously reported (Hollins et al., 2009), with the exception of replacing RLuc8 with NLuc. For masGRK3ct-NLuc, the previously assembled masGRK3ct constructs (amino acids 495–688 of bovine GRK3 with the myristic acid sequence MGSSKSKTSNS added to the N-terminus) and NLuc were copied from their original plasmids with polymerase chain reaction with appropriate overhangs for Gibson assembly into the EcoRV site in pCDNA3.1(+). A GCCACC Kozak sequence was added before the start codon of masGRK3ct. A GGG linker was incorporated into both overhang and both fragments. Next, pCDNA3.1(+) was digested with EcoRV, and the digested pCDNA3.1(+) was added along with the masGRK3ct and NLuc polymerase chain reaction products into an NEBuilder reaction. The reaction product was then transformed into XL10-Gold Ultracompetent E.Coli cells and colonies were screened for successful assembly. The mVenus(156-239)-Gβ1 and mVenus(1-155)-Gγ2 were cloned with an identical procedure with the incorporation of GGSGGG linker in the overhangs between the mVenus fragments and protein.

The human mGluR constructs (except mGluR6) were synthesized by GenScript in fragments and assembled in the laboratory. Each hmGluR coding sequence was domesticated by eliminating all BsaI, BbsI, BsmBI, SapI, and AarI restriction sites by introducing silent mutations. Each coding sequence was then divided into two, with a GCCACC Kozak sequence being added to the first fragment immediately before the start codon; the native stop codon was changed to a TGA stop; and overhangs were added for Gibson assembly into the EcoRV site of pCDNA3.1(+). Each fragment was synthesized by GenScript, and, once received, the appropriate fragments were mixed with EcoRV digested pCDNA3.1(+) and subjected to a NEBuilder reaction.

HEK293T Cell Culture and Transfection

HEK293T cell cultures were maintained in growth media consisting of Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM L-alanyl-L-glutamine (1x GlutaMax), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% carbon dioxide. Cells were routinely harvested, counted, and replated every 2 to 3 days to prevent cultures from overgrowing. Prior to counting, cells were resuspended in Trypan Blue stain to allow for assessment of cell death, which was routinely under 10%. When conducting experiments cells were plated as described in the individual assay protocols in growth media 4 hours prior to transfection. To transfect cells, cDNA was combined polyethylenimine (PEI) in unsupplemented DMEM for 20 minutes before addition to cells. The amount of PEI added was adjusted based on the total amount of cDNA used for the transfection using 4μL of 7.5 mM PEI per 1μg of cDNA. For some assays, the media on cells was changed to DMEM supplemented with 2% FBS only immediately prior to addition of the transfection mixture.

NanoBRET Experiments Using the Gβγ-masGRK3ct Sensor

For NanoBRET experiments, a modified standard protocol based on previously published protocols (Masuho et al., 2015a) or the mGluR optimized protocol described here was conducted. The day before the assay, HEK293T cells were plated into six well plates at 2 million cells per well in 1.5 mL of growth media. Four hours after plating transfections containing 200 ng masGRK3ct-NL, 200 ng mVenus(156-239)-Gβ1, 200 ng mVenus(1-155)-Gγ2, 400 ng EAAT3, 600 ng Gα protein, and 800 ng of receptor was assembled in 500 uL of supplemented DMEM with an appropriate amount of PEI. After 20 minutes, the transfection mixture was added to the cells dropwise. If the mGluR optimized protocol was being used, the media on the cells was changed to 1.5 mL of DMEM with 2% FBS immediately prior to adding the transfection. Cells were then allowed to transfect overnight.

The following day, the cell media was removed, and the wells were washed once with PBS with no calcium or magnesium. The PBS was then aspirated and PBS with 5 mM EDTA was added. Cells were then incubated in PBS with EDTA at 37°C for 5 minutes. Cells were then harvested by titration and collected into microcentrifuge tubes. Cells were then pelleted and washed three times with imaging buffer consisting of 136 mM NaCl, 560 μM MgCl2, 4.7 mM KCl, 1 mM Na2HPO4, 1.2 mM CaCl2, 10 mM HEPES, and 5.5 mM glucose. Experiments conducted with low Cl- buffer used the same imaging buffer except the NaCl was replaced with 136 mM sodium gluconate. After the third wash, the cells were resuspended in appropriate volume of imaging buffer and 25 μL of cells were transferred into each well of opaque, flat bottom, white 96-well plate. For the standard procedure, cells were than assayed immediately. For the mGluR optimized protocol, cells were allowed to incubate in the plate for 1 hour before being assayed.

