Operant sensation seeking requires metabotropic glutamate receptor 5 (mGluR5)

Pharmacological and genetic studies have suggested that the metabotropic glutamate receptor 5 (mGluR5) is critically involved in mediating the reinforcing effects of drugs of abuse, but not food. The purpose of this study was to use mGluR5 knockout (KO), heterozygous (Het), and wildtype (WT) mice to determine if mGluR5 modulates operant sensation seeking (OSS), an operant task that uses varied sensory stimuli as a reinforcer. We found that mGluR5 KO mice had significantly reduced OSS responding relative to WT mice, while Het mice displayed a paradoxical increase in OSS responding. Neither KO nor Het mice exhibited altered operant responding for food as a reinforcer. Further, we assessed mGluR5 KO, Het and WT mice across a battery of cocaine locomotor, place preference and anxiety related tests. Although KO mice showed expected differences in some locomotor and anxiety measures, Het mice either exhibited no phenotype or an intermediate one. In total, these data demonstrate a key role for mGluR5 in OSS, indicating an important role for this receptor in reinforcement-based behavior.     

A new series of pyridinyl-alkynes as antagonists of the metabotropic glutamate receptor 5 (mGluR5)

Synthesis and some structure-activity relationships for a new series of propargyl ethers as mGluR5 antagonists are reported.     

The metabotropic glutamate receptor mGluR5 is endocytosed by a clathrin-independent pathway

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.     

The role of metabotropic glutamate receptor (mGluR) ligands in parkinsonian muscle rigidity

Summary. It has been shown that the primary striatal dopaminergic hypofunction which is at the origin of Parkinson's disease, results in a secondary hyperactivity of glutamatergic neurotransmission. In the search for a therapy of Parkinson's disease, ionotropic, mainly NMDA, receptor antagonists were found to have moderately beneficial, yet also some undesirable side-effects. Therefore the present study was aimed at determining whether some metabotropic glutamate receptor (mGluR) ligands may have antiparkinsonian effects in the haloperidol-induced muscle rigidity. To this end three mGluR ligands were used: the potent and selective mGluR I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), the mixed group II agonist/group I antagonist (S)-4-carboxy-3-hydroxyphenyl-glycine ((S)-4-C3HPG), and the potent group II agonist (+)-2-aminobicyclo[3.1.0.]hexane-2,6,-dicarboxylic acid (LY354740). Only LY354740 penetrated the brain from the periphery; for this reason other drugs were injected bilaterally into the rostral striatum or nucleus accumbens. The muscle tone was recorded by a mechanomyographic/electromyographic (MMG/EMG) method which measured the resistance of a rat's hind foot and the EMG reflex response of its muscles to passive movements. (S)-4C3HPG (5 and 15 μg/0.5 μl) and LY354740 (5 and 10 mg/kg i.p.) diminished the muscle rigidity induced by haloperidol (1 mg/kg i.p.). AIDA (0.5–15 μg/0.5 μl) injected into the striatum was only slightly effective in the highest dose used. However, when injected into the nucleus accumbens AIDA (15 μg/0.5 μl) significantly and strongly counteracted the haloperidol-induced muscle rigidity. Our results suggest that stimulation of group II striatal mGluRs seems to play a major role in diminution of parkinsonian-like muscle rigidity. However, it seems that the antagonism of group I mGluRs located in the nucleus accumbens may also be of importance to the antiparkinsonian effect. Received August 31, 1999 Accepted September 3, 1999     

Importance of Shank3 protein in regulating metabotropic glutamate receptor 5 (mGluR5) expression and signaling at synapses

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.     

Permissive role of adenosine A2A receptors on metabotropic glutamate receptor 5 (mGluR5)-mediated effects in the striatum

The metabotropic glutamate receptors 5 (mGlu5Rs) and the adenosine A2A receptors (A2ARs) have been reported to functionally interact in the striatum. The aim of the present work was to verify the hypothesis that the state of activation of A2A Rs could influence mGlu5R-mediated effects in the striatum. In electrophysiological experiments (extracellular recording in rat corticostriatal slices), the ability of the selective mGlu5R agonist CHPG to potentiate the reduction of the field potential amplitude induced by NMDA was prevented not only by the selective mGlu5R antagonist MPEP, but also by the selective A2AR antagonist ZM 241385. Analogously, the application of CHPG potentiated NMDA-induced toxicity (measured by LDH release) in cultured striatal neurons, an effect that was abolished by both MPEP and ZM 241385. Finally, the A2AR agonist CGS 21680 potentiated CHGP effects, an action that was reproduced and abolished, respectively, by forskolin (an activator of the cAMP/protein kinase A, PKA, pathway) and KT 5720 (a PKA inhibitor). The results indicate that A2ARs exert a permissive role on mGlu5R-induced effects in the striatum. Such an interaction may represent an additional target for the development of therapeutic strategies towards striatal disorders.     

Depolarization- and agonist-regulated expression of neuronal metabotropic glutamate receptor 1 (mGluR1).

