Glutamate is the major excitatory neurotransmitter in the CNS, and it binds two different type of receptors, the ionotropic glutamate receptors (iGlu), and metabotropic glutamate receptors (mGluRs).208,209 The mGluRs belong to the Class C GPCRs, and they are grouped in three main families, mGluRI, mGluRII, and mGluRII, for a total of eight receptor subtypes (mGluR1-8).210 Among them the mGluR2 belonging to the mGluRII group received particular attention as pharmaceutical target for the treatment of CNS disease.201–
203 mGluR2 is negatively coupled to adenylate cyclase via Gi/0 protein (Figure 21). As discussed in the Orthosteric and Allosteric Binding site section such receptors are characterized by an orthosteric binding site at the VFT domain, and an allosteric binding site at the HTM bundle level (Figure 21). Both orthosteric and allosteric sites represent
druggable targets, especially for the treatment of mental disorders.115,203,211–213 Potent mGluR2 orthosteric agonists were identified,203,214,215 but due to the high sequence homology at the VFT level with the close-related mGluR3 subtype it was difficult to reach subtype selectivity. Only with the use of mGluR2/3 knockout animal models it was possible to identify mGluR2 as the major responsible of the behavioral control.216,217 Based on these assumptions new pharmaceutical strategies devoted their efforts to discover selective mGluR2 allosteric modulators. In this context to reach allosteric subtype selectivity is easier than orthosteric subtype selectivity, because of the low sequence similarity at the allosteric binding site level (see Orthosteric and Allosteric Binding Sites section). As a matter of fact, based on their pharmacological effects it has been possible to identify positive or negative allosteric modulators selective for the mGluR2 (PAM and NAM respectively),115,211–213 and mutagenesis studies for antagonist modulators were reported.213 Finally, it has been also reported that mGluR2 signaling status is strongly affected by the membrane composition, in particular by the cholesterol concentration.184 As discussed in the Serotoninergic Receptor Subtype 2A section, low cholesterol levels were also associated to mental disorders.206,207
Figure 21. Cartoon-snake representation of mGluR2, highlighted in purple the VFT domain, in green the CRD, in blue the HTM, in red a general orthosteric ligand binding at the orthosteric cleft, in yellow the allosteric binding site.
Homo- and Heteromer as Pharmaceutical Targets
As discussed above, the orthosteric binding site of the 5-HT2A receptor represents the target site of several compounds, showing pharmacological activity at CNS level (such as the so called HCs, and NHCs).195,196,205 It is also well known that the 5-HT2A receptor is involved in a large number of physiological processes (such as smooth muscle contraction, platelet aggregation, food intake).188 Recently, it has been reported that 5-HT2A participates to an intricate network of functional crosstalks,142,148–150,166,170–173,218 with intriguing consequences on the mechanism of action of antipsychotic drugs. Finally, the 5-HT2A
receptor represents a well-known example of functional selectivity, for which (i) different agonists (HCs, NHCs) activate different signaling pathways,148,149,195,196,205,219 and (ii) different antagonists induce different effect on the 5-HT2A trafficking and signaling.166,220 In the next sections of this chapter I introduce two GPCR dimers involving the 5-HT2A
receptor, namely 5-HT2A-5-HT2A homomer, and the 5-HT2A-mGluR2 heteromer. Both dimers meet the criteria proposed by Pin et al.,143 and represent key examples in which GPCR allosterism and GPCR functional selectivity concur to define the nature of the intracellular signaling. Furthermore, the 5-HT2A-5-HT2A homomer, and the 5-HT2A -mGluR2 heteromer represent intriguing pharmaceutical targets, since disrupted functional and physical crosstalks of these two GPCR dimers has seen implicated in CNS diseases, such as schizophrenia, depression and anxiety.142,148,149,166
1.8.1 The 5-HT2A-5-HT2A Homomer Complex
In 2009 Brea et al. reported that 5-HT2A homomerized in cells, displaying a dynamic equilibrium between its monomeric and dimeric forms.166 In this work the authors proposed that atypical and typical antipsychotic drugs, acting at the 5-HT2A orthosteric binding site, antagonized the 5-HT2A receptor in a pathway-specific manner.166 The 5-HT2A
receptor activation induces inositol-phosphate (PP) accumulation through a PLC-correlated pathway, and arachidonic acid (AA) release through a PLA2-correlated pathway. 5-HT2A agonists show functional selectivity discriminating between these two
downstream signaling pathways.221 The authors performed co-immunoprecipitation, and FRET experiments, showing that 5-HT2A forms homomers in live cells. Furthermore, the authors were able to show that typical and atypical antipsychotic drugs differentiate themselves by blocking 5-HT2A responses by two distinct mechanisms. On the basis of the aforementioned observations, Brea et al. proposed the Three-State Receptor Dimer Model,166 which hypothesizes that the receptor exists in equilibrium between an inactive dimeric form (R2), and two distinct active states (R2*, and R2**). The model explains the differential functional antagonist profiles by assuming different receptor active conformations for each pathway, for instance the R2* state for the IP induction, and R2** for the AA release (Figure 22). In summary, the authors proposed that 5-HT2A antagonists are characterized by functional selectivity, deactivating GPCR responses in a ligand- and pathway-related manner, thus providing evidences of a functional antagonist-dependent cooperativity for both atypical and typical antipsychotic drugs.