Cell responses were assayed using a PolarStar Omega multimodal plate reader (BMG Labtech) equipped with dual emission photomultiplier tubes and two compound injectors. To select for NLuc and mVenus light, a 485/15 and a 535/30 filter were used, respectively. Luminescent signals were integrated for 200 millisecond time bins, with the gain for both detectors set to 2000. Injectors were loaded with either NanoGlo reagent (Promega, 1:250 dilution in imaging buffer) or test compounds and addition was automated by the plate reader. Injections were done at a speed of 430 μL per sec. For kinetic concentration-response experiments, 60-second time courses were used with 25 μL of NanoGlo being injected at –19 seconds and 50 μL of 2x test compound being added at t = 0 seconds. For Gα profiling experiments, 120-second time courses were used, with injections at the same time points. Experiments reported in Figs. 1 − 5 were conducted in kinetic mode, as illustrated. Concentration-response experiments reported in Fig. 6 were mainly conducted in endpoint mode (NanoGlo reagent, then glutamate added manually and data points recorded subsequently for ∼90 seconds for most G proteins or ∼210 seconds for αz and α16 to account for slower activation kinetics). The exception was the experiments involving the group I mGluRs, which were acquired in kinetic mode due to the acute desensitization that was particularly evident with mGluR5 (see Fig. 2).

Fig. 1.

Fig. 1.

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Evaluation of the mGluR-optimized protocol for all human mGluRs. (A) Dendrogram illustrating homology of all eight human mGluRs and their distribution into three groups. (B) Schematic illustrating the setup of the NanoBRET system. Panel B was made with BioRender using an institutional license to T.W.M., agreement #YO25L7S2XM. (C and D) Example protocol of cells expressing mGluR2-GoA prepared under the standard protocol (C) or mGluR optimized protocol (D) to 100 μM glutamate (green), 100 μM LY341495 (red), or the combination of the two (blue). The stimulus was delivered at time = 0 as indicated by the arrow. Summary data describing the basal BRET ratio (E), the LY341495 response (F), the glutamate response (G), and responses to Glu+LY34 (H) of all eight mGluRs in the experiments as shown in (C) and (D). Bars describe the average ± S.E.M. Statistics show the results of a two-way ANOVA with Holm-Šídák post hoc test, * P < 0.05, ** P < 0.005, *** P < 0.0005, **** P < 0.0001.

Fig. 5.

Fig. 5.

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Group III (mGluR7 and mGlur8) signaling profiles. Signaling profiles of mGluR7 (A–C), and mGluR8 (D–F), through a panel of 14 Gα proteins in response to 1 mM glutamate. Each stimulus was delivered at time = 0. The dark solid line indicates the average response of three biologic replicates and the light shading indicates the S.E.M. for each trace. Maximum ΔBRET induced by glutamate (B and E) and initial rates (C and F) for mGluR1 and 5, respectively, are displayed as the average ± S.E.M. Raw measurements for each replicate are shown as open circles (○) in each bar graph. Gray = not determined.

Fig. 6.

Fig. 6.

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Glutamate dose response curves illustrating relative efficacy and potency of responses of each mGluR through each responding G protein. (A) Glutamate dose response curves for the indicated Gα proteins when coexpressed with mGluRs1–8. Note that mGluR7 only showed responses to GoA and only at glutamate concentrations above 1 mM, so accurate efficacy and potency estimates were not possible. Heat maps are also shown, illustrating calculated EC50 values (B) and Hill coefficients (C) for the indicated mGluR homodimer with each responding Gα protein in the NanoBRET assay.

Fig. 2.

Fig. 2.