In established 8-12-day-old primary cultures of differentiated rat cerebellar granule neurons the level of metabotropic glutamate receptor 1 (mGluR1) mRNA and the sensitivity of cultures to the agonist-stimulated inositol phosphate (IP) formation was reversibly modified by changing the depolarizing properties of the medium, i.e. the medium KCl concentration. The mGluR1 mRNA content was suppressed by increasing the medium KCl content and elevated by decreasing it. The mGluR agonist quisqualate inhibited the mGluR1 expression. This is the first direct demonstration of a differential expression of neuronal mGluR1 in an established neuronal culture. The model can be used to study the molecular mechanism of neuronal plasticity.     

Structure-activity relationships for the linker in a series of pyridinyl-alkynes that are antagonists of the metabotropic glutamate receptor 5 (mGluR5)

Studies of structure-activity relationships for the linker in a new series of metabotropic glutamate receptor 5 antagonists are presented together with in vitro and in vivo pharmacokinetic data.     

Native presynaptic metabotropic glutamate receptor 4 (mGluR4) interacts with exocytosis proteins in rat cerebellum

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.     

Allosteric activation of metabotropic glutamate receptor 5

Abstract

Metabotropic glutamate receptor 5 (mGluR5) is a class C G protein-coupled receptor (GPCR) with both an extracellular ligand binding site and an allosteric intrahelical chamber located similarly to the orthosteric ligand binding site of Class A GPCRs. Ligands binding to this ancestral site of mGluR5 can act as positive (PAM), negative (NAM) or silent (SAM) allosteric modulators, and their medicinal chemistry optimization is notoriously difficult, as subtle structural changes may cause significant variation in activity and switch in the functional response. Here we present all atom molecular dynamics simulations of NAM, SAM and PAM complexes formed by closely related ligands and analyse the structural differences of the complexes. Several residues involved in the activation are identified and the formation of a continuous water channel in the active complex but not in the inactive ones is recognized. Our results suggest that the mechanism of mGluR5 activation is similar to that of class A GPCRs.

Communicated by Ramaswamy H. Sarma     

Dipyridyl amides: potent metabotropic glutamate subtype 5 (mGlu5) receptor antagonists

The mGlu5 receptor has been implicated in a number of CNS disorders. Herein, we report on the discovery, synthesis, and biological evaluation of dipyridyl amides as small molecules mGluR5 antagonists.     

Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg(78), Ser(159), or Thr(182) to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist L-[(3)H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser(157) and Ser(160)) or a relatively modest effect (Ser(181)) on ligand affinity compared with the Arg(78), Ser(159), and Thr(182) mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog L-serine O-phosphate (L-SOP) was "docked" into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser(159) and Thr(182) of mGluR4 form hydrogen bonds with the alpha-carboxyl and alpha-amino groups on L-SOP, respectively, whereas Arg(78) forms an electrostatic interaction with the acidic side chains of L-SOP or glutamate. The conservation of Arg(78), Ser(159), and Thr(182) in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.     

The suppressive effect of metabotropic glutamate receptor 5 (mGlu5) inhibition on hepatocarcinogenesis

Metabotropic glutamate receptors (mGlus) are G-protein-coupled receptors playing an important role in the central nervous system (CNS). Recently, mGlus have been identified in peripheral tissues, and aberrant expression or inhibition of the receptors functions in the development of certain cancers. However, the correlation of mGlu activity with hepatocellular carcinoma (HCC) remains unknown. In this study, we analyzed the effects of inhibiting mGlu5 activity in hepatocarcinoma cell lines and a xenograft model. Inactivation of mGlu5 with 2-Methyl-6-(phenylethyl)-pyridine (MPEP), a specific antagonist of the receptor, caused inhibition of cell growth, migration, and invasion of HepG2 and Bel-7402 cells, assessed by MTT assay, ATP production, wound healing, and Boyden chamber assay, respectively. Moreover, inhibition of tumor growth and the potential metastasis of hepatocellular carcinoma were also found in nude mice. Furthermore, mGlu5-mediated extracellular signal-regulated kinase (ERK) phosphorylation has been found to be partially involved in cell growth and migration, as detected by stimulation of (S)-3,5-Dihydroxyphenylglycine (DHPG), an agonist of the receptor, and blockage of MPEP and U0126, an inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK). These data indicate that inhibiting the activity of mGlu5 has the molecular potential to suppress oncogenic actions by blocking downstream effector molecules. The study suggests that mGlu5 activity may contribute to understanding the development of HCC.     

Synthesis and SAR of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor 4 (mGluR4)

This Letter describes the synthesis and SAR of the novel positive allosteric modulator, VU0155041, a compound that has shown in vivo efficacy in rodent models of Parkinson’s disease. The synthesis takes advantage of an iterative parallel synthesis approach to rapidly synthesize and evaluate a number of analogs of VU0155041.     

