Metabotropic Glutamate Receptors Notes

Metabotropic Glutamate Receptors: From the Workbench to the Bedside

This review discusses the discovery, structure, activation mechanisms, and clinical development of metabotropic glutamate receptors (mGluRs).

Historical Background

mGluRs were discovered in the mid-1980s.
They were initially described as glutamate receptors coupled to polyphosphoinositide hydrolysis.
Almost 6500 articles have been published since their discovery.
Subtype-selective mGlu receptor ligands are under clinical development for treating various disorders.
Prof. Erminio Costa challenged the belief that glutamate could only activate ionotropic receptors.
Sladeczek et al. (1985) demonstrated that quisqualate and glutamate stimulated inositol phosphate formation in cultured striatal neurons.
Ibotenic acid was found to stimulate inositol phosphate formation in hippocampal slices (Nicoletti et al., 1986a,b).
Glutamate was able to stimulate polyphosphoinositide (PI) hydrolysis to a great extent (as much as 20 fold in some experiments) in slices from 7- to 9-day old rats (Nicoletti et al., 1986a).
L-2-amino-4-phosphonobutanoate (L-AP4) and L-serine-O-phosphate (L-SOP) reduced excitatory synaptic transmission at mossy fibre/CA3 pyramidal cell synapses of the guinea pig hippocampus (Cotman et al., 1986).
In 1987, Prof. Sugyiama and his colleagues introduced the terminology of “metabotropic glutamate receptors” (Sugiyama et al., 1987).
The cloning of the first mGlu subtype (mGluR1) in Prof. Nakanishi's lab (Masu et al., 1991) was a milestone for the field.
The eight mGlu receptor subtypes are divided into three groups: Group I (mGlu1 and mGlu5), Group II (mGlu2 and mGlu3), and Group III (mGlu4, mGlu6, mGlu7, and mGlu8).
mGlu1 and mGlu5 receptors are coupled to Gq/G11, whereas other subtypes are coupled to Gi/Go.
Quisqualate and ibotenic acid were the first molecules reported as active at mGlu receptors coupled to PI hydrolysis.
Trans-1-amino-cyclopentanedicarboxylic acid (trans-ACPD) could activate PI hydrolysis in brain slices with no effect at ionotropic glutamate receptors (Schoepp et al., 1991).
One of the isomers of trans-ACPD, 1S,3R-ACPD, activates both group-I and group-II mGlu receptors (reviewed by Schoepp et al., 1999).
L-AP4, L-SOP and L-2-amino-3-phosphonopropionic acid (L-AP3) were described as antagonists of mGlu receptors coupled to PI hydrolysis in brain slices (Nicoletti et al., 1986a,b,c; Schoepp and Johnson, 1989).
Jeff Watkins and his associates introduced phenylglycine derivatives as pharmacological tools (Birse et al., 1993).
R,S-α-methyl-4-carboxyphenylglycine (MCPG) has been widely used as a non-subtype-selective mGlu receptor antagonist (Eaton et al., 1993).
(2S,3S,4S)-2-(Carboxycyclopropyl)glycine (L-CCG-I) and (2S, 1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) have been used as potent mGlu2/3 receptor agonists.
(1S,2S,5R,6S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740) and (1R,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) show nanomolar potency as mGlu2/3 receptor agonists.
Positive and negative allosteric modulators (PAMs and NAMs) of individual mGlu receptor subtypes are also available.
No endogenous ligands (“endocoids”) of these modulatory sites have been identified.
After 25 years from the discovery of mGlu receptors, subtype-selective ligands are under clinical development.

