Modulatory action of metabotropic glutamate receptor (mGluR) 5 on mGluR1 function in striatal cholinergic interneurons

Within basal ganglia, group I metabotropic glutamate receptor subtypes (mGluR1 and 5) frequently co-localize in the same neuron. However, little is known about how these receptors functionally interact. We addressed this issue by means of electrophysiological recordings of striatal cholinergic interneurons, a neuronal subtype that co-express both group I mGluRs. The group I non-selective agonist 3,5-DHPG induced a membrane depolarization/inward current that was prevented by co-application of LY 367385, a selective mGluR1 antagonist, and SIB 1757 or MPEP, blockers of mGluR5 subtype. The reversal potential for the response to 3,5-DHPG was close to the equilibrium potential for potassium channels. Repeated bath or focal applications of 3,5-DHPG induced a progressive decline in the amplitude of the membrane depolarization, suggesting that group I mGluRs undergo receptor desensitization. Interestingly, in the presence of the mGluR5 blocker, SIB 1757, this event was not observed, whereas it occurred in LY 367385. PKC blockers chelerythrine and calphostin C mimicked the inhibitory effect of SIB 1757. In a subset of interneurons, in MPEP or SIB 1757, 3,5-DHPG induced a 0.5-1 Hz oscillatory response, that was prevented by L-type Ca2+ channel blockers, and by the tyrosine kinase inhibitors genistein and lavendustin. Together, these data suggest that mGluR5 modulates mGluR1 activity to shape cell excitability.     

Constitutive endocytosis of the metabotropic glutamate receptor mGluR7 is clathrin-independent

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are widely expressed throughout the brain and are involved in synaptic development, transmission, and plasticity. The endocytosis of several members of the GPCR superfamily of receptors, such as beta-adrenergic receptors, has been studied extensively. In contrast, the mechanisms regulating mGluR endocytosis and intracellular trafficking remain poorly defined. We describe here for the first time a distinct endocytic and intracellular sorting pathway utilized by mGluR7. We show that mGluR7 constitutively internalizes via a non-clathrin mediated pathway in heterologous cells and in neurons. Unlike clathrin-mediated NMDAR endocytosis, mGluR7 traffics via an Arf6-positive endosomal pathway, similar to other well-characterized proteins such as major histocompatibility complex class I (MHC I) and the GPI-anchored protein CD59. Thus constitutive endocytosis of mGluR7 in neurons is not regulated by clathrin-dependent mechanisms, and this clathrin-independent pathway ultimately determines the amount of receptor present on the plasma membrane available to bind and respond to glutamate.     

Subtype-selective noncompetitive modulators of metabotropic glutamate receptor subtype 1 (mGluR1)

This article describes recent advances in the development of subtype-selective, noncompetitive modulators of metabotropic glutamate receptor subtype 1 (mGluR1). mGluR1 plays an important role in modulating synaptic transmission and neuronal excitability via intracellular signal transduction pathways and has been implicated in a number of CNS disorders. Allosteric modulation of mGluR1 by potentiation and antagonism occurs through binding to the seven transmembrane domain. In addition to blocking agonist-dependent responses, many of the antagonists also show inverse-agonist activity by blocking constitutive receptor activity. Highly potent and selective mGluR1 radioligands have been used to evaluate receptor binding in vivo and in vitro. Some of these novel agents have demonstrated high levels of CNS exposure and receptor occupancy in vivo, as well as efficacy in a number of preclinical models of neurological disorders.     

Positive allosteric modulators of the metabotropic glutamate receptor subtype 2 (mGluR2)

This article describes recent medicinal chemistry progress toward selective potentiators of the metabotropic glutamate receptor 2 (mGluR2). Groups at Lilly and Merck have identified new classes of potentiators that exhibit selectivity for mGluR2 over the seven other subtypes of mGluRs. Structure-activity relationships as well as pharmacokinetic properties and in vivo activity are reviewed.     

