The Role of Metabotropic Glutamate Receptor 1 Dependent Signaling in Glioma Viability.

Glioma refers to malignant central nervous system tumors that have histologic characteristics in common with glial cells. The most prevalent type, glioblastoma multiforme, is associated with a poor prognosis and few treatment options. On the basis of reports of aberrant expression of mGluR1 mRNA in glioma, evidence that melanoma growth is directly influenced by glutamate metabotropic receptor 1 (mGluR1), and characterization of ß-arrestin-dependent prosurvival signaling by this receptor, this study investigated the hypothesis that glioma cell lines aberrantly express mGluR1 and depend on mGluR1-mediated signaling to maintain viability and proliferation. Three glioma cell lines (Hs683, A172, and U87) were tested to confirm mGluR1 mRNA expression and the dependence of glioma cell viability on glutamate. Pharmacologic and genetic evidence is presented that suggests mGluR1 signaling specifically supports glioma proliferation and viability. For example, selective noncompetitive antagonists of mGluR1, CPCCOEt and JNJ16259685, decreased the viability of these cells in a dose-dependent manner, and glutamate metabotropic receptor 1 gene silencing significantly reduced glioma cell proliferation. Also, results of an anchorage-independent growth assay suggested that noncompetitive antagonism of mGluR1 may decrease the tumorigenic potential of Hs683 glioma cells. Finally, data are provided that support the hypothesis that a ß-arrestin-dependent signaling cascade may be involved in glutamate-stimulated viability in glioma cells and that ligand bias may exist at mGluR1 expressed in these cells. Taken together, the results strongly suggest that mGluR1 may act as a proto-oncogene in glioma and be a viable drug target in glioma treatment.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells.

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Chloride is an Agonist of Group II and III Metabotropic Glutamate Receptors.

The elemental anion chloride is generally considered a passive participant in neuronal excitability, and has never been shown to function as an agonist in its own right. We show that the antagonist-mediated, glutamate-independent inverse agonism of group II and III metabotropic glutamate (mGlu) receptors results from inhibition of chloride-mediated activation. In silico molecular modeling, site-directed mutagenesis, and functional assays demonstrate (1) that chloride is an agonist of mGlu3, mGlu4, mGlu6, and mGlu8 receptors with its own orthosteric site, and (2) that chloride is not an agonist of mGlu2 receptors. Molecular modeling-predicted and site-directed mutagenesis supported that this unique property of mGlu2 receptors results from a single divergent amino acid, highlighting a molecular switch for chloride insensitivity that is transduced through an arginine flip. Ultimately, these results suggest that activation of group II and III mGlu receptors is mediated not only by glutamate, but also by physiologically relevant concentrations of chloride.

Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism.

The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, ß-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function.

A real-time method for measuring cAMP production modulated by Gαi/o-coupled metabotropic glutamate receptors.

Group II and group III metabotropic glutamate (mGlu) receptors are G protein-coupled receptors (GPCRs) that inhibit adenylyl cyclase via activation of Gαi/o. The purpose of this study was to design a universal method that overcomes previous challenges in consistently measuring group II and group III mGlu-receptor (mGluR) activation in stably transfected systems. In Chinese hamster ovary (CHO) cells stably transfected with the GloSensor cAMP biosensor, we optimized conditions for simple and highly reproducible (<5% S.E.M.) measurements of cAMP in real time. The GloSensor cAMP biosensor is a recombinant firefly luciferase conjugated to a cAMP-binding domain, where cAMP binding promotes a conformational shift within the GloSensor protein, inducing luciferase activity; cAMP levels are positively correlated with light output resulting from the luciferase-mediated breakdown of d-luciferin. Each group II and group III mGluR was then stably transfected into the CHO-GloSensor cell line, and experimental conditions were optimized for each receptor. During assay optimization, we observed ion sensitivity of several receptors and inverse agonist activity of the antagonist, LY341495 [2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-d-alanine]. Although these phenomena have been previously reported, they remain poorly understood, emphasizing the GloSensor assay as an important tool with which to study group II and group III mGlu receptors. Our results highlight many advantages of using the GloSensor method for measuring activation of group II and group III mGlu receptors, and they further suggest that corresponding methods designed to measure activation of any Gαi/o- or Gαs-coupled GPCR will be similarly advantageous.