Structure and Activation Mechanism of mGluRs

mGlu receptors belong to class CG-protein coupled receptors (GPCRs).
These receptors have a large extracellular domain made of a Venus Fly Trap (VFT) domain.
The VFT domain is similar to the periplasmic bacterial leucine/isoleucine/valine binding protein (LIVBP).
The VFT domain is linked to the first transmembrane (TM) domain via a cysteine-rich domain (CRD).
The glutamate binding domain is structurally similar to LIVBP-like proteins.
The VFT domain is composed of two lobes, each made of α-helices around a large β-sheet.
The glutamate binding site is located in the cleft between the two lobes.
Structural analysis revealed 5 residues that directly contact the amino acid moiety and are conserved in a variety of amino acid binding LIVBP-like proteins.
The CRD is made of two subdomains, each composed of 4 β-strands stabilized by 2 disulfide bridges.
The 7-TM domain of mGlu receptors shows low sequence similarity with the 7-TM domain of other GPCRs.
The 7-TM domains of mGlu receptors and other GPCRs share a similar general fold, with a relatively central helix 3, and a possible amphipathic helix 8.
The 7-TM domain alone can be directly activated by ligands known as positive allosteric modulators (PAMs).
An ionic interaction between residues at the intracellular side of helices 3 and 6 is important to maintain the receptor in an inactive conformation.
Intracellular loops 2 and 3, as well as helix 8, are important in G-protein coupling.
mGlu receptors form dimers stabilized by an intersubunit disulfide bond.
mGlu receptors form dimers but not larger complexes.
mGlu receptor subtypes can form heterodimers within the same group (e.g., mGlu1 with mGlu5, mGlu2 with mGlu3).
Group-II and group-III mGlu receptors can form intergroup heterodimers (e.g., mGlu2 with mGlu4).
No heterodimers can be formed between group-I and group-II/III mGlu receptors.
Different mGlu receptor subunits have been colocalized in specific subdomains in neurons.
A single agonist per dimer is sufficient for receptor activation, although two agonist molecules lead to a 3-fold higher coupling efficiency.
Ligand binding stabilizes the VFT domain in a closed conformation.
Structures with bound agonists are in the closed form, while antagonist-bound structures correspond to open VFTs.
Removing the steric or ionic hindrance that prevents VFT closure was found sufficient to convert group-III mGlu receptor antagonists into full agonists.
The fully activated state of the related GABAB receptor was obtained by inserting a disulfide bond expected to lock the VFT in its closed state (Kniazeff et al., 2004b).
An important reorientation of the VFTs in the dimer was observed when at least one VFT was in a closed state.
It is not possible to have both 7-TM domains of a dimeric mGlu receptor to be in close proximity according to the solved structure of the mGlu3 receptor extracellular domain composed of both the VFT and the CRD.
A relative movement between the mGlu receptor subunits during activation is supported by FRET measurements between their intracellular sides.
Agonist binding leads to the activation of only one 7TM within the dimer.

Detailed Description of mGlu Receptor Subtypes

mGlu1 Receptor

Gene name: GRM1 (human); Grm1 (rat, mouse).
Accession numbers: NP000829 (human); NP058707 (rat); NP_058672 (mouse).
Chromosomal location: 6q24 (human); 1p13 (rat); 10A2 (mouse).

Splice Variants

mGlu1α (mGlu1a): Longest isoform (1199 and 1194 amino acids in rodents and humans, respectively).
mGlu1β1 (mGlu1b): 906 amino acids.
mGlu1β2 (mGlu1f): 906 amino acids.
mGlu1γ (mGlu1d): 908 amino acids.
mGlu1δ (mGlu1E55): 321 amino acids (possible secreted protein).

Receptor Signalling and Interacting Proteins

Primarily coupled to Gq/G11 proteins.
Stimulates phospholipase Cβ (PLCβ), leading to the formation of inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) and diacylglycerol (DAG).
Ins-1,4,5-P3 releases Ca2+ from intracellular stores, whereas DAG activates protein kinase C.
Stimulation of cAMP formation, arachidonic acid release, the mitogen-activated protein kinase (MAPK) pathway, and L-type voltage-sensitive Ca2+ channels has also been reported.
Negatively modulates K+ channels, including M-type voltage-gated K+ channels and the “tandem-pore” TASK and TREK K+ channels.
Negatively modulates voltage-sensitive Ca2+ channels via mechanisms that involve pertussis toxin (PTX)-sensitive and PTX-insensitive G proteins and are regulated by Homer proteins.
Different isoforms of G-protein coupled receptor kinases (GRKs), including GRK2, GRK4, and GRK5, mediate homologous desensitization of mGlu1 receptors.
The Regulator of G-protein Signalling, RGS-4, inhibits mGlu1 receptor signalling.
Homer proteins bind to a proline-rich motif in the C-terminal tail of mGlu1a and mGlu5 receptors.
Homer proteins form a linking bridge between mGlu1α receptors and other proteins that are involved in receptor signalling.
mGlu1α receptors can also activate the phosphatidylinoisol-3-kinase (PtdIns-3-K) pathway via the interaction with Homer protein, and the PtdIns-3-K enhancer, PIKE-L.
These interactions are disrupted by the short Homer isoform, Homer1a.
mGlu1α receptors can also associate with tamaline, the cytoskeletal protein, 4.1G, and the ubiquitin ligase, Siah-1A (Seven In Ansentia Homologue-1A).
mGlu1 receptor co-immuno-precipitates and functionally interacts with ephrin-B2.

