Heteromeric Assembly of mGluR2 and 2AR Enhances Glutamate-Elicited Gi Signaling and Reduces Gq Signaling
2AR is a Gq/11- (or simply Gq-) coupled GPCR that responds to the neurotransmitter serotonin (5-HT) (González-Maeso and Sealfon, 2009
), whereas mGluR2 is a Gi/o- (or simply Gi-) coupled, pertussis toxin-sensitive GPCR that responds to the neurotransmitter glutamate (Glu) (Moreno et al., 2009
Similarly to what we showed previously by mRNA in situ
hybridization (González-Maeso et al., 2008
), 2AR and mGluR2 immunoreactivity co-localized in mouse cortical slices and neuronal primary cultures ( and Figure S1 F
). In addition, the two receptors were co-immunoprecipitated in mouse frontal cortex membrane preparations (). In order to investigate the signaling properties of the mGluR2/2AR heterocomplex, we utilized Xenopus
oocytes, a heterologous system widely used for functional expression of recombinant ion channels (Barela et al., 2006
). We expressed each of these GPCRs alone or together, but always with ion channels that served as reporters of GPCR-mediated signaling. We used inhibition of the IRK3 (Kir2.3) current to monitor Gq activity ( - left) (Du et al., 2004
), and activation of the GIRK4* (or Kir3.4*) current to monitor Gi activity ( - right) (He et al., 1999
; He et al., 2002
) (see Experimental Procedures section).
Heteromeric Assembly of 2AR and mGluR2 enhances Glu-induced Gi Signaling and Reduces 5-HT-induced Gq Signaling
First, we pursued the question of how the signaling properties of the mGluR2/2AR heteromeric complex differ from the signaling properties of the homomeric receptors. We quantified the signaling elicited by Glu and 5-HT, the endogenous ligands of the mGluR2 and 2AR receptors, respectively, and compared the Gi and Gq activities in the absence or presence of the heteromeric receptor partner (). We found that co-expression of mGluR2 with 2AR reduced the Gq activity elicited by 5-HT to approximately 50% of the activity of the 2AR expressed alone (-left panel and ). In contrast, co-expression of mGluR2 with 2AR increased the Gi activity elicited by Glu to approximately twice of that with the mGluR2 expressed alone (-right panel and ). As anticipated, 5-HT and Glu did not affect mGluR2 or 2AR signaling, respectively, when the two receptors were expressed individually ().
The metabotropic glutamate receptor 3 (mGluR3), which shares a high degree of homology with mGluR2, does not form a receptor heterocomplex with the 2AR (González-Maeso et al., 2008
). Exchanging the transmembrane domains required for mGluR2 heteromerization with 2AR either disrupts (mGluR2Δ) or rescues (mGluR3Δ) receptor complex formation and Gi cross-signaling upon 2AR receptor activation (González-Maeso et al., 2008
). Control experiments showed that mGluR2Δ did not exhibit the effects of heteromerization with 2AR on Gi or Gq signaling, whereas activation of mGluR3Δ, which forms a receptor complex with the 2AR, induced an increase in Gi- and decrease in Gq- dependent signaling (Figure S1 A, B, C and D
). Each of the mGluR chimeras, when expressed as homomers, showed intact Gi signaling (Figure S2 A, B and C
) and cell surface localization (Figure S2 D
We summarized the difference between Gi signaling and Gq signaling achieved by the formation of the complex using a new parameter: the balance index (BI). The BI combines the change in Gi activity (ΔGi) and the change in Gq activity (ΔGq) according to the formula (BI= ΔGi - ΔGq) (). Normalizing all the values to the homomeric Gi and Gq signaling, the formation of the complex alone yielded a BI of 1.45, which we shall refer to as the reference BI level (BIr).
We obtained the largest BIr
when expressing mGluR2/2AR mRNAs in a 1:2 ratio (Figure S3 D
). As shown in Figure S3 C
, this ratio of mRNAs yielded a cell surface localization of receptor protein levels (compared to the total protein in the cell – see Figure S3 A, B
) that suggested a higher order oligomeric complex between mGluR2 and 2AR.
Drugs that Bind 2AR Alter the Balance Between Gi and Gq Signaling
We next asked whether drugs bound to one receptor of the heteromer could affect the other receptor's signaling response to its endogenous neurotransmitter. We first investigated the effects of 2AR ligands (a neutral antagonist, a strong agonist, and an inverse agonist) on Glu-elicited Gi signaling by mGluR2. We define a “strong
” agonist to be one that results in greater signaling than the endogenous agonist (e.g
., DOI causing greater Gq signaling through 2AR than 5-HT – see , red bars and Figure S4 A
). In control experiments, the neutral antagonist (methysergide), the strong agonist (DOI) and the inverse agonist (clozapine) (Weiner et al., 2001
) worked as expected to stimulate, or reduce 5-HT-induced Gq signaling, respectively ().