Triple threat treatment: Exploiting the dependence receptor properties of metabotropic glutamate receptor 1 against melanoma.

Melanoma cells that express metabotropic glutamate 1 (mGlu1) receptors depend on glutamate for their survival and proliferation. The dependence receptor properties of mGlu1 allow us to propose and justify three promising approaches for melanoma treatment: glutamate depletion, mGlu1 receptor antagonism, and targeting of mGlu1 receptor signaling.

Two newly identified exons in human GRM1 express a novel splice variant of metabotropic glutamate 1 receptor.

To date, five human metabotropic glutamate (mGlu) 1 receptor splice variants (1a, 1b, 1d, 1f, and 1g) have been described, all of which involve alternative C-terminal splicing. mGlu1a receptor contains a long C-terminal domain (341 amino acids), which has been shown to scaffold with several proteins and contribute to the structure of the post-synaptic density. However, several shorter mGlu1 receptor splice variants lack the sequence required for these interactions, and no major functional differences between these short splice variants have been described. By using RT-PCR we have shown that two human melanoma cell lines express both mGlu1a and mGlu1b receptors. In addition, using 3'RACE, we identified three previously unknown mGlu1 receptor mRNAs. Two differ in the length of their 3' untranslated region (UTR), and encode the same predicted protein as mGlu1g receptor-the shortest of all mGlu1 receptor splice variants. The third mRNA, named mGlu1h, encodes a predicted C-terminal splice variant of 10 additional amino acids. mGlu1h mRNA was observed in two different melanoma cell lines and is overexpressed, compared with melanoma precursor cells, melanocytes. Most importantly, this new splice variant, mGlu1h receptor, is encoded by two previously unidentified exons located within the human GRM1 gene. Additionally, these new exons are found exclusively within the GRM1 genes of higher primates and are highly conserved. Therefore, we hypothesize that mGlu1h receptors play a distinct role in primate glutamatergic signaling.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish ß-arrestin-mediated from G protein-mediated signaling.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ß-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the ß-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for ß-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward ß-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate-induced PI hydrolysis. Taken together, these data indicate that ligand bias at mGlu1 receptors is attributable to different modes of receptor-glutamate interactions, which are differentially coupled to PI hydrolysis and ß-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

Heat shock enhances CMV-IE promoter-driven metabotropic glutamate receptor expression and toxicity in transfected cells.

In CHO-K1 cells, heat shock strongly activated reporter-gene expression driven by the cytomegalovirus immediate-early (CMV-IE) promoter from adenoviral and plasmid vectors. Heat shock treatment (2h at 42.5 °C) significantly enhanced the promoter DNA-binding activity in nuclear extracts. In CHO cells expressing mGluR1a and mGluR5a receptors under the control of the CMV promoter, heat shock increased receptor protein expression, mRNA levels and receptor function estimated by measurement of PI hydrolysis, intracellular Ca²+ and cAMP. Hyperthermia increased average amplitudes of Ca²+ responses, the number of responding cells, and revealed the toxic properties of mGluR1a receptor. Heat shock also effectively increased the expression of EGFP. Hence, heat shock effects on mGluR expression and function in CHO cells may be attributed to the activation of the CMV promoter. Moreover, this effect was not limited to CHO cells as heat shock also increased EGFP expression in PC-12 and HEK293 cells. Heat shock treatment may be a useful tool to study the function of proteins expressed in heterologous systems under control of the CMV promoter. It may be especially valuable for increasing protein expression in transient transfections, for enhancing receptor expression in drug screening applications and to control the expression of proteins endowed with toxic properties. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

The protective signaling of metabotropic glutamate receptor 1 Is mediated by sustained, beta-arrestin-1-dependent ERK phosphorylation.