Functional Anatomy

Extensive expression in cerebellar Purkinje cells and in the mitral/tufted cells of the olfactory bulb.
Strong expression in the pars compacta of the substantia nigra, lateral septum, globus pallidum, and thalamic relay nuclei.
Neuroendocrine regions of the hypothalamus express higher levels of mGlu1β than mGlu1α receptors.
Located at postsynaptic specialization of excitatory synapses, concentrated in perisynaptic and extrasynaptic areas.
Required for the induction of long-term depression (LTD) of excitatory neurotransmission at parallel fibre-Purkinje cell synapses.
Leads to formation of diacylglycerol, which is then cleaved by diacylglycerol lipase into 2-arachidonylglycerol (2-AG).
Mice lacking mGlu1 receptors impair LTD and show a defect in the elimination of supranumerary climbing fibers innervating Purkinje cells.
Also mediate a slow EPSP at synapses made between parallel fibers and Purkinje cells.
The mGlu1-receptor mediated EPSC is subject to pronounced synaptic plasticity.

Pharmacology

3,5-Dihydroxyphenylglycine (DHPG) binds to the glutamate recognition site of mGlu1 receptors, but also activates mGlu5 receptors.
No orthosteric agonists selective for mGlu1 receptors.
Selective mGlu1 receptor PAMs: (S)-2-(4-Fluoro-phenyl)-1-(toluene-4- sulfonyl)-pyrrolidine (Ro67-7476), (9H-xanthene-9-carbonyl)-carbamic acid butyl ester (Ro-674853), diphenyl-acetyl-carbamic acid ethyl ester (Ro-01-6128), and 4-nitro-N-(1,4- diphenyl-1H-pyrazol-5-yl) benzamide (VU71).
Orthosteric antagonists of mGlu1 receptors: 4-(Amino-carboxy-methyl)-3-methyl-benzoic acid (LY367385), 1-aminoindane-1,5-dicarboxylic acid (AIDA), and α-amino-5-carboxy-3-methyl-2- thiopheneacetic acid (3-MATIDA).
Negative allosteric modulators (NAMs) of mGlu1 receptors: 7-(Hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), (3aS,6aS)-hexahydro-5-methylene-6a-(2-naphthalenylmethyl)-1H-cyclopenta[c]furan-1-one (BAY367620), and (3,4-dihydro-2H-pyrano[2,3- b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone (JNJ16259685).

Relevance to Human Disorders

Inherited Ataxia and Autoimmune Cerebellar Disorders

Ataxic transgenic mice show mutations of the Grm1 gene or a reduced expression/activity of mGlu1 receptors.
No causative mutations in the GRM1 gene have been found in children with idiopathic early- onset ataxia.
Cerebellar ataxia has been associated with the presence of autoantibodies directed against mGlu1 receptors.
mGlu1 receptor PAMs are potential candidates for the treatment of ataxia associated with defective mGlu1 receptors.
Mice developing experimental autoimmune encephalomyelitis (EAE) show a reduced expression of mGlu1α receptors in Purkinje cells and an impaired motor coordination, which is corrected by a systemic treatment with a selective mGlu1 receptor PAM.

Malignant Melanomas

Insertional mutant mice predisposed to develop malignant melanomas had a deletion of 70 kb in intron 3 of Grm1, resulting into an ectopic expression of mGlu1 receptors in melanoma cells.
Mutant mice in which expression of the Grm1 gene is driven by the promoter of dopachrome tautomerase also develop multiple melanomas.
Expression of mGlu1 receptors has been detected in human melanomas, but not in benign nevi.
The oncogenic activity of mGlu1 receptors in melanoma cells depends on the activation of multiple transduction pathways.
Treatment of human melanoma cells with mGlu1 receptor NAMs or with riluzole reduces cell proliferation.