Drugs that Target 2AR: Integrative Effects on Gi and Gq Signaling
Occupancy of the 2AR receptor by each of the three ligands influenced uniquely Glu-elicited signaling through mGluR2. (blue bars) depicts the effects of methysergide, DOI, and clozapine on Glu-elicited Gi signaling through the complex. All values were normalized to the homomeric response of mGluR2 to Glu. As was shown in , the formation of the complex doubled the extent of Gi signaling (200%). Although neither 5-HT nor methysergide affected Glu-elicited Gi signaling, DOI bound to 2AR decreased Gi signaling back to homomeric levels, and clozapine increased it by approximately 40% (240% greater than homomeric levels). In summary, while the neutral antagonist (methysergide) had no effect on Gi signaling, the strong agonist (DOI) decreased Gi levels back to homomeric levels, while the inverse agonist (clozapine) significantly increased Gi signaling.
(red bars) summarizes the Gq activity associated with methysergide, DOI, and clozapine together with Glu in oocytes co-expressing mGluR2 and 2AR (note the difference with where 5-HT is also present). Since these three drugs target 2AR, Gq signaling was only detected when the agonists 5-HT and DOI were applied.
Using the results obtained for both the Gi ( - blue bars) and Gq signaling ( and - red bars), we calculated BI values for the three ligands in the co-presence of the endogenous ligands (summarized in ). All effects were disrupted when replacing mGluR2 by mGluR2Δ (see Supplemental Table S1
), rescued by mGluR3Δ, and present when the Gq pathway was blocked by the regulator of G protein signaling subunit 2 (RGS2) (Figure S5 D and E
). The 2AR ligand with the largest overall BI was the inverse agonist clozapine (BI=2.30; 140% increase in Gi and 100% decrease in Gq).
Could these ligands exert their effects by stabilizing different conformations of the receptor complex? We next investigated at the molecular level the conformational changes induced by the three 2AR ligands (i.e
. the neutral agonist methysergide, the strong agonist DOI, and the inverse agonist clozapine) in atomistic representations of 2AR either alone or interacting with mGluR2 within an explicit lipid-water environment. To be able to observe large conformational changes in relatively short timescales, we used a combination of adiabatic biased molecular dynamics (ABMD) and metadynamics simulations (see “Experimental Procedures”, under “Computational Methods”). This approach has been recently validated on a prototypic GPCR (Provasi et al., 2011
). First, we studied the effects of the three ligands (methysergide, DOI, and clozapine) on the activation free-energy profile of a protomeric 2AR (see - top) and identified the most energetically favorable 2AR state for each ligand. In agreement with known efficacies of these ligands, the clozapine-bound 2AR conformation is inactive (i.e
., 2RH1-like), the DOI-bound 2AR conformation is active (i.e
., 3P0G-like), and the methysergide-bound conformation adopts an inactive state that is structurally different from the inactive state stabilized by clozapine.
To provide a structural context for the crosstalk between 2AR and mGluR2, we studied further the effects of the three 2AR ligands on mGluR2 conformations in the dimeric complex. The location of the dimeric interface was obtained for each ligand-specific 2AR conformation using an implicit membrane model Monte Carlo search (see Computational Methods). The reconstructed free-energy in the bottom part of shows that when clozapine is bound to 2AR, the mGluR2 equilibrium is shifted toward a more activated conformation (i.e., 3DBQ-like), consistent with the functional up-modulation in Gi signaling. In contrast, methysergide and DOI bound to 2AR stabilized inactive (i.e., 1U19-like) states of mGluR2. Although no significant energetic and structural differences were noted between the TM regions of these inactive states of mGluR2, the functional down-modulation of Gi signaling induced by DOI, but not by methysergide, may be ascribed to possible different interactions between the receptor loop regions and the G-protein, which are not taken into account in our simulations. This computational approach that predicts the functional results () can be used to guide structure-based rational discovery of novel `biased' drugs that are capable of selectively activating specific signaling pathways.
Taken together, these results indicate that, in the heteromer, the Gi response elicited by Glu through mGluR2 can be modulated by ligands that bind the 2AR. While drugs such as the strong 2AR agonist DOI can greatly stimulate Gq signaling and decrease Gi signaling (henceforth referred to as dominant agonists), inverse agonists, such as clozapine, have the opposite effect, namely they abolish Gq signaling and increase Gi signaling.
