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. 2011 Nov 23;147(5):1011-23.
doi: 10.1016/j.cell.2011.09.055.

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs

Miguel Fribourg  1 , José L Moreno, Terrell Holloway, Davide Provasi, Lia Baki, Rahul Mahajan, Gyu Park, Scott K Adney, Candice Hatcher, José M Eltit, Jeffrey D Ruta, Laura Albizu, Zheng Li, Adrienne Umali, Jihyun Shim, Alexandre Fabiato, Alexander D MacKerell Jr, Vladimir Brezina, Stuart C Sealfon, Marta Filizola, Javier González-Maeso, Diomedes E Logothetis
Affiliations

Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs

Miguel Fribourg et al. Cell. .

Abstract

Atypical antipsychotic drugs, such as clozapine and risperidone, have a high affinity for the serotonin 5-HT(2A) G protein-coupled receptor (GPCR), the 2AR, which signals via a G(q) heterotrimeric G protein. The closely related non-antipsychotic drugs, such as ritanserin and methysergide, also block 2AR function, but they lack comparable neuropsychological effects. Why some but not all 2AR inhibitors exhibit antipsychotic properties remains unresolved. We now show that a heteromeric complex between the 2AR and the G(i)-linked GPCR, metabotropic glutamate 2 receptor (mGluR2), integrates ligand input, modulating signaling output and behavioral changes. Serotonergic and glutamatergic drugs bind the mGluR2/2AR heterocomplex, which then balances Gi- and Gq-dependent signaling. We find that the mGluR2/2AR-mediated changes in Gi and Gq activity predict the psychoactive behavioral effects of a variety of pharmocological compounds. These observations provide mechanistic insight into antipsychotic action that may advance therapeutic strategies for disorders including schizophrenia and dementia.