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that enhances the hydrolysis of membrane phosphoinositides. In addition to its role in synaptic transmission and plasticity, mGlu1 has been shown to be involved in neuroprotection and neurodegeneration. In this capacity, we have reported previously that in neuronal cells, mGlu1a exhibits the properties of a dependence receptor, inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence (Pshenichkin, S., Dolinska, M., Klauzinska, M., Luchenko, V., Grajkowska, E., and Wroblewski, J. T. (2008) Neuropharmacology 55, 500-508). Here, using CHO cells expressing mGlu1a receptors, we show that the protective effect of glutamate does not rely on the classical mGlu1 signal transduction. Instead, mGlu1a protective signaling is mediated by a novel, G protein-independent, pathway which involves the activation of the MAPK pathway and a sustained phosphorylation of ERK, which is distinct from the G protein-mediated transient ERK phosphorylation. Moreover, the sustained phosphorylation of ERK and protective signaling through mGlu1a receptors require expression of beta-arrestin-1, suggesting a possible role for receptor internalization in this process. Our data reveal the existence of a novel, noncanonical signaling pathway associated with mGlu1a receptors, which mediates glutamate-induced protective signaling.

Dual neurotoxic and neuroprotective role of metabotropic glutamate receptor 1 in conditions of trophic deprivation - possible role as a dependence receptor.

Group I metabotropic glutamate receptors have been often implicated in various models of neuronal toxicity, however, the role played by the individual receptors and their putative mechanisms of action contributing to neurotoxicity or neuroprotection remain unclear. Here, using primary cultures of rat cerebellar granule cells and mouse cortical neurons, we show that conditions of trophic deprivation increased mGlu1 expression which correlated with the developing cell death. The inhibition of mGlu1 expression by specific siRNA attenuated toxicity, while adenovirus-mediated overexpression of mGlu1 resulted in increased cell death, indicating a causal relationship between the level of receptor expression and neuronal survival. In pharmacological experiments selective mGlu1 antagonists failed to protect from mGlu1-induced cell death, instead, neuronal survival was promoted by glutamate acting at mGlu1 receptors. Such properties are characteristics of a novel heterogeneous family of dependence receptors which control neuronal apoptosis. Our findings indicate that increased expression of mGlu1 in neurons creates a state of cellular dependence on the presence of its endogenous agonist glutamate. We propose a new role and a new mechanism for mGlu1 action. This receptor may play a crucial role in determining the fate of individual neurons during the development of the nervous system.

Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists.

Metabotropic glutamate receptors mGluR1 and mGluR5 stimulate phospholipase C, leading to an increased inositol trisphosphate level and to Ca(2+) release from intracellular stores. Cyclothiazide (CTZ), known as a blocker of AMPA receptor desensitization, produced a non-competitive inhibition of [Ca(2+)](i) increases induced by mGluR agonists in HEK 293 cells transfected with rat mGluR1a but had no effect on the [Ca(2+)](i) signals in cells expressing rat mGluR5a. In cells expressing mGluR1, CTZ also inhibited phosphoinositide hydrolysis, as well as cAMP accumulation and arachidonic acid release induced by mGluR1 agonists, indicating a direct inhibition of the receptor and not of a particular signal transduction system. However, CTZ failed to antagonize cAMP inhibition stimulated by rat mGluR2, -3, -4, -6, -7 and -8 receptors confirming its selectivity for mGluR1. The use of chimeric receptors with substituted N-terminal domains showed that CTZ did not interact with the N-terminal mGluR1a domain. Instead, mutation analysis revealed that CTZ interacts with the Thr-815 and Ala-818 residues, located at the 7th transmembrane domain, similarly as the mGluR1-selective antagonist CPCCOEt. In primary cultures of cerebellar granule neurons, expressing native metabotropic and ionotropic glutamate receptors, the final outcome of CTZ effects depended on its combined ability to potentiate AMPA receptors and inhibit mGluR1 receptors.