Drugs that Bind mGluR2 Alter the Balance Between Gi and Gq Signaling
We proceeded to test how drugs bound to mGluR2 as part of the heteromeric complex affected the 5-HT-elicited Gq signaling of 2AR. The neutral antagonist ethylglutamic acid (eGlu), the strong agonist (LY37) (Figure S4 B
) and the inverse agonist (LY34) [for full names, see “Experimental Procedures”, under “Drugs”] worked as expected to reduce, stimulate or abolish Glu-elicited Gi signaling through mGluR2, respectively (). Consistent with its inverse-agonist properties, LY34 not only completely abolished Gi signaling, but also was able to reduce the basal Gi activity of the mGluR2 receptor even in the absence of Glu (Figure S4 C
Drugs that Target mGluR2: Integrative Effects on Gi and Gq Signaling
Occupancy of the mGluR2 receptor by each of the three ligands uniquely influenced the 5-HT-elicited signaling through 2AR. (red bars) depicts the effects of eGlu, LY37, and LY34 on 5-HT-elicited Gq signaling through the complex. As before, the formation of the complex reduced to half the extent of Gq signaling (relative to the homomeric 2AR levels shown by the dotted line). Although neither Glu nor eGlu affected 5-HT-elicited Gq signaling, LY37 bound to mGluR2 decreased it even further (around 35%), while LY34 increased it almost back to homomeric levels (83%).
(blue bars) summarizes the Gi activity associated with eGlu, LY37, and LY34 (in the absence of Glu) in response to 5-HT in oocytes co-expressing mGluR2 and 2AR. Since these three drugs target mGluR2, Gi signaling was only detected when the agonists Glu and LY37 were applied.
Using the data shown for Gi ( and , blue bars) and Gq signaling (, red bars), we calculated the BI values for the three ligands (). Similarly to 2AR drugs, the neutral antagonist eGlu only affected the mGluR2 side of signaling. The strong agonist LY37, however, affected both types of signaling through the complex and showed a dominant-agonist behavior as defined previously. Furthermore LY37, like DOI, was able to cross signal and elicit Gq signaling in the absence of Glu and 5-HT, respectively (Figure S4, F(5) and G(5)
). The inverse agonist LY34 had the opposite two effects: it blocked Gi, but also potentiated Gq signaling, achieving almost 2AR homomeric levels (83%). All effects were disrupted when replacing mGluR2 by mGluR2Δ (see Supplemental Table S1
), rescued by mGluR3Δ, and present when the Gi pathway was blocked by pertussis toxin (PTX) (Figure S5 A, B, and C
). The largest overall signaling difference between Gi and Gq was obtained by the dominant agonist LY37 (BI=2.10).
In summary, our results thus far indicate that formation of the heteromeric complex favors Gi over Gq (i.e., increasing Gi and decreasing Gq) signaling by endogenous ligands. Dominant agonists of each receptor enhance signaling of the receptor they bind as part of the complex and inhibit signaling of their heteromeric receptor partner. On the other hand, inverse agonists of each receptor inhibit signaling of the receptor they bind as part of the complex, while enhancing signaling of their heteromeric receptor partner.
The Balance Index (BI) Predicts the Anti- or Pro-Psychotic Activity of Drugs Targeting mGluR2 or 2AR
Clozapine is superior to other antipsychotics not only with regard to extrapyramidal side effects, but also in several other clinical respects (Grunder et al., 2009
). LY37 belongs to the class of the newly described Glu antipsychotics (Moreno et al., 2009
; Patil et al., 2007
). Our results thus far ( and ) indicate that although they act on different receptors, both drugs achieve through the mGluR2/2AR complex a similar effect on the relative Gi to Gq signaling activity, namely an increase in Gi activity concomitant with a decrease in Gq activity. We proceeded to test whether the extent of the difference between Gi-activity and Gq-activity elicited by different drugs was correlated with their psychoactive effects.
On the mGluR2 side, in addition to the dominant agonist LY37, we tested the neutral antagonist eGlu and the inverse agonist LY34, a drug that has been shown to increase locomotor activity and exploratory behavior in mice and might be considered pro-psychotic (Bespalov et al., 2007
). On the 2AR side, besides the inverse agonist clozapine, we tested the following drugs: risperidone, another widely used atypical antipsychotic like clozapine; ritanserin, an antidepressant also used as an adjuvant therapeutic for schizophrenia; methysergide, a drug mainly used for migraines; and DOI, a propsychotic drug with lysergic acid diethylamide (LSD)-like effects.