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Group I mGluR signaling profiles. Signaling profiles of mGluR1 (A–C), and mGluR5 (D–F), through a panel of 14 Gα proteins in response to 1 mM glutamate. Each stimulus was delivered at time = 0. The dark solid line indicates the average response of three biologic replicates and the light shading indicates the S.E.M. for each trace. Maximum ΔBRET induced by glutamate (B and E) and initial rates (C and F) for mGluR1 and 5, respectively, are displayed as the average ± S.E.M. Raw measurements for each replicate are shown as open circles (○) in each bar graph.

Data Analysis

For BRET experiments, the BRET ratio was calculated by dividing the mVenus signals (luminescence in the 535 channel) by the NLuc signals (luminescence in the 485 channel):

graphic file with name molpharm.123.000795_e1.jpg

Reponses are analyzed as ΔBRET, which is the average basal BRET ratio subtracted from the average stimulated BRET ratio:

graphic file with name molpharm.123.000795_e2.jpg

For endpoint assays, all three basal reading were averaged for the average basal BRET ratio, and all five readings post stimulation were averaged for the average stimulated BRET ratio. For kinetic experiments the basal BRET ratio was calculated as the average BRET ratio for 5 seconds immediately prior to test compound injection. For the average stimulated BRET ratio, the average BRET ratio for the last 10 seconds of the trace was used for nondesensitizing signals. For desensitizing signals, the average BRET ratio of a 10-second window centered at the signal’s peak was used.

To analyze the kinetics of BRET curves, the upstroke of each individual response was fit in GraphPad Prism (v. 9.3.1) using one of two models. The first model used was Pharmechanics’ “Baseline then rise to steady state time course” equation (Hoare et al., 2020) (a single-phase exponential association model):

graphic file with name molpharm.123.000795_e3.jpg

where Y is the response, SteadyState is plateau of the response, K is the rate constant, Baseline is the average baseline of the signal, and X0 is the time the response initiates, and X is time. The second model (when a single exponential fit was inadequate) used was a custom programmed two-phase exponential association model based on the previous equation:

graphic file with name molpharm.123.000795_e4.jpg

where Y is the response, SteadyState is plateau of the response, Kfast is the faster rate constant, Kslow is the slower rate constant, Baseline is the average baseline of the response, PercentFast is the percent contribution of the fast component to the response, X0 is the time the response initiates, and X is time. Calculation of the initial rates was then conducted by multiplying the SteadyState by the rate constant K for single association curves:

graphic file with name molpharm.123.000795_e5.jpg

or by multiplying the SteadyState by the weighted average of Kfast and Kslow for two-phase associations:

graphic file with name molpharm.123.000795_e6.jpg

To analyze concentration-response curves, data were imported into GraphPad Prism. Individual concentration-response curves were fitted with the built-in four-parameter logistic equation:

graphic file with name molpharm.123.000795_e7.jpg

where Y is the response, base is the response baseline, max is the maximum response (Emax), and EC50 is the concentration of drug that produces a 50% response. To aggregate responses from multiple replicates from the same conditions, the fit parameters were copied to Microsoft Excel and average values and standard errors were calculated for each condition.

Statistics

All statistical analysis was conducted in GraphPad Prism. Results are reported in the figure legends in [P = (P value)] format. Details of the test (type of test, results, significance levels) are indicated in the figure legend. The cartoon in Fig. 1B was made using BioRender (Toronto, ON, Canada).