Neurodegeneration/Neuroprotection

mGlu1 receptor antagonists protect hippocampal neurons against “post-ischemic” degeneration.
Pharmacological blockade of mGlu1 receptors enhances GABAergic transmission in the hippocampus.
Activation of mGlu1 receptors mediates the mechanism of ischemic tolerance observed with the paradigm of “ischemic preconditioning”.
In cultured neurons challenged with excitotoxins, mGlu1 receptor antagonists are consistently neuroprotective.
Michel Baudry and his associates have shown that mGlu1α receptors activate two pathways that differentially affect neurodegeneration.
mGlu1α may produce dual neuroprotective and neurotoxic signaling in cerebellar and cortical neurons.

Schizophrenia

Inhibition of mGlu1 receptors could be a novel treatment for schizophrenia.
mGlu1 receptor antagonists selectively improve prepulse inhibition in DBA/2J mice.
Deficits in PPI were also observed in mice with gene-targeted deletion of the mGlu1 receptor.
Allosteric mGlu1 receptor antagonists reduced methamphetamine-induced hyperlocomotion.

mGlu5 Receptor

Gene name: GRM5 (human); Grm5 (rat, mouse).
Accession numbers: NP000833 (human); NP058708 (rat); NP_001074883 (mouse).
Chromosomal location: 11q14.2 (human); 1q32 (rat); 7D3 (mouse).

Splice variants

mGlu5a: 1212 amino acids in humans; 1203 amino acids in rats; highly and diffusely expressed in the rat brain during early postnatal development.
mGlu5b: A variant that contains a 32 amino acid fragment inserted into the cytoplasmic tail 50 residues after the 7th TM; more abundant in the adult brain.
mGlu5d: Identified in human cerebellum and hippocampus; C-terminus domain 267 amino acids shorter than human mGlu5a receptors.

Receptor signalling and interacting proteins

The mGlu5 receptor is coupled to Gq/G11 protein and its activation stimulates PI hydrolysis.
In recombinant cells, activation of mGlu5 receptors induces oscillatory increases in intracellular $Ca^{2+}$ release, as a result of a PKC-mediated receptor phosphorylation.
Both mGlu5a and mGlu5b receptors are characterized by a long C-terminus domain that allows the interaction with Homer proteins similarly to mGlu1α receptors.
In the postsynaptic elements, mGlu5 receptors are physically linked to the NR2 subunit of NMDA receptors via a chain of interacting proteins, which include PSD-95, Shank, and Homer.
Calcium ions that enter the cell via the NMDA-gated ion channel activate protein phosphatase 2B, which dephosphorylates mGlu5 receptors thereby limiting mGlu5-receptor desensitization.
Activation of mGlu5 receptors enhances NMDA receptor function.
The neuron-specific protein, Norbin, physically interacts with mGlu5 receptors in vivo and increases both membrane localization and signalling of mGlu5 receptors.
The mGlu5 receptors are selectively desensitized by members of the GRK2 family (GRK2 and GRK3), through a mechanism that involves phosphorylation of the Threo 840 residue.

Functional anatomy

The mGlu5a receptor is highly expressed in the CNS during early postnatal life.
The mGlu5b receptor is abundant in the adult hippocampus, corpus striatum, and cerebral cortex.
mGlu5 receptors are involved in the regulation of synaptic plasticity.
Mice with genetic deletion of mGlu5 receptors show a mild deficit in long-term potentiation (LTP) in the hippocampus and an impaired spatial learning.
Pharmacological activation of mGlu5 receptors induces a robust form of LTD at the Schaffer collateral-CA1 pyramidal cell synapse in the hippocampus.
Different signaling mechanisms have been found to be involved in mGluR-LTD in the hippocampus, including the activation of p38 MAPK and tyrosine phosphatases.
mGlu5 receptors have been demonstrated to play a crucial role in some forms of metaplasticity.
The mechanisms by which activation of mGlu5 results in metaplasticity are not fully understood but might involve both $Ca^{2+}$ /calmodulin- dependent protein kinase II (CaMKII) and PKC.
mGlu5 receptors are also expressed in non-neuronal cells, including astrocytes, oligodendrocytes, and microglia, stem-progenitor cells, and a variety of peripheral cells.
Mice expressing a mutated form of human superoxide dismutase associated with amyotrophic lateral sclerosis (ALS) show astrocytic degeneration in the ventral horns of the spinal cord, which likely depends on the activation of mGlu5 receptors by endogenous glutamate.Blocking mGlu5 receptors in vivo delays the onset of motor neuron disease and increases survival of these mice (Rossi et al., 2008).
Embryonic stem (ES) cells grown under proliferating conditions express mGlu5 receptor and no other mGlu receptor subtypes.
Mice lacking mGlu5 receptors have a lower number of proliferating neuroprogenitors in zones of active neurogenesis of the adult brain.
mGlu5 receptors are found in the liver, and hepatocytes lacking mGlu5 receptors are less sensitive to hypoxic damage.
mGlu5 receptors are expressed in many other peripheral cells, including cells of the male germinal line, insulinoma cell lines, immune cells, and brain endothelial cells.