Discovery of positive allosteric modulators of metabotropic glutamate receptor subtype 5 (mGluR5)

This review provides an overview of the drug discovery process used to identify, develop and characterize the first positive allosteric modulators of the metabotropic glutamate receptor (mGluR) subtype 5 (mGluR5). Discovery and optimization of three series of positive allosteric modulators are described, each using different approaches. The symmetric benzaldazine series was discovered and optimized from samples already existing in our sample collection without an active synthetic program to further elucidate SAR. This series yielded a family of highly selective pharmacological tools that produced positive, negative and neutral allosteric modulation of mGluR5 activity. The original compound in the benzamide series was discovered from screening and this series was optimized using an iterative library synthesis approach to explore SAR in each of three regions of the molecule. This series produced more potent positive allosteric mGluR5 modulators than the benzaldazine series which could be evaluated for their effect on mGluR5 in brain slice electrophysiological studies. The pyrazole series used a fragment library approach based on small structural motives from the benzamide series to discover lead compounds and establish SAR. This series produced still more potent positive allosteric mGluR5 modulators with improved pharmacokinetic and physical properties. These modulators showed efficacy in animal behavioral models in which other antipsychotic drugs were active. Evaluation of assay data in mathematical models of allosterism to constrain possible mechanisms of action is briefly discussed. Other reviews of this emerging field with different emphases have been published recently [1-3].     

The metabotropic glutamate receptor 7 (mGluR7) allosteric agonist AMN082 modulates nucleus accumbens GABA and glutamate, but not dopamine, in rats

The group III metabotropic glutamate receptor 7 (mGluR7) has been implicated in many neurological and psychiatric diseases, including drug addiction. However, it is unclear whether and how mGluR7 modulates nucleus accumbens (NAc) dopamine (DA), L-glutamate or gamma-aminobutyric acid (GABA), important neurotransmitters believed to be involved in such neuropsychiatric diseases. In the present study, we found that systemic or intra-NAc administration of the mGluR7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082) dose-dependently lowered NAc extracellular GABA and increased extracellular glutamate, but had no effect on extracellular DA levels. Such effects were blocked by (R,S)-alpha-methylserine-O-phosphate (MSOP), a group III mGluR antagonist. Intra-NAc perfusion of tetrodotoxin (TTX) blocked the AMN082-induced increases in glutamate, but failed to block the AMN082-induced reduction in GABA, suggesting vesicular glutamate and non-vesicular GABA origins for these effects. In addition, blockade of NAc GABAB receptors by 2-hydroxy-saclofen itself elevated NAc extracellular glutamate. Intra-NAc perfusion of 2-hydroxy-saclofen not only abolished the enhanced extracellular glutamate normally produced by AMN082, but also decreased extracellular glutamate in a TTX-resistant manner. We interpret these findings to suggest that the increase in glutamate is secondary to the decrease in GABA, which overcomes mGluR7 activation-induced inhibition of non-vesicular glutamate release. In contrast to its modulatory effect on GABA and glutamate, the mGluR7 receptor does not appear to modulate NAc DA release.     

Tyrosine phosphorylation of the metabotropic glutamate receptor mGluR5 in striatal neurons

Protein phosphorylation, controlled by the coordinated actions of phosphatases and kinases, is an important regulatory mechanism in synaptic transmission and other neurophysiological processes. Ionotropic glutamate receptors are known targets of phosphorylation on serine, threonine and tyrosine residues, with functional consequences for cell excitability, plasticity and toxicity. While phosphorylation of metabotropic glutamate receptors (mGluRs) also impacts critical cellular processes, there has been no evidence for direct tyrosine phosphorylation of mGluRs. In the present study, anti-phosphotyrosine and specific mGluR antibodies were used to detect tyrosine-phosphorylated mGluRs in rat brain. In particular, we found that mGluR5 is an abundant phosphotyrosine protein in vivo as well as in primary striatal neurons and tissue slices in vitro. The protein phosphatase inhibitor pervanadate robustly increased the amount of tyrosine-phosphorylated mGluR5, suggesting the receptor is subject to an endogenous, active cycle of phosphorylation and dephosphorylation. Furthermore, NMDA treatment also increased the amount of tyrosine-phosphorylated mGluR5, suggesting these endogenous phosphorylation regulatory mechanisms can be used to mediate crosstalk between synaptic glutamate receptors. While mGluR5-stimulated phosphoinositide hydrolysis appears to be unaltered by pervanadate treatment, tyrosine phosphorylation of mGluR5 may be important in trafficking, anchoring, or signaling of the receptor through G protein-independent pathways.     

Blockade of the metabotropic glutamate receptor subtype 5 (mGluR5) produces antiparkinsonian-like effects in rats.

The aim of the present study was to examine a potential beneficial effect of the blockade of metabotropic glutamate receptor subtype 5 (mGluR5) by the selective non-competitive antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), in models of parkinsonian symptoms in rats. Haloperidol, 0.25, 0.5 and 1mg/kg ip, was used to induce hypolocomotion, catalepsy and muscle rigidity, respectively. The locomotor activity was estimated by an open-field test, the catalepsy -- by a 9-cm cork test. The muscle rigidity was measured as an increased resistance of a hind leg to passive extension and flexion at the ankle joint. Additionally, increases in the electromyographic activity were recorded in the gastrocnemius and tibialis anterior muscles. MPEP (1.0-10mg/kg ip) inhibited the muscle rigidity, electromyographic activity, hypolocomotion and catalepsy induced by haloperidol. MPEP administered alone (5mg/kg ip) did not induce catalepsy, nor did it influence the muscle tone or locomotor activity in rats. The present results suggest that blockade of mGluR5 receptors may be important to amelioration of both parkinsonian akinesia and muscle rigidity.     