We calculated the BI values of these drugs at either a 10 μM (BI10
, ) or at 50 μM concentrations (BI50
, ; Table S2
summarizes the BI values obtained for each of the drugs we tested). Since, in the presence of endogenous ligands and absence of drugs, the difference between Gi and Gq signaling is naturally kept in a balance that favors Gi over Gq (BIr
), we converted the balance index scale in terms of the percentage recovery or loss of the Gi-Gq balance, taking a fractional occupancy of the receptor complex by the drug of 0.5 as a reference level for comparison (see Gi-Gq recovery/loss calculation in the Experimental Procedures section). Results from drugs that target mGluR2 (blue icons), versus those that target 2AR (red icons) were plotted in .
Use of BI Index to Classify Anti-/Pro-psychotic Propensity of Drugs Targeting the mGluR2/2AR Complex
As can be appreciated from (and Table S1
), drugs with the most effective antipsychotic properties, regardless of the receptor they target (2AR: clozapine, risperidone; mGluR2: LY37), show the highest BI values. In contrast, drugs with the most effective propsychotic properties (2AR: DOI; mGluR2: LY34) show the lowest BI values. All of these drugs are either dominant agonists (antipsychotic for mGluR2 and propsychotic for 2AR) or inverse agonists (antipsychotic for 2AR and propsychotic for mGluR2). Neutral antagonists that target either receptors lie in between the dominant agonists and inverse agonists (between 25% recovery and 25% loss of the Gi-Gq signaling balance) and have not been shown to affect psychotic states.
Inverse-Agonist Up-Modulation Occurs in Mouse Frontal Cortex
To study the relevance of the effects obtained in Xenopus
oocytes, we examined the pattern of G protein coupling in mouse frontal cortex, a region that plays an important role in schizophrenia and antipsychotic action (González-Maeso and Sealfon, 2009
). We first measured the mGluR2/2AR complex-dependent up-modulation of Gi signaling by a 2AR inverse agonist. Membrane preparations from mouse frontal cortex were incubated with the inverse agonist clozapine or the neutral antagonist methysergide (Figure S4 D and E
), together with DCG-IV, a selective mGluR2/3 agonist. In oocytes, DCG-IV acted like the endogenous ligand Glu in stimulating Gi activity and in failing to down-regulate Gq signaling, unlike the dominant agonist LY37 (data not shown). Clozapine increased the DCG-IV-mediated Gi signaling (), while methysergide did not significantly affect Gi signaling (). Furthermore, clozapine failed to increase the DCG-IV-mediated Gi signaling in frontal cortex membrane preparations from 2AR knockout (KO) mice (Figure S6 C
; see also Figure S6 A and B
for LY37-dependent activation of Gq in wild-type but not in 2AR-KO mouse frontal cortex).
Up-Modulation of Gq Signaling by LY34 and Gi Signaling by Clozapine in Mouse Frontal Cortex
We next tested the mGluR2 inverse agonist up-modulation of Gq signaling in cortical primary cultures. Stimulation of Gq signaling in neurons is known to elicit a transient increase of intracellular calcium (via an IP3
release from ER) that can be recorded using fluorescent calcium-sensitive dyes (Pichon et al., 2010
). We used the calcium dye fura-2 to monitor changes in free intracellular calcium in response to 5-HT, and 5-HT with the mGluR2 inverse agonist LY34. As predicted, LY34 was able to boost the 5-HT response approximately five times (), while by itself it showed no response. In contrast, co-application of the mGluR2 neutral antagonist eGlu with 5-HT, did not elicit a significant increase in intracellular calcium ().
These data suggest that the effects of inverse agonists that bind 2AR or mGluR2 and boost their heteromeric partner receptor's signaling seen in vitro (see , , above) also occur in cortical neurons in vivo ().