Pharmacology

There are no selective orthosteric agonists of mGlu5 receptors with the exception of (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), which displays low affinity and is active in the high micromolar range .
A number of mGlu5-receptor enhancers include [(3-fluorophenyl) methylene]hydrazone-3-fluorobenzaldehyde (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3- dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydrox-ybenzamide (CPPHA), (S)-(4- fluorophenyl)-(3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]piperidin-1-yl)methanone (ADX47273), 3-cy-ano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), 4-nitro-N- (1,3-diphenyl-1H-pyrazol-5-yl)benzamide (VU29), and ADX63365 behave as mGlu5-receptor enhancers.
mGlu5 receptors NAMs include MPEP, 3-((2- methyl-1,3-thiazol-4-yl)ethynyl) pyridine hydrochloride (MTEP), 6-methyl-2- (phenylazo)-3-pyridinol (SIB-1757), and (E)-2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), fenobam, AFQ056, AZD2516, AZD2066, STX107, ADX10059, and ADX48621.

Relevance to human disorders and clinical perspectives

Fragile-X syndrome (FXS)

FXS is the most frequent inherited cause of mental retardation caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X mental retardation protein (FMRP).
Symptoms include cognitive dysfunction, autism, behavioural problems, seizures, dysmorphic features, and macroorchidism.
Fmr1 knockout mice show a selective amplification of mGlu5-receptor mediated LTD.
FMRP is one of the dendritic proteins synthesized in response to mGlu5- receptor activation and has the specific function of acting as a translational suppressor other LTD-associated proteins.
A series of recent elegant studies have characterized the signaling pathways leading to dendritic protein synthesis in response to mGlu5-receptor activation, such as ERK/MAPK-interacting kinase and PtdIns-3-K/mammalian target of rapamycin.
The lack of one of the two Grm5 as well as systemic treatment with MPEP or other mGlu5-receptor antagonists corrects most of the phenotypes associated with the lack of FMRP.
mGlu5-receptor NAMs are under clinical development for the treatment of FXS.
Non-sedative doses of MPEP block repetitive self-grooming behavior in BTBR T + tfJ mice, which show multiple behavioral traits with face validity for the diagnostic symptoms of autism suggesting forms of autism other than FXS may be amenable of treatment with mGlu5-receptor antagonists.

Parkinson’s disease and L-DOPA-induced dyskinesia

mGlu5 receptors are highly expressed in the basal ganglia motor circuit and are involved in the regulation of motor behavior.
Seminal work have shed lights into the role of group-I mGlu receptors in processes of striatal synaptic plasticity underlying habit memory and motor learning.
The progressive loss of nigro-striatal neurons lead to motor dysfunction.
Dopamine replacement therapy with L-3,4-dihydroxyphenylalanine (L-DOPA treatment is complicated by loss of efficacy and the occurrence of stereotyped involuntary movements.
mGlu5 receptors are expressed in the neostriatum by medium spiny projection neurons and interneurons.
Growing body of evidence indicates that systemic treatment with mGlu5-receptor NAMs is highly effective in relieving motor symptoms and L-DOPA- induced dyskinesias.
The mGlu5 NAMs, AFQ056, AZD2516, and ADX48621, are currently in phase I/II of clinical development for the treatment of Parkinson’s disease and L-DOPA-induced dyskinesias.
Genetic deletion of mGlu5 receptors or systemic treatment with mGlu5-receptor blockers is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine mouse models of toxicological parkinsonism.

