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.

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, β-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response.

Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas.

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability.

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.

Ligand bias at metabotropic glutamate 1a receptors: molecular determinants that distinguish β-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 β-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.

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.

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.

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.

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.

Synthesis of N(1)-substituted analogues of (2R,4R)-4-amino-pyrrolidine-2,4-dicarboxylic acid as agonists, partial agonists, and antagonists of group II metabotropic glutamate receptors.

The chemical synthesis of a series of N(1)-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid [(2R,4R)-APDC] as constrained analogues of gamma-substituted glutamic acids is described. Appropriate substitution of the N(1)-position results in agonist, partial agonist, or antagonist activity at mGluR2, mGluR3, and/or mGluR6.

1-amino-APDC, a partial agonist of group II metabotropic glutamate receptors with neuroprotective properties.

The synthesis of the 1-amino derivative of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (1-amino-APDC), a selective metabotropic glutamate ligand, is disclosed. This compound acts as a partial agonist of the group II mGluRs and shows pronounced neuroprotective properties in the NMDA model of cell toxicity.

Synthesis and biological evaluation of two analogues of (S)-alpha-methyl-3-carboxyphenylalanine.

Two analogues 2, 3 of (S)-alpha-methyl-3-carboxylphenylalanine 1 were synthesized to test their activity for metabotropic glutamate receptors (mGluRs). Both compounds 2 and 3 were inactive as antagonist for mGluRs, but 2 showed weak agonistic activity for GluR6 in contrast to that reported by Kemp and co-workers.

alpha-substituted quisqualic acid analogs: new metabotropic glutamate receptor group II selective antagonists.

Syntheses of both the alpha-methyl and benzyl analogs of quisqualic acid are described. Testing of these compounds for their activity at excitatory amino acid receptors revealed a striking change in activity in comparison to quisqualic acid. This structural modification results in the loss of quisqualate's potent agonist action at both non-NMDA ionotropic glutamate receptors as well as at group I mGluRs, while allowing these analogs to acquire antagonist properties with relative selectivity for group II metabotropic glutamate receptors.

Synthesis and metabotropic glutamate receptor activity of a 2-aminobicyclo[3.2.0]heptane-2,5-dicarboxylic acid, a molecule possessing an extended glutamate conformation.

A photochemical approach to the synthesis of the aminobicycloheptane 6a is reported. This compound assumes an extended glutamate conformation, and for this reason was created to further probe the structural features relevant to achieving selectivity for the subtypes of the metabotropic glutamate family of receptors.

Synthesis and biology of the conformationally restricted ACPD analogue, 2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I, a potent mGluR agonist.

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.

N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells.

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 mGluR1-6 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-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD) and L-2-amino-4-phosphonobutyrate (L-AP4).

Okadaic acid stimulates nerve growth factor production via an induction of interleukin-1 in primary cultures of cortical astroglial cells.

Neonatal rat cortical astroglial cells in primary culture synthesize and secrete interleukin-1 beta (IL-1) and nerve growth factor (NGF). Treatment of astrocytes with okadaic acid (OA), an inhibitor of phosphoprotein phosphatases, dramatically increased both IL-1 and NGF mRNA content (about 50-fold) with maximal induction seen at 20-30 nM OA. The induction of IL-1 mRNA preceded that of NGF mRNA and was maximal after 9 h of treatment.

Okadaic acid increases nerve growth factor secretion, mRNA stability, and gene transcription in primary cultures of cortical astrocytes.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF) in response to cytokines, growth factors, and activators of protein kinases. To further implicate a protein phosphorylation mechanism in the regulation of NGF expression, astrocytes were treated with okadaic acid and calyculin A, inhibitors of phosphoprotein phosphatases 1 and 2A. Okadaic acid dramatically increased both NGF mRNA content (50-fold) and NGF secretion (100-fold) in astrocytes, while calyculin A, which has a spectrum of phosphatase inhibitory activity different from okadaic acid, failed to augment NGF expression.

Transcriptional and posttranscriptional mechanisms involved in the interleukin-1, steroid, and protein kinase C regulation of nerve growth factor in cortical astrocytes.

Neonatal rat cortical astrocytes in primary culture synthesize and secrete nerve growth factor (NGF). Treatment of astrocytes with interleukin-1 beta (IL-1) or the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) increased NGF mRNA content by six- to 10-fold, followed in time by increases in cell content and cell secretion of NGF. Dexamethasone potently inhibited the effects of IL-1 and TPA on astroglial cell NGF expression.

[The normalizing effect of dalargin on the glucocorticoid and opioid levels of the blood in CBA and C57Bl/6 mice undergoing footshock stress].

Radioreceptor investigations showed that it is impossible to interpret changes in the activity of opioid receptor ligands of mu and delta types in plasma of mice under footshock stress (FSS) as activation or depletion of opioid system (OS). There occurred qualitative changes characterized by interstrain differences (ID). These are also typical for dynamics of development of FSS effects on blood corticosterone levels.

[The effect of dalargin on stress-induced changes in the 5'-nucleotidase activity of the macrophages and in the level of endogenous blood cortisol in mice].

The action of dalargin, synthetic analogue of leuenkephaline, on stress-induced changes in 5'-nucleotidase activity and endogenous hydrocortisone levels was investigated in mice. It was found that there is a direct relation between the activity of 5'-nucleotidase and the level of hydrocortisone in CBA mice. For C57Bl/6 mice the relation was inverse.