Results

Optimizing the NanoBRET Assay for mGluR Signaling

Our goal was to comprehensively examine receptor-G protein coupling profiles of each of the eight human mGluR homodimers (Fig. 1A) with members of each family of G proteins. To accomplish this, we employed an optimized version of a Gβγ-based BRET assay (Hollins et al., 2009; Masuho et al., 2015a). This assay detects the interaction between the Gβγ binding region of GRK3 fused to NLuc on its C-terminus and to a myristic acid sequence on its N-terminus (MAS-GRK3-NLuc), and a complemented YFP-tagged Gβγ that is sequestered when inactive by a heterologously expressed Gα (“NanoBRET”; Fig. 1B). Each construct (see Materials and Methods), along with the indicated receptor, was expressed in HEK293T cells (Fig. 1B). However, because HEK cells secrete micromolar concentrations of glutamate into the extracellular space (Hlavackova et al., 2012), assay conditions needed to be optimized compared with those originally published (Masuho et al., 2015a). This helps to reduce ambient glutamate levels that could potentially produce basal activation of mGluRs, which could produce high apparent basal BRET signals and reduce the observed ΔBRET, as shown in Fig. 1C, using mGluR2 and GoA, which shows high basal BRET signals and small ΔBRET upon application of 1 mM glutamate. The dramatic reduction in BRET signal in this experiment when the competitive antagonist LY341495 [(2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid] was applied with or without glutamate (Fig. 1C, red and blue, respectively) demonstrates that the high basal BRET signal was likely due to ambient glutamate in the well. To address the elevated glutamate in the bath, a combination of amino acid transporter expression (Goudet et al., 2004; Hlavackova et al., 2005), washing, and timing of the experiments was used. Conditions were optimized to 1) reduce the basal BRET ratio, 2) reduce the responsiveness to a pan-mGluR antagonist (LY341495) in the absence of exogenous agonist, and 3) maximize the ΔBRET signal generated by glutamate application. Fig. 1D shows responses of mGluR2/GoA to the optimized protocol (also see Materials and Methods). Note the lower basal BRET value, the reduced effect of antagonist (red), and the strengthened ΔBRET signal upon application of 1 mM glutamate (green). A summary of the basal BRET levels (Fig. 1E), the change in BRET with LY341495 (Fig. 1F), and the ΔBRET upon glutamate application (Fig. 1G) or glutamate +LY34 (Fig. 1H) is also shown using the standard (red) and optimized (gray) NanoBRET protocols. Effects of null receptor conditions are also shown (“-/oA” and “-/q”), illustrating that no responses are seen in the absence of heterologous mGluR expression. To be certain that each Gα protein expressed in the NanoBRET assays was expressed and functional, control experiments were performed with receptors that canonically couple to all of the G protein families to be tested. For these experiments, we used the H1 histamine receptor (Gi/o and Gq/11), the lysophosphatidic acid receptor 2A (Gi/o, Gq/11, and G12/13) (Avet et al., 2022), and the D5R (the Gs family). The combined results of these experiments (Supplemental Fig. 1) show that positive results can be obtained with each of the Gα proteins expressed in our NanoBRET assays.

Group I mGluR Profiles

To begin to assess mGluR-G protein coupling, each group I mGluR (1 and 5) was expressed in combination with the optimized NanoBRET system with a panel of 14 Gα proteins spanning all four major families. In each experiment, 1 mM glutamate was added at 0 seconds. Fig. 2A shows averaged, time-resolved ΔBRET traces for 14 Gα proteins in cells expressing mGluR1, which responded to each member of the Gi/o family except for Gz, and as expected responded to the Gq/11 family of Gα proteins with the notable exception of G14. Gq appeared to be activated with the highest efficacy (Fig. 2, A and B), while G11, GoA, and GoB also responded with high efficacy and Gi1-3, and G16 responded somewhat strongly as well. No responses were observed indicating coupling of mGluR1 with the Gs family members Sshort, Slong, or Olf (Fig. 2, A and B). Kinetics of activation of each responding G protein were also assessed by calculating the initial rate of activation for each (Fig. 2C; also see Materials and Methods).

The profile of mGluR5 was qualitatively similar to mGluR1 but with a notable apparent desensitization of responses to Gq and G11 (Fig. 2D), which has been documented previously (Kammermeier and Ikeda, 2002; Dhami and Ferguson, 2006). This desensitization may have hindered measurement of the full efficacy of these responses. As such, mGluR5 coupled most strongly to Goa (Fig. 2, D–F).

Because previous reports have indicated that group I mGluRs can initiate cAMP accumulation and may therefore couple to Gs proteins (Kubo and Tateyama, 2005), and a recent study has suggested that purified, truncated, mGluR5 is capable of activating Gs in the presence of the agonistic positive allosteric modulator VU0424465 (VU042) (Nasrallah et al., 2018), we re-examined the coupling profile of mGluR5 in the presence of glutamate alone, VU042 alone, and glutamate + VU042 together (Supplemental Fig. 2). While we observed some differences in maximum BRET with glutamate compared with glutamate + VU042 when coupling to Gi1, Gi3, and GoB (Supplemental Fig. 2), we did not observe mGluR5 coupling to any Gs family member in any condition. Together, these data confirm that the group I mGluRs are dual-coupled receptors that can couple with high efficacy to the Gi/o and Gq/11 families.