Chronic pain

Chronicization of pain reflects the development of “nociceptive sensitization”.
All mGlu receptor subtypes (with the exception of mGlu6 receptors) are widely distributed in pain centers of the brain and spinal cord, and are implicated in the induction, expression, and maintenance of nociceptive sensitization.
In peripheral nociceptors (the peripheral terminals of dorsal root ganglia neurons) mGlu1 and mGlu5 receptors respond to ambient glutamate by triggering a cascade of reactions leading to sensitization of TrpV1 channels.
In nociceptive neurons of the dorsal horns of the spinal cord, the mGlu5 receptor is activated by glutamate released from primary afferent fibers or by local excitatory interneurons expressing PKC-γ, leading to increased neuronal excitability.
mGlu5 receptors are also found in supraspinal brain centers, such as dorsal raphe nuclei and amygdala, where their activation sustains chronic pain.
Pharmacological blockade of mGlu5 receptors is analgesic in models of inflammatory and neuropathic pain.
Based on these preclinical data, mGlu5-receptor NAMs such as AZD2516 and AZD2066 are under clinical development for the treatment of neuropathic pain.
In a phase IIa clinical trial, the mGlu5-receptor NAM, ADX100159, has shown better efficacy than placebo in the treatment of acute pain associated with migraine.

Drug addiction

Drug addiction or “substance dependence” is a chronic, relapsing disorder characterized by compulsive drugs seeking behavior, loss of control in taking the drug, and emergence of a negative emotional state when access to the drug is prevented
It helpful to subdivide the addiction process into three phases: (i) an “intoxication phase”; (ii) an abstinence phase, in which mechanisms of negative reinforcement sustain drug craving; and (iii) a phase of enduring vulnerability to drug intake.
Activation of the dopaminergic mesolimbic system, mediates the reinforcing properties of drugs of abuse
Chiamulera et al. (2001) have shown that mice lacking mGlu5 receptors fail to self-administer cocaine without showing a defect in the ability of cocaine to increase extracellular dopamine levels in the nucleus accumbens.
Data obtained with MPEP indicate that activation of mGlu5 receptors is required for incentive motivation for the reinforcer.
mGlu5-receptor NAMs exert anti-reinforcing effects at multiple levels .
Accordingly, cocaine-mediated synaptic potentiation is absent in VTA neurons from mGlu5-defient mice.
mGlu5-receptor blockade in the nucleus accumbens is effective in preventing relapse to drug seeking as well as neurochemical changes associated with drug reinstatement.
mGlu5-receptor NAMs hold promise in the treatment of drug addiction, and AFQ056 is under clinical development for the treatment of tobacco smoking.

Anxiety

*Drugs that block mGlu5 receptors consistently show anxiolytic-like activity in a variety of animal models such as fear potentiated startle, contextual fear conditioning, elevated plus maze, conflict tests, and stress-induced hyperthermia Mouse lacking mGlu5 receptors reduced stress-induced hyperthermia and clinically validated anxiolytic drug, behaves as potent and selective mGlu5 receptor NAM.

A critical issue on interactions between mGlu5-receptor NAMs and drug metabolizing enzymes development of mGlu5-receptor NAMs as novel anxiolytic agents warrants further investigations.

Schizophrenia

The interest for mGlu5 receptors in schizophrenia stems from the evidence that mGlu5-receptor knockout mice show a disruption in PPI, which reflects a defect in sensory-motor gating.
mGlu5-receptor blockade enhances the psychotomimetic effect of phencyclidine and mGlu5 and NMDA receptors may act synergistically in regulating neuronal activity in the prefrontal cortex.
The prediction that amplification of mGlu5-receptor function could be beneficial in the treatment of schizophrenia stimulation of mGlu5receptor PAMs in preclinical models (CDPPB and ADX47273), are active and ADX63365 is currently developed for the treatment of schizophrenia.