Type 2 metabotropic glutamate receptor (mGluR2) fails to negatively couple to cGMP in stably transfected cells

The group II metabotropic glutamate receptors 2 and 3 (mGluR2 and mGluR3) share sequence homology, common pharmacology and negative coupling to cAMP. We recently discovered that mGluR3 also is negatively coupled through a G-protein to the cGMP transduction pathway in rat cerebellar granule cells and astrocytes. To test the hypothesis that mGluR2 also has access to the cGMP pathway, C6 glioma cells were stably transfected with mGluR2 and mGluR3 cDNA and their coupling to cGMP levels was characterized. In contrast to many other cell lines, C6 has a robust cGMP response that makes it attractive in the study of receptor coupling to this second messenger pathway. Consistent with prior studies, the mGluR3 receptor was negatively coupled to cGMP and this coupling was blocked by PTX. In contrast, mGluR2 agonists failed to reduce sodium nitroprusside stimulated cGMP levels in transfected cell lines where the receptor was negatively coupled to cAMP. These data provide further support for the functional divergence between these two closely related receptors.     

Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5)

Novel in vitro mGlu₅ positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC₅₀ 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC₅₀ 156.3 nM). Derivation of a second-generation of mGlu₅ PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC₅₀ 50.1 nM) as a potent and soluble mGlu₅ PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.     

Cyclohexenyl- and dehydropiperidinyl-alkynyl pyridines as potent metabotropic glutamate subtype 5 (mGlu5) receptor antagonists

Structure-activity relationship studies leading to the discovery of novel mGlu5 receptor antagonists are described. These compounds show high in vitro potency, have good in vivo receptor occupancy, and a reasonable intravenous pharmacokinetic profile.     

Protein kinase C phosphorylation of the metabotropic glutamate receptor mGluR5 on Serine 839 regulates Ca2+ oscillations

The activation of Group 1 metabotropic glutamate receptors, mGluR5 and mGluR1alpha, triggers intracellular calcium release; however, mGluR5 activation is unique in that it elicits Ca2+ oscillations. A short region of the mGluR5 C terminus is the critical determinant and differs from the analogous region of mGluR1alpha by a single amino acid residue, Thr-840, which is an aspartic acid (Asp-854) in mGluR1alpha. Previous studies show that mGluR5-elicited Ca2+ oscillations require protein kinase C (PKC)-dependent phosphorylation and identify Thr-840 as the phosphorylation site. However, direct phosphorylation of mGluR5 has not been studied in detail. We have used biochemical analyses to directly investigate the phosphorylation of the mGluR5 C terminus. We showed that Ser-839 on mGluR5 is directly phosphorylated by PKC, whereas Thr-840 plays a permissive role. Although Ser-839 is conserved in mGluR1alpha (Ser-853), it is not phosphorylated, as the adjacent residue (Asp-854) is not permissive; however, mutagenesis of Asp-854 to a permissive alanine residue allows phosphorylation of Ser-853 on mGluR1alpha. We investigated the physiological consequences of mGluR5 Ser-839 phosphorylation using Ca2+ imaging. Mutations that eliminate Ser-839 phosphorylation prevent the characteristic mGluR5-dependent Ca2+ oscillations. However, mutation of Thr-840 to alanine, which prevents potential Thr-840 phosphorylation but is still permissive for Ser-839 phosphorylation, has no effect on Ca2+ oscillations. Thus, we showed that it is phosphorylation of Ser-839, not Thr-840, that is absolutely required for the unique Ca2+ oscillations produced by mGluR5 activation. The Thr-840 residue is important only in that it is permissive for the PKC-dependent phosphorylation of Ser-839.     

The metabotropic glutamate G-protein-coupled receptors mGluR3 and mGluR1a are voltage-sensitive

G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K(+) currents and mGluR1a-induced Ca(2+)-activated Cl(-) currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [(3)H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric Galpha subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.     

Ligand binding to the amino-terminal domain of the mGluR4 subtype of metabotropic glutamate receptor

The metabotropic glutamate receptor (mGluR) 4 subtype of metabotropic glutamate receptor is a presynaptic receptor that modulates neurotransmitter release. We have characterized the properties of a truncated, epitope-tagged construct containing part of the extracellular amino-terminal domain of mGluR4. The truncated receptor was secreted into the cell culture medium of transfected human embryonic kidney cells. The oligomeric structure of the soluble truncated receptor was assessed by gel electrophoresis. In the presence of high concentrations of a reducing agent, the truncated receptor migrated as a monomer; at lower concentrations of the reducing agent, only higher molecular weight oligomers were observed. Competition binding experiments using the radiolabeled agonist [3H]L-2-amino-4-phosphonobutyric acid revealed that the rank order of potency of metabotropic ligands at the truncated receptor was similar to that of the full-length membrane-bound receptor. However, the truncated receptor displayed higher affinities for agonists and lower affinities for antagonists compared with the full-length receptor. Deglycosylation produced a shift in the relative molecular weight of the soluble protein from Mr = 71,000 to Mr = 63,000; deglycosylation had no effect on the binding of [3H]L-2-amino-4-phosphonobutyric acid, indicating that the asparagine-linked carbohydrates are not necessary for agonist binding. These results demonstrate that although the primary determinants of ligand binding to mGluR4 are contained within the first 548 amino acids of the receptor, additional amino acids located downstream of this region may influence the affinity of ligands for the binding site.