Group II Profiles

When assayed in the optimized NanoBRET system, mGluR2 yielded detectable coupling to each member of the Gi/o family (Gi1-3, Goa, GoB, Gz) as well as the promiscuous G16. The kinetics of Gz and G16 activation by mGluR2 were dramatically slower, however. Note that the S1 subunit of pertussis toxin was coexpressed (Ikeda et al., 1999) with each pertussis toxin-insensitive Gα protein, including Gz, to prevent a small but detectable signal presumably carried by endogenous Gi/o proteins in these and all subsequent experiments. Similar results were seen with the other group II member, mGluR3 (Fig. 3), although because of the high potency of glutamate on this receptor and its apparent consequent basal activation leading to high basal BRET ratios even under optimized conditions (Fig. 1F), it was necessary to reduce extracellular Cl- levels to right-shift glutamate/mGluR3 potency (Tora et al., 2018) to obtain meaningful data (see later discussion). Reassaying mGluR2’s signaling profile under low Cl- conditions showed a similar coupling profile as in the standard high Cl- buffer, which justifies using this method for mGluR3 measurements. Supplemental Fig. 3 shows the maximum BRET amplitude and activation kinetics with mGluR2 (Supplemental Fig. 3D) as well as correlations (Supplemental Fig. 3, E and F) of these measurements for each G protein in low and high Cl- conditions. Correlations show a consistent shift to higher potencies in low Cl- but maintain the slopes, indicating that the rank order of G protein coupling remains unaltered. Thus, examining the full profile of mGluR3 in low Cl- reveals that, like mGluR2, mGluR3 couples almost exclusively to the Gi/o family, although it favors coupling to GoA and GoB versus Gi proteins (Fig. 3B). Like with mGluR2, mGluR3 showed a weaker, slower activation of Gz but, in contrast, no detectable coupling to G16. As expected, neither group II receptor coupled to any members of the Gq or Gs families.

Fig. 3.

Fig. 3.

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Group II mGluR signaling profiles. Signaling profiles of mGluR2 (A–C), and mGluR3 (D–F), through a panel of 14 Gα proteins in response to 1 mM glutamate. Each stimulus was delivered at time = 0. The dark solid line indicates the average response of three biologic replicates and the light shading indicates the S.E.M. for each trace. Maximum ΔBRET induced by glutamate (B and E) and initial rates (C and F) for mGluR1 and 5, respectively, are displayed as the average ± S.E.M. Raw measurements for each replicate are shown as open circles (○) in each bar graph.

Group III Profiles

G protein coupling was also similarly assessed with the group III receptors mGluR4-8 (Figs. 4 and 5). As with mGluR2, each of the Gi/o family members were activated by mGluR4, although no detectable activation of G16 was observed and coupling to GZ was very weak (Fig. 4). Similar profiles were observed with all of the group III mGluRs, mGluR6 (Fig. 4), as well as 7 and 8 (Fig. 5), confirming that these receptors are exclusively coupled to the Gi/o family and demonstrating variable GZ coupling within the group III mGluRs. In this group, mGluR7 was somewhat anomalous, only showing weak coupling to GoA and GoB (Fig. 5, A and B). Likely due to its high constitutive activity (Kammermeier, 2015) and activation by glutamate with very low potency, this receptor exhibited a high basal BRET signal (Fig. 1) and required 10 mM glutamate to detect its comparatively poor activation (Fig. 5). It is possible mGluR7 may be capable of coupling to other G proteins, but our assay would only sense this if more efficient activation of mGluR7 could be achieved. In apparent contrast to the GABAB receptor (Wang et al., 2021), none of the mGluRs showed detectable activation through G13, and none showed activation of G14 or the Gs family.

Fig. 4.

Fig. 4.

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Group III (mGluR4 and mGluR6) signaling profiles. Signaling profiles of mGluR4 (A–C), and mGluR6 (D–F), through a panel of 14 Gα proteins in response to 1 mM glutamate. Each stimulus was delivered at time = 0. The dark solid line indicates the average response of three biologic replicates and the light shading indicates the S.E.M. for each trace. Maximum ΔBRET induced by glutamate (B and E) and initial rates (C and F) for mGluR1 and mGluR5, respectively, are displayed as the average ± S.E.M. Raw measurements for each replicate are shown as open circles (○) in each bar graph.