Synthesis and structure-activity relationships of 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine analogues as potent, noncompetitive metabotropic glutamate receptor subtype 5 antagonists; search for cocaine medications.

Recent genetic and pharmacological studies have suggested that the metabotropic glutamate receptor subtype 5 (mGluR5) may represent a druggable target in identifying new therapeutics for the treatment of various central nervous system disorders including drug abuse. In particular, considerable attention in the mGluR5 field has been devoted to identifying ligands that bind to the allosteric modulatory site, distinct from the site for the primary agonist glutamate. Both 2-methyl-6-(phenylethynyl)pyridine (MPEP) and its analogue 3-[(2-methyl-4-thiazolyl)ethynyl]pyridine (MTEP) have been shown to be selective and potent noncompetitive antagonists of mGluR5. Because of results presented in this study showing that MTEP prevents the reinstatement of cocaine self-administration caused by the presentation of environmental cues previously associated with cocaine availability, we have prepared a series of analogues of MTEP with the aim of gaining a better understanding of the structural features relevant to its antagonist potency and with the ultimate aim of investigating the effects of such compounds in blunting the self-administration of cocaine. These efforts have led to the identification of compounds showing higher potency as mGluR5 antagonists than either MPEP or MTEP. Two compounds 19 and 59 exhibited functional activity as mGluR5 antagonists that are 490 and 230 times, respectively, better than that of MTEP.

Neuroprotective effects of the agonist of metabotropic glutamate receptors ABHxD-I in two animal models of cerebral ischaemia.

The neuroprotective efficacy of 2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I (ABHxD-I), a rigid agonist of metabotropic glutamate receptors, was studied using a 3-min global cerebral ischaemia model in Mongolian gerbils and the hypoxia/ischaemia model in 7-day-old rats. The effects on brain damage of ABHxD-I (30 mg/kg, intraperitoneally or 7.5 microg intracerebroventricularly) administered 30 min before global ischaemia or 30 min after hypoxia/ischaemia was evaluated 14 days after the insults. Treatment of adult gerbils with ABHxD-I injected i.c.v. but not systemically, prevented post-ischaemic hyperthermia and substantially reduced brain damage. These effects may reflect low permeability of the adult blood-brain barrier to ABHxD-I, and the role of reduced body and brain temperature in neuroprotection after its i.c.v. administration. ABHxD-I given either i.p. or i.c.v. to developing rats reduced brain damage by 55 and 37%, respectively, without affecting the body temperature. Due to immaturity and increased post-ischaemic permeability of the blood-brain barrier in developing rats, ABHxD-I may induce neuroprotection by direct interference with brain metabotropic glutamate receptors.

Functionalization at position 3 of the phenyl ring of the potent mGluR5 noncompetitive antagonists MPEP.

We described the synthesis and biological evaluation of MPEP analogs functionalized at the position 3 of the phenyl ring. The results point out the limitation in the choice of a functional group at this position; the only substituents leading to retention of activity are NO(2) (IC(50)=13 nM) and CN (IC(50)=8 nM).

Synthesis and receptor assay of aromatic-ethynyl-aromatic derivatives with potent mGluR5 antagonist activity.

Noncompetitive antagonists of the human metabotropic glutamate receptor subtype 5 (mGluR5) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and drug addiction. To discover novel noncompetitive antagonists to the mGluR5, we initiated an SAR study around the known lead compounds MPEP and M-MPEP. Our results pointed out the critical role of the para position of the two aromatic rings, which leads to inactive products and permitted the discovery of potent mGluR5 antagonists (e.g., 16, 25, 28, 34 IC(50) = 13.5, 11.9, 21, 15 nM, respectively).

NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR.

Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP.

Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents.