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Figures

Figure 1
Figure 1. Heteromeric Assembly of 2AR and mGluR2 enhances Glu-induced Gi Signaling and Reduces 5-HT-induced Gq Signaling
2AR and mGlu2 co-localize and form a receptor complex in mouse frontal cortex. (A) Representative micrographs showing co-expression of endogenous 2AR (red) and mGluR2 (green) in mouse frontal cortex (left panels) and mouse cortical primary neurons (right panels). (Scale bar 25 μm). (See also Figure S1 E) (B) Mouse frontal cortex membrane preparations were immunoprecipitated (IP) with anti-2AR antibody. Immunoprecipitates were analyzed by western blot (WB) with anti-mGluR2 antibody (lower blot). Mouse frontal cortex membrane preparations were also directly analyzed by WB with anti- 2AR antibody (upper blot) or anti-mGluR2 antibody (middle blot). 2AR-KO and mGluR2-KO mouse frontal cortex tissue samples were processed identically and used as negative controls. Frontal cortex tissue samples from 2AR-KO and mGluR2-KO mice were also homogenized together (mixed) and processed identically for immunoprecipitation and WB. (C) (Left) Representative barium-sensitive traces of IRK3 currents obtained in response to 1 μM 5-HT in oocytes expressing 2AR alone, mGluR2 and 2AR together, or mGluR2 alone. (Right) Representative barium-sensitive traces of GIRK4* currents obtained in response to 1 μM Glu in oocytes expressing mGluR2 alone, mGluR2 and 2AR together, or 2AR alone. Barium (Ba) inhibited IRK3 and GIRK4* currents and allowed for subtraction of IRK3 and GIRK4*-independent currents. For illustrative purposes, traces with similar basal currents were chosen. (D) Summary bar graphs of Gq activity measured as IRK3 current inhibition (mean ± SEM) following stimulation with 5-HT and (E), of Gi activity measured as GIRK4* current activation (mean ± SEM) following stimulation with Glu. IRK3 current inhibition was measured relative to basal currents and was normalized relative to that obtained by stimulating 2AR alone with 5-HT (100% or 1). GIRK4* current activation was measured relative to the basal currents and was normalized relative to that obtained by stimulating mGluR2 alone with Glu (100% or 1) (F) Calculation of the balance index (BI) as the difference of the increase in Gi-signaling in response to Glu from the mGluR2 homomeric level (ΔGi) and the decrease of Gq-signaling in response to 5-HT from the 2AR homomeric level (ΔGq). A reference BI (Bir=1.45) was calculated for the mGluR2/2AR complex in response to 1 μM Glu and 1 μM 5-HT using mean values (** p<0.01, *** p < 0.001). See also Figures S1, S2, and S3.
Figure 2
Figure 2. Drugs that Target 2AR: Integrative Effects on Gi and Gq Signaling
(A) Summary bar graphs of Gi activity (mean ± SEM) measured in oocytes expressing mGluR2/2AR following stimulation with 1μM 5-HT alone, or together with 10μM methysergide, 10μM DOI, or 10μM clozapine. Gq activity was normalized relative to that obtained by stimulation of 2AR alone with 5-HT (100% or 1, dotted line). (B) Summary bar graphs of Gi activity (top) and Gq activity (bottom) (mean±SEM) measured in oocytes expressing mGluR2/2AR following stimulation with 1 μM Glu alone, or together with 10μM methysergide, 10μM DOI, or 10μM clozapine. Gi and Gq activity were normalized relative to the response to Glu and 5-HT respectively (100% or 1, dotted line). (C) ΔGi referenced to the homomeric mGluR2 response to 1 μM Glu and ΔGq referenced to the homomeric 2AR response to 1 μM 5-HT together with 10 μM methysergide, 10 μM DOI, or 10 μM clozapine (** p<0.01, *** p<0.01, n.s. not significant) (D) Metadynamics-based mechanistic interpretation of functional crosstalk between 2AR and mGluR2. (Top) Activation profile of 2AR in the presence of different ligands. Free-energy of the 2AR bound to the inverse agonist clozapine (green), the neutral antagonist methysergide (purple), and the dominant agonist DOI (orange), as a function of the position along the path connecting the inactive (s=1) to the active (s=8) states. (Bottom) Activation profile of mGluR2 in the presence of the different ligand-specific 2AR conformations. The three lines correspond to the activation free-energy profile of mGluR2 in dimeric complex through a TM4-TM4 interface with 2AR bound to the inverse agonist clozapine (green line), a TM4,5-TM4,5 interface with 2AR bound to the neutral antagonist methysergide (purple), and a TM4,5-TM4,5 interface with 2AR bound to the dominant agonist DOI (orange line). The most energetically stable states are indicated by a star, and the chemical structures of the three drugs are also shown.
Figure 3
Figure 3. Drugs that Target mGluR2: Integrative Effects on Gi and Gq Signaling