Figure 22. Representation of the Three-State Receptor Dimer Model.
1.8.2 The mGluR2-5-HT2A Heteromer Complex
In 2008 Gonzalez-Maeso et al. reported that the metabotropic glutamate receptor subtype 2 and the serotoninergic receptor subtype 2A physically interact, at pyramidal neurons of prefrontal cortex structures, to form functional heteromer complex.149 The existence of a functional crosstalk between mGluR2 and 5-HT2A receptors have long been hypothesized by different research groups,222–224 but only in 2008 a physical crosstalk was proved.149 Furthermore, the existence of a functional complex between the mGluR2 and 5-HT2A
receptors has been associated with important functional consequences related to the mechanism of action of antipsychotic drugs, for which both receptor subtypes are considered validated targets.200–203 These findings allowed the reconciliation of the serotoninergic and glutamatergic hypothesis of the schizophrenia.200–203 In a further work Moreno et al. showed that the mGluR2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonist (Figure 23a).150 In this context, mGluR2 behaves as an allosteric modulator of the 5-HT2A intracellular signaling pathway. Finally, in 2011 Fribourg et al. showed that mGluR2 and 5-HT2A
receptors are indeed necessary for the pharmacological and behavioral effects induced by both HCs, NHCs, and antipsychotic drugs.148 In this work, the authors investigated the role of the mGluR2-5-HT2A heteromer complex in altering the G protein signaling. The authors measured the influence of dimerization on signaling of mGluR2 through Gi/0
protein, and 5-HT2A receptor through Gq/11. They reported that ligand binding to both mGluR2 and 5-HT2A protomers alters the Gq/11- Gi/0 balance (Figure 23b). Based on their findings, they propose a new metric, named balance index (BI), to describe the balance between the Gi/0 and Gq/11 signaling. This study suggests that both functional and physical mGluR2-5-HT2A crosstalk induce cellular and phenotypic behavior effects different from those produced by mGluR2 and 5-HT2A receptors separately. According to the BI metric, the Gq/11- Gi/0 signaling balance is the dependent variable predicting the psychotic state.
Thus, they proposed that 5-HT2A inverse agonists, and/or mGluR2 strong agonists and/or PAM would be the most effective compounds in restoring the physiological BI balance for the treatment of schizophrenia. The observation that the close related mGluR3 receptor was not able to form heteromer complex with the 5-HT2A receptor149 provided an useful support to investigate which TM domains are involved in the physical interaction between
the mGluR2 and 5-HT2A receptors, so as to grasp the structural bases underlying the mGluR2 and 5-HT2A physical crosstalk the authors studied mGluR2/3 receptor chimera.149 On the basis of that study the authors proposed a TM4-TM5 heteromer interface arrangement (Figure 23a,b).
Taken together, all these observations indicate that mGluR2 behaves as an allosteric modulator of the 5-HT2A receptor through direct interaction at the HTM bundle, selecting particular conformational states of the serotoninergic receptor depending on the mGluR2-bound ligand (agonist, antagonist or allosteric modulators).142,148,149 The observations are also in agreement with the functional selectivity theory.46,136,139,140
The experimental evidences about the existence of a mGluR2/5-HT2A heteromer complex142,148,149 and its tissues specificity (pyramidal neurons of prefrontal cortex structures) provide a new and intriguing pharmaceutical target for the treatment of schizophrenia.142,148,149,219,225 As a consequence, compounds targeting the specific mGluR2-5-HT2A heteromer complex, and restoring a physiological BI are likely to have improved specificity and reduced side effects.
Figure 23. Schematic Representation of the mGluR2-5-HT2A cross-talk. (A) Different effects exerted by HCs in mGluR2 knockout mice (no psychotic effects), and in WT mice (psychotic effects). (B) Putative sites of action of antipsychotic and psychotic compounds, affecting the BI metric.