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  • Although the overall structures of mGlu and mGlu VFTs are

    2022-05-21

    Although the overall structures of mGlu2 and mGlu3 VFTs are highly similar, two major differences can be observed: (i) the number of functional Cl− binding sites, one in mGlu2 versus two in mGlu3 receptors and (ii) the loop 7 in lobe 2 is longer in mGlu3 than in the mGlu2 receptor. These two particularities of the mGlu3 receptor contribute to the building-up of a unique “Cl− lock” that greatly stabilizes the glutamate-induced closed conformation of its VFT. Indeed, as compared to mGlu2, the presence of Cl− in site 2 in mGlu3 receptor is coordinating a network of interactions between the two lobes, involving notably the twisted loop 7, that is tightening the closed VFT. As a result, the atypical high-basal activity of the mGlu3 receptor is not observed in the cognate mGlu2 receptor in which the Cl− lock is absent. The residue S152 seems to play a crucial role in Cl− binding and bridging between the lobes. This amino PIK-75 was previously reported to be involved in mGlu3 Ca2+-based basal activity (Kubo et al., 1998; Vafabakhsh et al., 2015), but no Ca2+ ion was found in crystal structures at this location. Regarding literature, Ca2+ seems to play a role in mGlu3 receptor activity, but its binding(s) site(s) as well as its contribution to the receptor's basal activity remains unclear. In contrast, in the mGlu2 receptor, the corresponding serine residue is mutated into an aspartate (D146), which disrupts Cl− binding at site 2, explaining divergence with mGlu3 receptors towards both Cl− sensitivity and basal activity. Prior to this study, D146 has been proposed to disrupt the cation-pi interaction and also the Cl− binding site 1, by engendering a flip of R271 (so called “arginine flip”), thus postulating there would be no Cl− binding sites in mGlu2 (DiRaddo et al., 2015). However, in recently released mGlu2 VFT crystal structure (Monn et al., 2015b), the cation-pi interaction is intact and only one Cl− ion is found in site 1, which is consistent with our previous study and the present data (Tora et al., 2015). In addition, it is worth to mention that another Cl− ion is localized near the cation-pi in this structure, but at a different site from site 2, as it is found in only one protomer and therefore may be a crystallization artefact. Additional inter-lobe connections may also participate to the closed VFT stabilization, such as R249 (lobe 2, loop 7), which can bind to G51 (lobe 1, loop 1) in mGlu3 receptors because of the unfolding of the α-helix 7 and a new conformation of loop 7 (Fig. 5C and D right panels). The H-bond between R249 and G51 carbonyl is weaker than the salt bridge with D102 that occurs in mGlu2 (Fig. 5C and D left panels), nevertheless this loss of stability is compensated by new interactions at the dimer interface between loop 7 and helix 6 of the other monomer (See Fig. 6 and Supplemental Fig. 5). This interaction network could possibly explain why the two mGlu3 VFT monomers are closer than those of mGlu2. One can speculate that this proximity may further increase the stability of the active dimer resulting in the basal activity of the receptor at low glutamate concentrations. However, further studies will be necessary to determine more precisely the dimer interface and its involvement in receptor activation. The specific pathophysiological roles of the mGlu3 receptor are still poorly understood. Until recently, these studies have been hampered by the lack of selective tools discriminating between the cognate mGlu2 and mGlu3 receptors. Indeed, the orthosteric binding sites of these two receptors are 100% identical, making the identification of selective orthosteric ligands highly challenging. Thus, the first ligands available were selective for the group II mGluRs over other groups, but failed to discriminate between mGlu2 and mGlu3 receptors (Celanire et al., 2015). Still, mGlu2/3 ligands have been very helpful to highlight the involvement and the therapeutic potential of these receptors in different CNS disorders, notably in anxiety, schizophrenia, addiction, depression and chronic pain. Recently, several selective mGlu3 negative allosteric modulators (Engers et al., 2015) or orthosteric ligands (Monn et al., 2015b, 2018) have been identified. The selectivity of these newly discovered orthosteric ligands lies behind their interaction with amino acids residing at the periphery of the glutamate binding site of the mGlu3 receptor (Monn et al., 2018). Indeed the crystal structure of LY2794193, a selective mGlu3 agonist, bound to mGlu3 VFT (PDB ID 6B7H) reveals that its methoxyphenyl substituent pushes away R277 but does not prevent the stabilization of the active dimer with the curled loop7 (Supplemental Fig. 7) (Monn et al., 2018). On the other hand, a similar perturbation of R271 in mGlu2 decreases significantly the receptor activation. Thus, it appears that targeting the Cl− binding sites of mGlu3 receptor provide an interesting way to overcome the difficulty of designing selective orthosteric drugs. Interestingly, this approach is also valid for other mGluRs. For example, the first mGlu4 receptor selective orthosteric agonist named LSP4-2022 is a dualsteric/bitopic ligand targeting both the glutamate- and the Cl− binding pockets of the mGlu4 receptor (Goudet et al., 2012; Selvam et al., 2018). Hopefully, new selective ligands will help to better define the particular roles of mGlu3 receptors.