Because of the need to test mGluR3 responses in low Cl- as described ealier, concentration-response curves were generated for each receptor (except mGluR7) in normal and low Cl- (Supplemental Fig. 4). Full concentration-response curves were generated using the Gα protein activated by glutamate with the highest potency against each receptor (Gq for the group I receptors and GoA for groups II and III) in high (144 mM) and low (7 mM) Cl-. Interestingly, reducing the Cl- concentration resulted in a right shift in glutamate potency against every receptor tested. Rescue of the mGluR3 responses in low Cl- is consistent with the interpretation that the low levels of basal extracellular glutamate present in these experiments is enough to activate and desensitize these receptors (Abreu et al., 2021) when measured at high Cl-, but low Cl- shifts the EC50 such that ambient glutamate is below the threshold of activation, therefore avoiding high basal activity and desensitization of this receptor in high Cl-. Net effects of Cl- changes on EC50 (Supplemental Fig. 4B) and efficacy (Supplemental Fig. 4C) are also shown. Finally, Supplemental Fig. 4, D and E illustrate that the rank order of G protein EC50 with mGluR2 is unaffected by the change in [Cl-], suggesting that low Cl- remains a reasonable modification to measure responses through mGluR3.

Potency of mGluR Homodimer Activation Through Different Gα Proteins with Glutamate

To assess the potency of glutamate activating mGluRs through each identified Gα protein signaling partner, we employed the NanoBRET system at a range of glutamate concentrations (Fig. 6). For each receptor, dose-response data were only obtained with Gα proteins that showed significant responses in the profiling assay (Figs. 2–5). The group I mGluRs, mGluR1 and mGluR5, were the only mGluRs that showed responses with Gq and G11, and both of these receptors responded with the highest potency with Gq activation by glutamate, which was slightly higher than G11 in each case (Fig. 6A). In both cases, Gq and G11 signaling was also slightly left shifted compared with signaling through Gi/o proteins, which all showed very similar EC50 values. The group II mGluRs showed a clear preference for GoA and GoB (Fig. 6A) in terms of glutamate potency, and mGluR2 showed intermediate responses with Gi1-3 and lowest potency activation of Gz and G16, while mGluR3 (in low Cl-) exhibited similar responses to Gi1-3, z, and G16. In general, the group III receptors were also activated by glutamate through GoA and GoB with the highest potency, followed by Gi1-3, and finally Gz (mGluRs4 and mGluR6; Fig. 6). Due to the very low potency of glutamate on mGluR7 through most G proteins, dose-response curves could only be obtained with GoA, and these could not be tested to saturation due to solubility as well as osmolarity issues at very high glutamate concentrations. Heat maps summarizing the EC50 values for each receptor with each Gα protein tested are shown in Fig. 6B, and the Hill coefficients for each condition tested are shown in Fig. 6C. Note that efficacy for this data set was normalized for each pathway to allow for easier comparison of potencies, but, in all cases, efficacy was similar to the Max ΔBRET values shown in Figs. 2 − 5.

