RESUMO
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.
Assuntos
Glioma/genética , Glioma/patologia , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromonas/farmacologia , Glioma/tratamento farmacológico , Ácido Glutâmico/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proto-Oncogene Mas , Quinolinas/farmacologia , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/genéticaRESUMO
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.
Assuntos
Melanoma/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Humanos , Ionomicina/farmacologia , Ácido Quisquálico/farmacologia , Receptores de Glutamato Metabotrópico/genética , Sesquiterpenos/farmacologia , Sesquiterpenos de Guaiano , Transdução de SinaisRESUMO
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.
Assuntos
Cerebelo/citologia , Neurônios/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Animais Recém-Nascidos , Arrestinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Hidrólise/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , beta-ArrestinasRESUMO
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.
Assuntos
Técnicas Biossensoriais/métodos , AMP Cíclico/análise , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Aminoácidos/farmacologia , Animais , Soluções Tampão , Células CHO , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/agonistas , AMP Cíclico/antagonistas & inibidores , AMP Cíclico/biossíntese , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Ácido Glutâmico/farmacologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Ensaio Radioligante , Receptores de Glutamato Metabotrópico/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Transfecção , Xantenos/farmacologiaRESUMO
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.
RESUMO
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.
Assuntos
Processamento Alternativo , Éxons , Receptores de Glutamato Metabotrópico/genética , Sequência de Bases , Linhagem Celular Tumoral , Sequência Conservada , Humanos , Melanoma/metabolismo , Dados de Sequência Molecular , Poliadenilação , Densidade Pós-Sináptica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de ProteínaRESUMO
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.
Assuntos
Arrestinas/fisiologia , Receptores de Glutamato Metabotrópico/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Ligantes , Receptores Acoplados a Proteínas G/fisiologia , beta-ArrestinasRESUMO
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'.
Assuntos
Resposta ao Choque Térmico/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Células CHO , Sobrevivência Celular , Cricetinae , Cricetulus , Vetores Genéticos/genética , Temperatura Alta , Proteínas Imediatamente Precoces/agonistas , Plasmídeos , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/genética , Transdução de Sinais , Testes de Toxicidade , TransfecçãoRESUMO
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.
Assuntos
Ácido Glutâmico/toxicidade , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Neurônios/efeitos dos fármacos , Cloreto de Potássio/toxicidade , Receptores de Glutamato Metabotrópico/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Córtex Cerebral/citologia , Cromonas/farmacologia , Dioxolanos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Purinas/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Receptores de Glutamato Metabotrópico/genética , Fatores de Tempo , TransfecçãoRESUMO
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.
Assuntos
Sítio Alostérico/efeitos dos fármacos , Benzotiadiazinas/farmacologia , Inibição Neural/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/genética , Análise de Variância , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Interações Medicamentosas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Mutagênese/fisiologia , Ratos , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Transfecção/métodosRESUMO
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).
Assuntos
Antagonistas de Aminoácidos Excitatórios/síntese química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Receptor de Glutamato Metabotrópico 5 , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
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.