G protein-specific mechanisms in the serotonin 5-HT2A receptor regulate psychosis-related effects and memory deficits

Abstract

G protein-coupled receptors (GPCRs) are sophisticated signaling machines able to simultaneously elicit multiple intracellular signaling pathways upon activation. Complete (in)activation of all pathways can be counterproductive for specific therapeutic applications. This is the case for the serotonin 2 A receptor (5-HT_2AR), a prominent target for the treatment of schizophrenia. In this study, we elucidate the complex 5-HT_2AR coupling signature in response to different signaling probes, and its physiological consequences by combining computational modeling, in vitro and in vivo experiments with human postmortem brain studies. We show how chemical modification of the endogenous agonist serotonin dramatically impacts the G protein coupling profile of the 5-HT_2AR and the associated behavioral responses. Importantly, among these responses, we demonstrate that memory deficits are regulated by G_αq protein activation, whereas psychosis-related behavior is modulated through G_αi1 stimulation. These findings emphasize the complexity of GPCR pharmacology and physiology and open the path to designing improved therapeutics for the treatment of stchizophrenia.

Fig. 1. Structural derivatives of the endogenous 5-HT_2AR agonist serotonin (5-HT).

Ligand binding affinities (pKi) are indicated for Nitro-I, Met-I, OTV1 and OTV2 obtained in [³H]ketanserin competition binding experiments in CHO cells (n = 3). The data represent the mean ± SD (see methods section).

Fig. 2. G protein coupling and β-arrestin recruitment for Nitro-I, Met-I, OTV1 and OTV2 in living cells.

A Schematic representation of enhanced bystander BRET (ebBRET) biosensors used to measure G protein activation and β-arrestin (βarr) recruitment in HEK-293 cells, created with BioRender. B–G Dose response curves depicting the activity of Nitro-I, Met-I, OTV1 and OTV2 at the different G protein signaling pathways and for the recruitment of βarr 1 and 2. The ligand-promoted BRET (ΔBRET) was further normalized with respect to the response of 5-HT (mean ± SEM; n = 3). H Bias factor for the G_αq, G_αi family and βarr1 and 2. The operational model was used for bias calculations (see method section in supplementary information).

Fig. 3. Antibody-capture [³⁵S]GTPγS binding scintillation proximity assay (SPA).

A Schematic representation of the SPA methodology, created using BioRender. SPA allows the determination of the activation level of different G_α subunit subtypes present in postmortem brain tissue thanks to the selective immunoprecipitation of each of them and their coupling to protein A-coated polyvinyltoluene SPA beads. Modulation of specific [³⁵S]GTPγS binding to G_αi1, G_αi2, G_αi3 and G_αq/11 proteins by 10 μM Nitro-I (B), Met-I (C), Otava 3575001 (OTV1) (D) and Otava 3736689 (OTV2) (E) in human prefrontal cortex both in the absence (colored bars) and in the presence (white bars) of the 5-HT_2AR antagonist MDL-11,939. Basal values of specific [³⁵S]GTPγS binding to the different G proteins are expressed as 100% and stimulatory/inhibitory effects on the respective basal are shown. Individual dots represent different assays 4–6 carried out for each subunit/condition performed in duplicate or triplicate. White dots represent data for the drug-alone condition, and black dots represent data for the drug + MDL condition (B–E). ^#p < 0.05 vs 100%; ^$p < 0.05 vs incubation in the presence of 1 µM MDL-11,939 (t-test). Modulation of specific [³⁵S]GTPγS binding to G_αi1-, G_αi2-, G_αi3- and G_αq/11 proteins by 10 _μM Nitro-I (F), Met-I (G), OTV1 (H) and OTV2 (I) in brain cortex tissue from WT (colored bars) and 5-HT_2AR KO mice (white bars). ^#p < 0.05 vs 100%; ^$p < 0^.05 WT vs 5-HT_2AR KO (t-test)^. Basal values of specific [³⁵S]GTPγS binding to the different G_α proteins are expressed as 100%, and stimulatory/inhibitory effects are expressed as % of the respective basal activity. Individual dots represent the different assays (3–5) for each subunit performed in duplicate or triplicate. White dots represent data for WT mice and black dots represent data for KO mice (F–I).

Fig. 4. Head twitch response (HTR) and long-term novel object recognition (NOR).

HTR in wild-type (WT) and 5-HT_2AR knockout (KO) mice following ICV administration of (A) Nitro-I, (B) Met-I, (C) OTV1, and (D) OTV2, or vehicle (VEH). The increase in HTR induced by Nitro-I and OTV2 at the dose of 0.05 µg/µl was absent in KO mice. E ICV administration of YM-254890 (16 µM) did not modulate the increase in HTR induced by OTV2 at the dose of 0.05 µg/µl. F ICV administration of specific antisense oligonucleotides (ODN), G_αi1-ODN, G_αi3-ODN, but not control random oligonucleotides (RDN) blocked HTR induced by OTV2 at the dose of 0.05 µg/µl. NOR memory test in WT and 5-HT_2AR KO mice following ICV administration of (G) Nitro-I, (H) Met-I, (I) OTV1, and (J) OTV2 or vehicle (VEH). In WT mice, Met-I and OTV1 induced memory deficits at the dose of 0.025 and 0.05 µg/µl, and OTV2 was effective only at the dose of 0.025 µg/µl. These effects were abrogated in KO mice. Nitro-I did not trigger memory deficits at any of the doses tested. K YM-254890 (16 µM) abrogated the memory deficits induced by OTV2 at the dose of 0.025 µg/µl. L ICV administration of G_αi1-ODN, G_αi3-ODN, or the control RDN sequence did not modulate the memory deficits induced by OTV2 at the dose of 0.025 µg/µl. The data represent mean ± SEM. The number of mice used in the experiments (n) corresponds to the individual points in the graph. **p < 0.01, ***p < 0.001, and ^###p < 0.001 (main effect of treatment). Two-way ANOVAs followed by Fisher’s post-hoc test.

Fig. 5. In silico modeling of the ligand-5-HT_2AR interactions for Nitro-I, Met-I, OTV1, and OTV2.

A 3D model of the ligand-5-HT_2AR interactions for Nitro-I, Met-I, OTV1, and OTV2. The ECL2 and included residues are highlighted in green. B The contact frequency (%) of residues in the transmembrane region of the receptor for each ligand. Contact frequencies have been computed for the main conformational cluster (see Methods) extracted from three replicates of 500 ns MD simulations (3 × 500 ns). C Accumulated contact frequency of residues in the ECL2 for each ligand computed for the main cluster extracted from three replicates of 500 ns MD simulations (3 × 500 ns).