Discussion

G Protein Coupling Profiles of mGluR Homodimers

We show here for the first time a comprehensive mGluR-G protein coupling profiling assessment with every homodimeric member of the human mGluR family against 14 Gα proteins, representing each G protein family. Heat maps summarizing all of the maximal responses and activation kinetics are shown in Fig. 7, A and B, respectively. These data show that the group I mGluRs, mGluR1 and mGluR5, couple to both the Gq/11 and Gi/o proteins, as previously suggested (McCool et al., 1998; Kammermeier and Ikeda, 1999). No evidence for group I mGluR coupling to members of the Gs family was seen, including in the presence of the mGluR5 positive allosteric modulator VU042 (Nasrallah et al., 2018). In addition, the group II and III mGluRs coupled almost exclusively to the Gi/o proteins, with the only exception being a novel but weak, slow activation of the promiscuous G16 by mGluR2 and to a lesser degree mGluR3. While G protein profiling has been examined to some extent on representative members of the mGluR family, to our knowledge this is the first comprehensive assessment of all of the mGluRs with a large set of Gα proteins. One recent study examined G protein profiles of many GPCRs using an effector translocation-based BRET assay and reported results on mGluR2, 4, 5, 6, and 8 largely consistent with those reported here (Avet et al., 2022) but did not assay all of the mGluRs or the kinetics of activation. One notable exception was a reported coupling between mGluR5 and α14, which we did not see. However, we would note that in that study, the authors similarly did not observe coupling of mGluR5 to members of the αs family. Our results were consistent with that finding. To some degree, these results are consistent with recent studies (Avet et al., 2022; Masuho et al., 2023), but the data presented here are more thorough in that they include all mGluRs or a more comprehensive Gα protein expression data set. Further, we detail a comprehensive protocol for optimization of the assay for glutamate receptors in general (Fig. 1) and for mGluR3 in particular (Supplemental Figs. 2 and 3) and report novel findings regarding G16 coupling and the kinetics of group II coupling to Go.

Fig. 7.

Fig. 7.

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Summary of mGluR-G protein responses of all mGluR homodimers. (A) Summary of signal amplitude data displayed as raw ΔBRET according to blue intensity scale shown below for each mGluR-G protein pair. (B) Initial rate data displayed as Log10 transformed initial rates according to the color scale shown below for each mGluR-G protein pair. Heatmaps in both (A) and (B) display the average value of three biologic replicates. Data are from the same experimental replicates shown in Figs. 2–5. Gray boxes in (B) indicate “not determined.”

G Protein Activation Kinetics

Comparing signaling of each of the members of the mGluR family, it is apparent that the efficacies in the NanoBRET assay are somewhat comparable. However, examination of the kinetics reveals that the initial rates of activation of different mGluR/G protein pairings to be quite variable (Fig. 7). Under some circumstances such as desensitization or differences in receptor expression level, maximal efficacy in this kind of assay may be misleading. Thus, activation kinetics can provide a more objective assessment of the efficiency of receptor-G protein coupling (Hoare et al., 2020). In general, we see the group II mGluRs are highly efficient receptors, activating G proteins at considerably faster rates compared with the group I or group III receptors (Fig. 7). The only major exception to this trend is with G16 signaling, where the bona fide Gq/11 coupling mGluR1 and mGluR5 show faster activation than the group II receptors. Additionally, most mGluRs showed faster kinetics through Go proteins than other Gα proteins, with only mGluR1 showing slightly faster kinetics through Gq. Although this finding is going to be largely influenced by the affinity of each individual Gα protein for the Gβγ used, receptor level effects are clearly present given the H1R was able to activate Gq/11 proteins with faster kinetics than the Go protein, and the D5R was able to activate Gs proteins with faster kinetics than Go proteins. These additional findings suggest that mGluRs favor signaling through Go over other αi/o family members.

Regarding GPCR-G protein coupling experiments, especially when using activation kinetics as a proxy for coupling efficiency, it is important to consider the expression of the regulators of G protein signaling (RGS) proteins in the cells assayed. This is important because while RGS proteins facilitate deactivation kinetics of Gα proteins by acting as GTPase activating proteins, they can also accelerate activation kinetics (Doupnik et al., 1997; Saitoh et al., 1997; Jeong and Ikeda, 2001; Choi et al., 2006). HEK293 cells have been suggested to express a wide array of RGS proteins (Laroche et al., 2010), but more recent work using RNA microarrays suggested that the expression may be more limited (Atwood et al., 2011). Still, while RGS protein expression may affect interpretation of specific details such as kinetics, it is unlikely that a different compliment of RGSs will yield coupling to a specific Gα protein in another system where none was observed here. It is also unlikely that the rank order of coupling efficiency of receptors would be altered with different RGSs due to them exerting their effects on the G protein level rather than the receptor level. For example, we observed coupling to αoA through the group II mGluRs to be more efficient than through the group III receptors. Since they all couple to the same family of G proteins, it is unlikely this relation will be different with other RGSs that also act as GTPase activating proteins through these same G proteins.

