Abstract:In previous studies, we demonstrated that the neuropeptide, N-acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluRi-6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans-i -aminocyclopentane-1,3-dicarboxylate (trans-ACPD) and L-2-amino-4-phosphonobutyrate (L-AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluRi, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans-ACPD, activated each of the transfected receptors, whereas L-AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluRl a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans-ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera. Key Words: Metabotropic glutamate receptors-Glutamate-N-Acetylaspartylglutamate-Cyclic AMP-mGluR3 agonist-Neuropeptide-Chimeric receptors. J. Neurochem. 69, i74-181 (1997).A Series of structurally related metabotropic glutamate receptors (mGluRs) have been cloned, characterized, and classified within one of three groups based on sequence homology and pharmacology (Houamed et al., 1991;Masu et al., 1991;Abe et al., 1992;pin et al., 1992, 1996 Tanabe et al., 1992; Minakami et al., 1993; Nakajima eta!., 1993; Thomsen et al., 1993; Okamoto et al., 1994;Saugstad et al., 1994;Duvoisin et al., 1995;Pin and Duvoisin, 1995; Kubokawa Ct a!., 1996). Cloned group I receptors (mUluR I and mGluR5) are coupled via phosphoinositide (PT) turnover to phospholipase C and activated by quisqualate, ibotenate, and (S)-3,5-dihydroxyphenylglycine. Cloned group II receptors (mGluR2 and mGluR3) are activated most effectively by (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC), trans-i -aminocyclopentane-I ,3-dicarboxylate (trans-ACPD), and (2S,1 'R,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl )glycine (DCG-IV), whereas L-2-amirlo-4-phosphonobutyrate (L-AP4) and O-phosphono-Lserine are selective agonists for group III receptors (mGluR4, mGluR6, mGluR7, and mGluR8). Group I! and III receptor activation ...

Introduction.The amino acid glutamate is present in high concentrations in the mammalian brain, and it acts as the major excitatory neurotransmitter in the CNS. Through its actions on both ionotropic and metabotropic receptors, glutamate plays an important role in a variety of physiological functions including learning, memory, and developmental plasticity. Excessive activation of glutamate receptors or disturbances in the cellular mechanisms that protect against the adverse consequences of physiological glutamate receptor activation have been implicated in the pathogenesis of a host of neurological disorders. Although several drugs designed to attenuate the pathological consequences of excessive glutamate activation have been shown to reduce injury in experimental models of cerebral ischemia, so far none of these compounds has proven to be effective in the clinical treatment of stroke. 1 N-Acetyl-L-aspartyl-L-glutamate (NAAG) is a peptide neurotransmitter that is widely distributed in the mammalian nervous system. 2 NAAG is both an agonist at metabotropic glutamate receptors (mGluR3) 3 and a mixed agonist/antagonist at the N-methyl-D-aspartate (NMDA) receptor. 4 NAAG is hydrolyzed by the neuropeptidase glutamate carboxypeptidase II (GCPII; also known as N-acetylated R-linked acidic dipeptidase, NAALADase, or NAAG peptidase) to liberate N-acetylaspartate and glutamate both in vitro and in vivo. 5 The role of this metalloprotease GCPII is thus thought to be twofold: (1) to terminate the neurotransmitter activity of NAAG and (2) to liberate glutamate which is then able to act at the various glutamate receptor subtypes. Alterations in the levels of GCPII and NAAG have been observed in disorders that are linked to abnormalities in glutamatergic neurotransmission. 6 As a consequence of these findings, it has been hypothesized that the inhibition of GCPII might provide an effective strategy for achieving neuroprotection in cases of cerebral ischemia by increasing the levels of

Metabotropic glutamate receptor 1 (mGlu1) is a G proteincoupled 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., Dolińska, M., Klauzińska, 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 ␤-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. Metabotropic glutamate (mGlu)3 receptors are a family of G protein-coupled receptors (GPCRs) that have been categorized into three groups based on sequence homology and pharmacology (2, 3). Structural features of these receptors include a large extracellular domain containing an agonist binding site (4), seven transmembrane-spanning domains, and a variable length intracellular C-terminal domain. The second intracellular loop and portions of the C terminus are responsible for binding of G proteins and therefore for the coupling of mGlu receptors to the different second messenger systems (5, 6). Group I mGlu receptors stimulate phospholipase C (PLC) via coupling to G q/11 (7,8), which results in the hydrolysis of membrane phosphoinositides (PI) followed by increased Ca 2ϩ release from intracellular stores. Furthermore, agonist stimulation of group I mGlu receptors more recently has been shown to cause a transient phosphorylation of extracellular signal-regulated kinase (ERK) (9, 10).Several studies indicate that activation of group I mGlu receptors promotes neuronal death. Such results have been demonstrated in an in vivo model of rat traumatic brain injury and in an in vitro model of traumatic injury of rat cortical neurons (11). Toxic effects of group I mGlu receptors appear to be mediated through mechanisms including the activation of protein kinase C (12) and potentiation of NMDA and AMPA currents (13-16). In contrast, several other studies indicate that in the presence of glutamate, mGlu1 induces signaling that facilitates growth and development as opposed to neurotoxicity. When stimulated with glutamate, mGlu1 ...