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells

Class C G-protein coupled receptors form obligatory dimers. Metabotropic glutamate receptors (mGluRs) are found commonly as homodimers. Alternative splicing of mGluR1 gene results in vivo in the expression of a long variant mGluR1a and at least two short variants mGluR1b and d. The amino acid sequences diverge within their carboxyl-termini six amino acid residues following RRKK motif. This four basic residue sequence was shown to have pronounced impact on function and trafficking of the short variants, while for mGluR1a the long C-terminus reduces the effects caused by presence of the RRKK motif. Here we investigated consequences of interactions between long mGluR1a and short mGluR1b variants. Our results show that mGluR1a interferes with mGluR1b trafficking to the cell surface in HEK293 transfected cells. Expression of a mGlu1a mutant incapable of activating G-proteins with mGluR1b mutated in the glutamate binding site led to the formation of a functional heterodimer. Moreover, we show that swapping long mGluR1a and/or short mGluR1b C-termini with corresponding regions in chimerical GB1 and GB2 gamma-amino butyric acid b (GABAb) receptor subunits do not exclude heterodimerization. These data reveal that the C-terminal ends of mGluR1 do not control subunit association, such that mGluR1 dimers with two distinct C-termini can form and function properly.     

Fragile X syndrome: a preclinical review on metabotropic glutamate receptor 5 (mGluR5) antagonists and drug development

Rationale

Fragile X syndrome (FXS) is considered the leading inherited cause of intellectual disability and autism. In FXS, the fragile X mental retardation 1 (FMR1) gene is silenced and the fragile X mental retardation protein (FMRP) is not expressed, resulting in the characteristic features of the syndrome. Despite recent advances in understanding the pathophysiology of FXS, there is still no cure for this condition; current treatment is symptomatic. Preclinical research is essential in the development of potential therapeutic agents.

Objectives

This review provides an overview of the preclinical evidence supporting metabotropic glutamate receptor 5 (mGluR5) antagonists as therapeutic agents for FXS.

Results

According to the mGluR theory of FXS, the absence of FMRP leads to enhanced glutamatergic signaling via mGluR5, which leads to increased protein synthesis and defects in synaptic plasticity including enhanced long-term depression. As such, efforts to develop agents that target the underlying pathophysiology of FXS have focused on mGluR5 modulation. Animal models, particularly the Fmr1 knockout mouse model, have become invaluable in exploring therapeutic approaches on an electrophysiological, behavioral, biochemical, and neuroanatomical level. Two direct approaches are currently being investigated for FXS treatment: reactivating the FMR1 gene and compensating for the lack of FMRP. The latter approach has yielded promising results, with mGluR5 antagonists showing efficacy in clinical trials.

Conclusions

Targeting mGluR5 is a valid approach for the development of therapeutic agents that target the underlying pathophysiology of FXS. Several compounds are currently in development, with encouraging results.     

Emerging signalling and protein interactions mediated via metabotropic glutamate receptors

Metabotropic glutamate receptors (mGlu) are GTP-binding (G) protein-coupled receptors (GPCRs) that are involved in learning and memory, cardiovascular control and motor function. Their structure and pharmacology has been reviewed recently in Current Drug Targets: CNS and Neurological Disorders (Vol. 1, Issue 3) where their roles in a variety of neurological disorders were highlighted. The present review focuses on the emerging evidence for interactions of mGlu receptors with other GPCRs in the CNS at the membrane interface and amongst signaling cascades in the cytosol (e.g. intracellular Ca(2+), cAMP and scaffolding proteins). While initially non-selective activity was thought to be responsible for many atypical responses, increasing evidence points to GPCR interactions in neurons and glia, with adrenoceptors, adenosine receptors, dopamine receptors and muscarinic receptors. For example, group II mGlu receptors were found to be required for group I mGlu receptor induction of long-term potentiation at the postsynaptic terminal. Increasing evidence demonstrates the intimate interaction of adenosine receptors and mGlu receptors, particularly in the regulation of neurotransmitter release. While adenosine itself can be released from astrocytes by co-activation of group II mGlu and beta-adrenergic receptors. Given the complexity of neurological disorders such as ischemic stroke, Alzheimer's disease and epilepsy, exploitation mGlu receptor-associated GPCR interactions may prove efficacious in the treatment of such disorders.     