These data highlight an interesting aspect of mGluR-G protein coupling across the family, specifically coupling efficiency. We found that the group II mGluRs exhibited the fastest signaling kinetics when coupled to Gi/o proteins and mGluR2 in particular when coupled to all members of the Gi/o family (Fig. 7). These results suggest that, in the physiologic context, when mGluRs reside in the synaptic environment and are likely to be exposed to saturating concentrations of glutamate for only brief periods of time, the group II mGluRs may play a dominant role in the modulation of synaptic transmission. Another interesting aspect of mGluR-α protein coupling is the differences in EC50 through which the receptors activate different Gα proteins. These differences probably reflect a combination of varying affinities that each Gα protein has with the active state of the receptors and the abundance of each Gα in cell. The group II and III receptors show a clear preference for the αo proteins, followed by αi1-3, with most also activating αz. By contrast, the group I receptors activate the αq/11 proteins with the lowest EC50, followed by members of the Gi/o family, which were activated at similar glutamate concentrations. Of course activation kinetics can be influenced to some degree by receptor expression, although kinetics will saturate with higher expression (Masuho et al., 2023). Unfortunately, it was not possible to directly compare expression across receptors in this study and even more difficult to measure levels of mGluRs expressed in subcellular compartments in vivo. It will be important in the future to test whether differences in mGluR-G protein coupling efficiency seen here, using heterologously expressed receptors in HEK293 cells, accurately predicts their behavior in a native physiologic context.

In this study, we examine mGluR activation kinetics. One recently published study examined intradimer conformational changes of several mGluR dimers using a fluorescence resonance energy transfer assay (Kukaj et al., 2023). There, authors reported that glutamate induced changes in fluorescence resonance energy transfer were measurable for five of the eight mGluR homodimers. Interestingly and seemingly at odds with the kinetics of G protein activation described here, they reported that the fastest on kinetics were associated with mGluR1 and the slowest with mGluR2. However, it should be noted that, in that study, what was measured was the movement of the subunits within each dimer that would lead to activation, while we measured the presence of active, “free” Gβγ, which can be considered a measure of the efficiency of catalysis of guanine nucleotide exchange of each active receptor, not the kinetics of the conformational changes of an inactive receptor transitioning to an active one. Comparing these values directly, inactive to active conformational changes of even the slowest receptor in that study was on the order of 10 to 20 milliseconds (Kukaj et al., 2023), still several orders of magnitude faster than the rates of guanine nucleotide exchange of all of the receptors in this study with which coupling was detected. Thus, from a physiologic perspective, it is still reasonable to consider the group II mGluRs as the most efficient activators of G proteins in the family.

Acknowledgments

The authors thank Cesare Orlandi (University of Rochester) for use of his plate reader and for helpful guidance.

Data Availability

Data and reagents generated for and reported on in this study will be made available upon request. Authors declare that all of the data supporting this study are included within this manuscript and in the Supplemental Material.

Abbreviations

BRET

bioluminescence resonance energy transfer

D5R

D5 dopamine receptor

DMEM

Dulbecco’s modified Eagle medium

mGluR

metabotropic glutamate receptor

NanoBRET

detection of G protein signaling by measurement of BRET between YFP-Gβγ and MAS-GRK3ct-NLuc

NLuc

nanoluciferase

PEI

polyethylenimine

RGS

regulators of G protein signaling

Authorship Contributions

Participated in research design: McCullock, Kammermeier.

Conducted experiments: McCullock, Cardani.

Contributed new reagents or analytic tools: McCullock, Kammermeier.

Performed data analysis: McCullock, Cardani.

Wrote or contributed to the writing of the manuscript: McCullock, Cardani, Kammermeier.

Footnotes

These studies were supported by the National Institutes of Health National Institute of Neurologic Disease and Stroke [Grants R21NS126779 and R03NS124987] and the National Institute of Mental Health [Grant R01MH125849] (to P.J.K.).

The authors report no actual or perceived financial conflicts of interests pertaining to the contents of this article.

1Current affiliation: Chemistry Graduate Program, Columbia University.

All authors have approved submission of this manuscript.

A preprint of this article was deposited in bioRxiv [https://doi.org/10.1101/2023.07.24.550373].

Inline graphicThis article has supplemental material available at molpharm.aspetjournals.org.

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