To better characterize the roles of metabotropic glutamate receptors (mGluRs) in physiological and pathophysiological processes, there is an important need to learn more about the structural features relevant to the design of novel, high-affinity ligands that are family and subtype specific. To date, many of the biological studies that have been conducted in the area of mGluR research have made use of the agonist (1S,3R)-ACPD. This compound has been shown to act as an agonist at both the group I and group II receptors while showing little selectivity among the four subtypes belonging to these two groups. Moreover, (1S,3S)-ACPD, the cis isomer, shows negligible activity at group I receptors and is a good agonist of mGluR2. Since ACPD is itself somewhat flexible, with four distinctive conformations being identified from molecular modeling studies for the trans isomer and five conformations for the cis isomer, we believed that it would be of interest to examine the activity of an ACPD analogue that has been constrained through the introduction of a single carbon atom bridge. Accordingly, we have prepared an aminobicyclo[2.1.1]hexanedicarboxylic acid (ABHxD-I) analogue of ACPD. The synthesis of this compound was accomplished by use of an intramolecular [2 + 2] photocycloaddition reaction, in which four distinct isomers were isolated. Of these four compounds, only a single isomer, ABHxD-I (6a), was found to be a potent agonist of the mGluRs. This compound, which expresses the fully extended glutamate conformation, was found to be more potent than ACPD at all six of the eight mGluR subtypes that were investigated and to be comparable to or more potent than the endogenous ligand, glutamate, for these receptors. Interestingly, despite its fixed conformation, ABHxD-I, like glutamate, shows little subtype selectivity. Through modeling studies of ABHxD-I (6a), ABHD-VI, LY354740, (1S,3R)-ACPD, (1S, 3S)-ACPD, and l-glutamate, we conclude that the aa conformation of l-glutamate is the active conformation for both group I and group II mGluRs. Moreover, the modeling-based comparisons of these ligands suggest that the selectivity exhibited by LY354740 between the group I and group II mGluRs is not a consequence of different conformations of L-glutamate being required for recognition at these mGluRs but rather is related to certain structural elements within certain regions having a very different impact on the group I and group II mGluR activity. The enhanced potency of ABHxD-I relative to trans-ACPD commends it as a useful starting point in the design of subtype selective mGluR ligands.

The metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G-protein-linked receptors that couple to multiple second messengers. These include the negative modulation of adenylate cyclase, activation of phosphoinositide-specific phospholipase C, and modulation of ion channel currents. 1 Three types of mGlu receptors have been identified: group I receptors couple to phosphoinositide hydrolysis and include mGluR1 and mGluR5; group II receptors couple to the inhibition of cyclic adenosine 5′-monophosphate (cAMP) formation and include mGluR2 and mGluR3; group III receptors (mGluR4, mGluR6, mGluR7, and mGluR8) are negatively coupled to cAMP. Each subtype is distinguished on the basis of its pharmacology and sequence homology. Excessive activation of glutamate receptors or disturbances in the cellular mechanisms that protect against the adverse consequences of physiological glutamate receptor activation have been implicated in the pathogenesis of a host of neurological disorders. These disorders include epilepsy, ischemia, central nervous system trauma, neuropathic pain, and chronic neurodegenerative diseases. Because of the ubiquitous distribution of glutamatergic synapses, mGluRs have the potential to participate in a wide variety of functions in the CNS. In addition, because of the wide diversity and heterogeneous distribution of the mGluR subtypes, the opportunity exists for the development of highly selective drugs that affect a limited number of CNS functions. The mGluRs therefore provide novel targets for the development of therapeutic agents that could have a dramatic impact on the treatment of CNS disorders.To date, almost all of the commonly used agonists and antagonists employed in biological studies of the mGluRs are amino acids, often embodying a structurally rigidified glutamate-like core. 2 During our efforts to identify potent and selective ligands acting at these receptors, we have discovered an mGluR3-selective agonist that contains only acid groups and that acts simultaneously as a potent inhibitor of NAAG peptidase.

The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with ␤-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 ␤-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 ␤-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 ␤-arrestinmediated 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 ␤-arrestin-mediated cytoprotective signaling, and they reveal the existence of new endogenous agonists acting at mGlu1 receptors.

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 characteristic 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.

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