Membrane trafficking processes regulate G protein–coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT2A receptors (5-HT2ARs) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments, and coexpression of 5-HT2AR with mGluR2 increased the intracellular distribution of the otherwise plasma membrane–localized mGluR2. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT2AR–mGluR2 heterocomplex alone, or together. In addition, overnight pharmacological 5-HT2AR blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT2AR and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT2AR–mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT2AR–dependent effects on mGluR2 subcellular distribution. The expression of 5-HT2AR also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

Highlights d Photoactivatable unnatural amino acids inform the structural interface of 5-HT 2A R-mGluR2 d TAG mGluR2 constructs were co-expressed with 5-HT 2A R for photo-crosslinking d UV-induced crosslinking only in cells co-expressing 5-HT 2A R and mGluR2-TAG 4.44 d 5-HT 2A R interacts with mGluR2 via the intracellular end of mGluR2's TM4

The serotonergic and glutamatergic neurotransmitter systems have both been implicated in the pathophysiology of schizophrenia, and there are multiple lines of evidence to demonstrate that they can interact in a functionally relevant manner. Particularly, it has been demonstrated that serotonin (5hydroxytryptamine) 2A (5-HT 2A ) receptors and metabotropic glutamate type 2 (mGlu2) receptors can assemble into a functional heteromeric complex and modulate each other's function. This heteromeric complex has been implicated in the mechanism of action of hallucinogens as well as antipsychotic agents, and its role has been demonstrated in both in vitro and in vivo systems. Additionally, the difference in the changes in G i/o and G q/11 protein activity when a ligand binds to the heteromeric complex can be used as an index to predict the pro-or antipsychotic properties of an agent. Signaling via the heteromer is dysregulated in postmortem human brain samples of schizophrenia subjects, which may be linked to altered cortical functions. Alternative routes for the functional crosstalk between mGlu2 and 5-HT 2A receptors include synaptic and epigenetic mechanisms. This Review highlights the advances made over the past few years in elucidating the structural and functional mechanisms underlying crosstalk between 5-HT 2A and mGlu2 receptors in preclinical models of schizophrenia.

Pharmacophore models for 5-HT2A receptor antagonists consist of two aromatic/hydrophobic regions at a given distance from a basic amine. We have previously shown that both aromatic/hydrophobic moieties are unnecessary for binding or antagonist action. Here, we deconstructed the 5-HT2A receptor antagonist/serotonin-dopamine antipsychotic agent risperidone into smaller structural segments that were tested for 5-HT2A receptor affinity and function. We show, again, that the entire risperidone structure is unnecessary for retention of affinity or antagonist action. Replacement of the 6-fluoro-3-(4-piperidinyl)-1,2-benz­[d]­isoxazole moiety by isosteric tryptamines resulted in retention of affinity and antagonist action. Additionally, 3-(4-piperidinyl)-1,2-benz­[d]­isoxazole (10), which represents less than half the structural features of risperidone, retains both affinity and antagonist actions. 5-HT2A receptor homology modeling/docking studies suggest that 10 binds in a manner similar to risperidone and that there is a large cavity to accept various N4-substituted analogues of 10 such as risperidone and related agents. Alterations of this “extended” moiety improve receptor binding and functional potency. We propose a new risperidone-based pharmacophore for 5-HT2A receptor antagonist action.

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Known classic psychedelic serotonin 2A receptor (5-HT 2A R) agonists retain a tryptamine or phenethylamine at their structural core. However, activation of the 5-HT 2A R can be elicited by drugs lacking these fundamental scaffolds. Such is the case of the Nsubstituted piperazine quipazine. Here, we show that quipazine bound to and activated 5-HT 2A R as measured by [ 3 H]ketanserin binding displacement, Ca 2+ mobilization, and accumulation of the canonical G q/11 signaling pathway mediator inositol monophosphate (IP 1 ) in vitro and in vivo. Additionally, quipazine induced via 5-HT 2A R an expression pattern of immediate early genes (IEG) in the mouse somatosensory cortex consistent with that of classic psychedelics. In the mouse head-twitch response (HTR) model of psychedelic-like action, quipazine produced a lasting effect with high maximal responses during the peak effect that were successfully blocked by the 5-HT 2A R antagonist M100907 and absent in 5-HT 2A R knockout (KO) mice. The acute effect of quipazine on HTR appeared to be unaffected by serotonin depletion and was independent from 5-HT 3 R activation. Interestingly, some of these features were shared by its deaza bioisostere 2-NP, but not by other closely related piperazine congeners, suggesting that quipazine might represent a distinct cluster within the family of psychoactive piperazines. Together, our results add to the mounting evidence that quipazine's profile matches that of classic psychedelic 5-HT 2A R agonists at cellular signaling and behavioral pharmacology levels.

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