Discovery of orally efficacious tetracyclic metabotropic glutamate receptor 1 (mGluR1) antagonists for the treatment of chronic pain

Metabotropic glutamate receptor 1 (mGluR1) plays important roles in the neurotransmission and pathogenesis of several neurological disorders, including chronic pain. Antagonists of mGlur1 are suggested to be useful for the treatment of pain. Herein, we report the discovery of a novel series of tetracyclic mGluR1 antagonists, such as 23c and 23e, with oral efficacy of ED50 of 8 and 5.1 mg/kg, respectively, in rat spinal nerve ligation neuropathic pain model.     

The group II metabotropic glutamate receptor 3 (mGluR3, mGlu3, GRM3): expression, function and involvement in schizophrenia

Group II metabotropic glutamate receptors (mGluRs) comprise mGluR2 (mGlu2; encoded by GRM2) and mGluR3 (mGlu3; encoded by GRM3) and modulate glutamate neurotransmission and synaptic plasticity. Here we review the expression and function of mGluR3 and its involvement in schizophrenia. mGluR3 is expressed by glia and neurons in many brain regions and has a predominantly presynaptic distribution, consistent with its role as an inhibitory autoreceptor and heteroceptor. mGluR3 splice variants exist in human brain but are of unknown function. Differentiation of mGluR3 from mGluR2 has been problematic because of the lack of selective ligands and antibodies; the available data suggest particular roles for mGluR3 in long-term depression, in glial function and in neuroprotection. Some but not all studies find genetic association of GRM3 polymorphisms with psychosis, with the risk alleles also being associated with schizophrenia-related endophenotypes such as impaired cognition, cortical activation and glutamate markers. The dimeric form of mGluR3 may be reduced in the brain in schizophrenia. Finally, preclinical findings have made mGluR3 a putative therapeutic target, and now direct evidence for antipsychotic efficacy of a group II mGluR agonist has emerged from a randomised clinical trial in schizophrenia. Together these data implicate mGluR3 in aetiological, pathophysiological and pharmacotherapeutic aspects of the disorder.     

Covalent and noncovalent interactions mediate metabotropic glutamate receptor mGlu5 dimerization

Some, perhaps all, G protein-coupled receptors form homo- or heterodimers. We have shown that metabotropic glutamate receptors are covalent dimers, held together by one or more disulfide bonds near the N terminus. Here we report how mutating cysteines in this region affect dimerization and function. Covalent dimerization is preserved when cysteines 57, 93, or 99 are mutated but lost with replacement at 129. Coimmunoprecipitation under nondenaturing conditions indicates that the C[129]S mutant receptor remains a dimer, via noncovalent interactions. Both C[93]S and C[129]S bind [3H]quisqualate, whereas binding to C[57]S or C[99]S mutants is absent or greatly attenuated. The C[93]S and C[129]S receptors have activity similar to wild-type when assayed by fura-2 imaging of intracellular calcium in human embryonic kidney cells or electrophysiologically in Xenopus laevis oocytes. In contrast, C[57]S or C[99]S are less active in both assays but do respond with higher glutamate concentrations in the oocyte assay. These results demonstrate that 1) covalent dimerization is not critical for mGlu5 binding or function; 2) mGlu5 remains a noncovalent dimer even in the absence of covalent dimerization; and 3) high-affinity binding requires Cys-57 and Cys-99.     

G protein-coupled receptor kinases regulate metabotropic glutamate receptor 5 function and expression

Metabotropic glutamate receptors (mGluR) serve important neuromodulatory roles at glutamatergic synapses to shape excitatory neurotransmission. Recent evidence indicates that the desensitization of mGluRs is an important determinant in regulating the functions of these receptors. The present results demonstrate that G protein-coupled receptor kinases (GRKs), which are known to regulate the desensitization of many G protein-coupled receptors, regulate both the expression and function of mGluR5 in a heterologous expression system. This regulatory event is limited to members of the GRK2 family since GRK4 family members do not elicit the same effects on mGluR5. Kinase activity is shown to be required for GRK-mediated regulation of mGluR5. Furthermore, the ability of GRK2 to regulate mGluR5 is dependent, at least in part, on the presence of threonine 840 in the carboxyl terminus of mGluR5. These studies identify novel roles for GRKs in regulating mGluR5 that may serve to further shape the function of these receptors in neurotransmission.