2AR and mGluR2 Are Both Necessary for Antipsychotic-Like Behavior in Mice
We proceeded to explore the involvement of 2AR and mGluR2 in behavioral responses induced by the two antipsychotic drugs LY37 and clozapine and to compare their effects in wild-type and receptor KO mice. We determined the effects of the mGluR2/3 agonist LY37 on the locomotor behavior induced by MK801 in wild-type and 2AR-KO mice (). Non-competitive NMDA receptor antagonists, such as phencyclidine (PCP) and ketamine are used as pharmacological models for schizophrenia in rodents because of their capacity in humans to evoke symptoms resembling those seen in this disease (Morris et al., 2005
; Mouri et al., 2007
; Patil et al., 2007
). The potent and selective non-competitive NMDA receptor antagonist MK801 (dizocilpine) can also elicit ketamine-like symptoms in healthy volunteers (Reimherr et al., 1986
). Activation of mGluR2, but not mGluR3, by LY37 has been shown to reduce the hyperlocomotion response induced by non-competitive NMDA antagonists in mouse models of schizophrenia (Woolley et al., 2008
). We found that the locomotor activity elicited by MK801 in wild-type and 2AR-KO mice was indistinguishable. Remarkably, the MK801-stimulated activity was significantly attenuated by LY37 in wild-type mice, but it was not affected in 2AR-KO mice (). As expected, the effect of LY37 on the MK801-locomotor response was absent in mGluR2-KO mice (Figure S7 B
We next tested the role of mGluR2 in the antipsychotic-like effect induced by the atypical antipsychotic clozapine. Since clozapine binds with high affinity to 2ARs, and with lower affinity to dopamine D2 receptors (Meltzer et al., 1989
), we first established the lowest dose of clozapine that induced an antipsychotic-like effect in mice. Clozapine significantly decreased the MK801-stimulated locomotor activity at doses ranging from 1.5 to 10 mg/kg (Figure S7 A
). The locomotor activity induced by MK801 was similar in wild-type and mGluR2-KO mice. Notably, pre-treatment with 1.5 mg/kg clozapine significantly decreased the MK801-stimulated locomotion in wild-type mice, but not mGluR2-KO mice (). The same dose of clozapine had no effect on 2AR-KO mice when compared to wild-type mice (Figure S7 C
; see also Figure S7 A, B, and C
for modulation of the MK801-induced locomotor activity by 10 mg/kg clozapine in wild-type, 2AR-KO and mGluR2-KO mice). The findings of were also consistent with the absence of antipsychotic-like behavioral effects of methysergide (Figures S7, D and E
). Our data do not exclude the possibility that the absence of antipsychotic-like effect of LY37 in 2AR-KO might also be affected by the lower expression of mGluR2 in 2AR-KO mice (González-Maeso et al., 2008
; Moreno et al., 2011
). Co-injection of LY37 and clozapine (1.5 mg/kg) did not affect the MK801-dependent locomotor response in either mGluR2-KO or 2AR-KO mice (data not shown).
Together, these findings demonstrate that the mGluR2-dependent antipsychotic-like behavioral response of LY37 requires the expression of the 2AR, and that the corresponding 2AR-dependent effect of clozapine requires the expression of the mGluR2.
A Drug Combination Approach To Control Psychotic-like Behavior
We established earlier that an optimal mGluR2/2AR ratio of expression (1 ng of mGluR2 mRNA and 2 ng of 2AR mRNA injected) yielded the largest difference in Gi and Gq signaling (BIr
=1.45) ( and Figure S3 D
). However, injection of either a greater amount (3 ng) or a lesser amount (1ng) of 2AR mRNA than mGluR2 mRNA (1ng) decreased the BI (Figure S3 D
). These findings may model the alterations in 2AR and mGluR2 expression that we have previously shown in postmortem human brain of untreated schizophrenic subjects (González-Maeso et al., 2008
). Recent preclinical findings suggest that co-administration of suboptimal doses of atypical and Glu antipsychotics results in robust therapeutic-like behavioral effects and reduced unwanted side effects (Uslaner et al., 2009
). We proceeded to test whether co-administration of clozapine (targeting 2AR) and LY37 (targeting mGluR2) could produce BI levels close to the BIr
and compensate for alterations in the signaling crosstalk between mGluR2 and 2AR, caused by sub-optimal expression ratios. shows the ΔGi and ΔGq values obtained in response to clozapine, LY37, and LY37 together with clozapine at concentrations of 50 μM. Co-administration of the two drugs increased significantly the BI in both suboptimal cases (left and right panels) compared to the optimal case (middle panel). These results reveal that co-administration of LY37 and clozapine could compensate for the loss in signaling capacity that is likely to result from decreased mGluR2/2AR heteromeric formation since cross signaling is decreased in suboptimal signaling receptor ratios.
Control of BI Through a Drug Combination Approach
Further behavioral experiments showed that administration of either clozapine or LY37 in mGluR2 or 2AR heterozygote mice did not affect the MK801-dependent locomotor response (). However, co-administration of both antipsychotics in mGluR2 or 2AR heterozygotes significantly decreased the MK801-stimulated locomotor activity ().
These results suggest that a combination of mGluR2 dominant agonists with 2AR inverse agonists are likely to synergize to achieve an optimal BI in cases where a suboptimal Gi – Gq signaling balance exists, as is potentially the case suggested by findings in postmortem human brain of schizophrenic subjects (González-Maeso et al., 2008