The neuropeptidase glutamate carboxypeptidase II (GCPII) hydrolyzes N-acetyl-L-aspartyl-L-glutamate (NAAG) to liberate N-acetylaspartate and glutamate. GCPII was originally cloned as PSMA, an M(r) 100,000 type II transmembrane glycoprotein highly expressed in prostate tissues. PSMA/GCPII is located on the short arm of chromosome 11 and functions as both a folate hydrolase and a neuropeptidase. Inhibition of brain GCPII may have therapeutic potential in the treatment of certain disease states arising from pathologically overactivated glutamate receptors. Recently, we reported that certain urea-based structures act as potent inhibitors of GCPII (J. Med. Chem. 2001, 44, 298). However, many of the potent GCPII inhibitors prepared to date are highly polar compounds and therefore do not readily penetrate the blood-brain barrier. Herein, we elaborate on the synthesis of a series of potent, urea-based GCPII inhibitors from the lead compound 3 and provide assay data for these ligands against human GCPII. Moreover, we provide data revealing the ability of one of these compounds, namely, 8d, to reduce the perception of inflammatory pain. Within the present series, the gamma-tetrazole bearing glutamate isostere 7d is the most potent inhibitor with a K(i) of 0.9 nM. The biological evaluation of these compounds revealed that the active site of GCPII likely comprises two regions, namely, the pharmacophore subpocket and the nonpharmacophore subpocket. The pharmacophore subpocket is very sensitive to structural changes, and thus, it appears important to keep one of the glutamic acid moieties intact to maintain the potency of the GCPII inhibitors. The site encompassing the nonpharmacophore subpocket that binds to glutamate's alpha-carboxyl group is sensitive to structural change, as shown by compounds 6b and 7b. However, the other region of the nonpharmacophore subpocket can accommodate both hydrophobic and hydrophilic groups. Thus, an aromatic ring can be introduced to the inhibitor, as in 8b and 8d, thereby increasing its hydrophobicity and thus potentially its ability to cross the blood-brain barrier. Intrathecally administered 8d significantly reduced pain perception in the formalin model of rat sensory nerve injury. A maximal dose of morphine (10 mg) applied in the same experimental paradigm provided no significant increase in analgesia in comparison to 8d during phase 1 of this pain study and modestly greater analgesia than 8d in phase 2. These urea-based inhibitors of GCPII thus offer a novel approach to pain management.

L-homocysteine sulfinic acid and other acidic homocysteine derivatives are potent and selective metabotropic glutamate receptor agonists.

Moderate hyperhomocysteinemia is associated with several diseases, including coronary artery disease, stroke, Alzheimer's disease, schizophrenia, and spina bifida. However, the mechanisms for their pathogenesis are unknown but could involve the interaction of homocysteine or its metabolites with molecular targets such as neurotransmitter receptors, channels, or transporters. We discovered that L-homocysteine sulfinic acid (L-HCSA), L-homocysteic acid, L-cysteine sulfinic acid, and L-cysteic acid are potent and effective agonists at several rat metabotropic glutamate receptors (mGluRs). These acidic homocysteine derivatives 1) stimulated phosphoinositide hydrolysis in the cells stably expressing the mGluR1, mGluR5, or mGluR8 (plus Galpha(qi9)) and 2) inhibited the forskolin-induced cAMP accumulation in the cells stably expressing mGluR2, mGluR4, or mGluR6, with different potencies and efficacies depending on receptor subtypes. Of the four compounds, L-HCSA is the most potent agonist at mGluR1, mGluR2, mGluR4, mGluR5, mGluR6, and mGluR8. The effects of the four agonists were selective for mGluRs because activity was not discovered when L-HCSA and several other homocysteine derivatives were screened against a large panel of cloned neurotransmitter receptors, channels, and transporters. These findings imply that mGluRs are candidate G-protein-coupled receptors for mediating the intracellular signaling events induced by acidic homocysteine derivatives. The relevance of these findings for the role of mGluRs in the pathogenesis of homocysteine-mediated phenomena is discussed.