(A) Summary bar graphs of Gi activity (mean ± SEM) measured in oocytes expressing mGluR2/2AR following stimulation with 1μM Glu alone, or together with 10μM eGlu, 10μM LY37, or 10μM LY34. Gi activity was normalized relative to that obtained by stimulation mGluR2 alone with 5-HT (100% or 1, dotted line) (B) Summary bar graphs of Gq activity (red) and Gi activity (blue) (mean± SEM) measured in oocytes expressing mGluR2/2AR following stimulation with 1μM 5-HT alone, or together with 10μM eGlu, 10μM LY37, or 10μM LY34. Gq and Gi activities were normalized relative to the response to 5-HT and Glu respectively (100% or 1, dotted line). (C) ΔGi referenced to the homomeric mGluR2 response to 1 μM Glu and ΔGq referenced to the homomeric 2AR response to 1 μM 5-HT together with 10 μM methysergide, 10 μM DOI, or 10 μM clozapine (** p<0.01, *** p<0.01, n.s. not significant) (see also Figures S4 and S5)
Figure 4
Figure 4. Use of BI Index to Classify Anti-/Pro-psychotic Propensity of Drugs Targeting the mGluR2/2AR Complex
Correlation maps between the balance index (BI) and percentage of Gi-Gq balance loss or recovery for different drugs assuming a fractional occupancy of the heteromer by the drug of 0.5 (see Experimental Procedures section). BIs were calculated for 10 μM (BI10) (A) and 50 μM (BI50) (B) concentrations of the drugs together with 1 μM Glu and 1 μM 5-HT and placed accordingly in the horizontal axis. BIr=1.45 corresponds to zero. Effects on the difference between Gi and Gq signaling is shown for drugs with known antipsychotic effects like clozapine, risperidone and LY37, for ritanserin, an antidepressant, for neutral antagonists methysergide and eGlu, for the psychedelic DOI, and for the propsychotic LY34 (see also Table S1).
Figure 5
Figure 5. Up-Modulation of Gq Signaling by LY34 and Gi Signaling by Clozapine in Mouse Frontal Cortex
DCG IV-stimulated [35S]GTPγS binding in mouse frontal cortex membranes followed by immunoprecipitation with anti-Gαi antibody in the presence or clozapine (A), methysergide (B), or vehicle. Activation of Gi was accomplished by DCG IV (10 μM). Data represent mean ± SEM (*p<0.05, **p<0.01, ***p<0.001, n.s. not significant) (see also Figure S6C). (C) Representative traces of 5-HT-evoked elevation of intracellular calcium in mouse frontal cortex neurons as detected by ratiometric Fura-2 measurements. Measurements were obtained with 200 μM LY34 alone, 100 μM 5-HT (5-HT) alone, 100 μM 5-HT together with 200 μM eGlu (mGluR2 neutral antagonist), and 100 μM 5-HT together with 200 μM LY34 (mGluR2 inverse agonist). (D) Bar graph summary of measured Fura-2 R340/380 change. Traces were normalized to the basal level, the steady-state fluorescence before perfusion of drugs. Data are mean ± SEM (* p<0.05, *** p<0.001, n.s. not significant). (E) Summary bar graphs (mean± SEM) of the total MK801-induced locomotion as a summation of horizontal activity from t = 30 min to t =120 min. Injection time was at t = 0 min. Wild-type (WT, left) and 2AR-KO (right) mice were administered LY37 (5 mg/kg), or vehicle followed by MK801 (0.5 mg/kg) or vehicle (N = 5 − 6). (F) (Left) Wild-type mice were administered clozapine (1.5 mg/kg) or vehicle, followed by MK801 (0.5 mg/kg) or vehicle. (Right) mGluR2-KO mice were administered clozapine (1.5 mg/Kg) or vehicle (* p<0.05, ** p<0.01, *** p<0.001, n.s., not significant).
Figure 6
Figure 6. Control of BI Through a Drug Combination Approach
(A) BI calculations at 50 μM ligand concentrations for BI. ΔGi referenced to the homomeric mGluR2 (1 ng of mRNA) response to 1 μM Glu and ΔGq referenced to the homomeric 2AR (2 ng of RNA) response to 1 μM 5-HT and 1μM Glu. Responses to a concentration of 50uM clozapine, LY37, or LY37 together with clozapine were measured in oocytes injected with 1 ng mGluR2 mRNA and 1 ng (left), 2 ng (center) and 3 ng (right) or 2AR mRNA respectively. (B) Summary bar graphs (mean± SEM) of the total MK801-induced locomotion as a summation of horizontal activity from t = 30 min to t =120 min. Injection time was at t = 0 min. mGluR2 heterozygotes (mGluR2 +/-) (left) and 2AR heterozygotes (right). Mice were administered vehicle, clozapine (1.5 mg/kg), LY37 (5 mg/kg), or both LY37 and clozapine, followed by MK801 (0.5 mg/kg) (N = 5 − 6). (* p<0.05, n.s. not significant).
Figure 7
Figure 7. Gi-Gq Balance Model of the Mechanism of Action of Antipsychotic and Psychedelic Drugs through the mGluR2/2AR Complex
Formation of the receptor complex establishes an optimal Gi-Gq balance in response to Glu and 5-HT (increase in Gi, decrease in Gq). A. Psychedelics (LY34 and DOI) invert the balance (strong Gi decrease, strong Gq increase). B. Disruption of the optimal balance in psychotic states (decrease in Gi, increase in Gq) can be compensated for by antipsychotics (LY37, clozapine, and risperidone) that recover the Gi-Gq balance (increasing Gi and decreasing Gq).
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