Gastroesophageal reflux disorder (GERD)

GERD is a common disorder caused by an abnormal reflux of stomach acid to the esophagus due to a transient relaxation of the lower esophageal sphincter (LES).
Preclinical studies have shown that mGlu5- receptor NAMs such as MPEP and MTEP increase basal LES pressure and inhibit LES relaxation.
In a clinical study, treatment with the mGlu5 NAM, ADX100159, reduced the number and duration of symptomatic reflux episodes as well as nocturnal and postprandial esophageal acid exposure. Modified release formulation of ADX100159 showed particular suitable for long-term treatments in patients with GERD.

mGlu2 and mGlu3 receptors

mGlu2 receptor

Gene name: GRM2 (human); Grm2 (rat, mouse).
Accession numbers: NP000830 (human); NP001099181 (rat); NP_001153825 (mouse).
Chromosomal location: 3q21.31 (human); 8q32 (rat); 9 (mouse).

mGlu3 receptor

Gene name: GRM3 (human); Grm3 (rat, mouse).
Accession numbers: NP000831 (human); NP001099182 (rat); NP_862898 (mouse).
Chromosomal location: 7q21.1–q21.2 (human); 4q32 (rat); 5A1-h (mouse).

Signal transduction and interacting proteins

mGlu2 and mGlu3 receptors are coupled to Gi/Go proteins in heterologous expression systems, inhibiting cAMP formation and voltage-sensitive $Ca^{2+}$ channels.
They can also activate the MAPK and PtdIns-3-K pathways without directly stimulating PI hydrolysis but amplify the stimulation of PI hydrolysis mediated by mGlu1/5 receptors in brain slices.
mGlu2 and mGlu3 receptors show a different sensitivity to processes of homologous desensitization mediated by GRK2/β-arrestin, with mGlu2 receptors being resistant to desensitization, when inhibition of cAMP formation is a read-out.
Group-II mGlu receptors interact with calmodulin, protein phosphatase 2C, and the Ran binding protein in the microtubule-organizing center (RanBPM).

Functional anatomy

mGlu2 and mGlu3 receptors are diffusely expressed in the CNS with mGlu2 receptors uniquely localized in neurons, presynaptically in axons, and mGlu3 receptors at both pre- and postsynaptic sites, as well as in glial cells.
Presynaptic mGlu2/3 receptors can be activated by synaptic glutamate or glutamate released from astrocytes via the cystine–glutamate membrane antiporter.
A major function of presynaptic mGlu2/3 receptor is to inhibit neurotransmitter release; both receptors regulate synaptic plasticity, particularly LTD.
In the mouse accessory olfactory bulb, activation of mGlu2 receptors relieves the GABAergic inhibition of mitral cells.
mGlu3 receptors were also found in embryonic stem cells and glioma-initiating cells, where their activation limits cell differentiation by negatively regulating type-4 bone- morphogenetic protein (BMP-4) receptor signaling

Pharmacology

No marketed orthosteric agonists or antagonists that can differentiate between mGlu2 and mGlu3 receptors, with the exception of N- acetylaspartateglutamate (NAAG), which selectively activates mGlu3 receptors (Wroblewska et al.,1997; but see also Chopra et al., 2009).
(−)-(1R,4S,5S,6S)-4-Amino-2-sulfonylbicyclo[3.1.0]hexane-4,6- dicarboxylic acid (LY404-039), which is pharmacologically similar to LY379268, is under clinical development for the treatment of schizophrenia.
(3-(3,4-Dichlorobenzyloxy)-2-amino-6-fluorobicyclo[3.1.0] hexane-2,6-dicarboxylic acid) MGS0039 (Nakazato et al., 2004), is a potent and selective mGlu2/3 receptor antagonist.
2,2,2-Trifluoro-N-[4-(2-methoxyphenoxy)phenyl]-N-(3-pyridinylmethyl) ethanesulfonamide hydrochloride (LY487379) and 3′-([(2-cyclo-pentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H- inden-5-yl)oxy]methy-l)biphenyl l-4-carboxylate (BINA) are prototypes of selective mGlu2 receptor PAMs which are also under development in the treatment of schizophrenia.
No mGlu3 receptor enhancers are currently available.

Relevance to human disorders and clinical perspectives

Anxiety

mGlu2/3 receptors regulates synaptic transmission and plasticity in the amygdala, a brain region that encodes fear memory and is critically involved in the pathophysiology of anxiety disorders.
Treatment with LY354740 is highly effective in several animal models of anxiety and panic disorders with no reductions of activity on repeated dosing.

Schizophrenia

The development of mGlu2/3 receptor agonists as antipsychotic drugs moved from the observation that LY354740 inhibits glutamate release, allowing for testing the hyperglutamatergic theory in schizophrenia.
A pioneer phase II clinical study has shown that the a 21-day treatment with LY214