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J Biol Chem. 2010 Aug 20; 285(34): 26041–26048.
Published online 2010 Jun 21. doi: 10.1074/jbc.M110.139899
PMCID: PMC2924003
PMID: 20566651

The Protective Signaling of Metabotropic Glutamate Receptor 1 Is Mediated by Sustained, β-Arrestin-1-dependent ERK Phosphorylation*

Andrew C. Emery,‡§,1 Sergey Pshenichkin,§ Guy Rodrigue Takoudjou,‡ Ewa Grajkowska,‡ Barry B. Wolfe,‡§ and Jarda T. Wroblewski‡§,2
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

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., 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 β-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.

Keywords: Cell Death, Cell Surface Receptor, ERK, G Protein-Coupled Receptors (GPCR), Glutamate, Glutamate Receptors Metabotropic, Inositol Phosphates, Receptor Endocytosis, Beta-Arrestin

Introduction

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 Gq/11 (7, 8), which results in the hydrolysis of membrane phosphoinositides (PI) followed by increased Ca2+ 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 has been shown to stimulate axon migration in the developing CNS (17) and outgrowth of dendritic spines in the developing hippocampus (18). We have described recently that mGlu1a produces dual neuroprotective and neurotoxic signaling in cerebellar and cortical neurons (1). Thus, mGlu1a exhibits the properties of a dependence receptor (19, 20), inducing apoptosis in the absence of glutamate, while promoting neuronal survival in its presence. However, the mechanisms of this neuroprotective signaling are unknown.

Like other G protein-coupled receptors, mGlu1a is internalized both constitutively and in response to agonist stimulation (21). In the presence of agonists, mGlu1a undergoes rapid homologous desensitization and can be internalized in a β-arrestin-dependent manner (21, 22). In the process of β-arrestin-mediated endocytosis, mGlu1a may be phosphorylated by several G protein-coupled receptor kinases (23). The association of receptors with β-arrestin also has been shown to induce receptor-mediated transient phosphorylation of ERK (23). ERK functions in the MAPK pathway downstream of MEK1/2 and the phosphorylation of ERK is a critical step in signal transduction from the membrane to the nucleus and usually causes protective or mitogenic cellular responses (24). Although a relatively recently described signal transduction pathway, numerous GPCRs have been shown to couple to ERK phosphorylation in a β-arrestin-dependent manner (25). However, ERK can be activated by both β-arrestin-dependent and Gq/11-dependent signaling pathways (26). In several systems, including PAC1 and VPAC receptors, these parallel pathways have been described where ERK phosphorylation due to G protein activation is transient, whereas phosphorylation is sustained over time when ERK is bound to β-arrestin (27).

The aim of this study was to identify the signal transduction pathway through which glutamate causes protective signaling in cells expressing mGlu1a receptors. We have determined that glutamate, but not quisqualate, stimulates a sustained phosphorylation of ERK through mGlu1a receptors. This phenomenon was unique to mGlu1a and required the expression of β-arrestin-1. Moreover, inhibition of ERK phosphorylation and silencing of β-arrestin-1 expression abolished the protective effects of glutamate. We conclude that the protective signaling of mGlu1a receptors does not rely on the classical, G protein-mediated, mechanism of signal transduction, but, instead involves a β-arrestin-dependent receptor internalization and ERK phosphorylation.

EXPERIMENTAL PROCEDURES

Materials

DMEM and fetal bovine serum for cell cultures were purchased from Invitrogen. Receptor agonists glutamate and quisqualate, antagonists CPCCOEt, YM 298198, and JNJ16259685, and inhibitors U73122, U0126, PD98059, and dynasore were obtained from Tocris Cookson (Ellisville, MO). All other chemicals were purchased from Sigma.

Cell Cultures

CHO cells were stably transfected with mGlu receptor cDNA in pcDNA-3.1 vector (Invitrogen) using Effectene transfection reagent (Qiagen, Hilden, Germany). Individual cell lines were isolated and cultured in DMEM supplemented with 10% fetal bovine serum, 2 mm glutamine, and 0.8 mg/ml G-418 for selection (Invitrogen). For viability assays, confluent cells were treated in media not containing serum or l-glutamine.

Treatment of Cells with β-Arrestin-1 shRNA

SureSilencing shRNA targeted against rat β-arrestin-1 (insert sequence, ATGGAGGAAGC-TGATGATACT) and scrambled control were contained in the pGeneClip hygromycin vector (SABiosciences, Frederick, MD). CHO cells stably expressing mGlu1a were transfected with plasmid-based shRNA and selected in 0.8 mg/ml hygromycin B. Knockdown of β-arrestin-1 was confirmed by Western blotting. Refractory to the effects of shRNA targeted against rat β-arrestin-1, human β-arrestin-1, which was subcloned in pcDNA 6.2, was used in rescue experiments.

Western Blots

Cells grown and treated in 35-mm dishes were collected in 25 mm Tris-HCl buffer, pH 7.5, containing Halt Protease and Phosphatase Inhibitor Cocktails with 1 mm EDTA (Pierce). Proteins were solubilized in Laemmli buffer containing 50 mm Dithiothreitol, and equal amounts of sample protein were resolved on 8% polyacrylamide gels (Invitrogen). Proteins were transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA) and were probed with antibodies against mGlu1a (BD Biosciences), β-arrestin-1 (Abcam, Inc., Cambridge, MA), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), and total p44/42 MAPK (ERK1/2) (Cell Signaling Technology, Danvers, MA). Proteins were visualized by incubation with goat anti-rabbit secondary antibodies coupled to horseradish peroxidase (Pierce) followed by exposure to chemiluminescent HRP substrate SuperSignal West Femto (Pierce). Films were quantified by densitometry using ImageJ software (Rasband, ImageJ).

Receptor Internalization Assays

Receptor endocytosis was assayed as described previously (28). Cells grown in 35-mm dishes were washed in PBS and labeled for 30 min with cleavable Sulfo-NHS-SS-Biotin (Pierce). For 20 min, excess biotin was quenched with 100 mm glycine. Labeled cells were then incubated for 30 min in the absence or presence of glutamate at 37 °C. Stimulation was halted by washing and chilling the cells in cold PBS. Extracellular biotin was then cleaved by two washes with 50 mm glutathione, 75 mm NaCl, 5 mm NaOH, and 10% FBS in water for 15 min. Excess glutathione was then quenched for 30 min in PBS containing 50 mm iodoacetamide and 1% BSA. Cells were then lysed in radioimmune precipitation assay buffer (0.5% Triton X-100, 10 mm Tris-HCl, pH 7.5, 120 mm NaCl, 25 mm KCl, and Halt protease inhibitor mixtures) and clarified, and equal biotinylated proteins were precipitated with NeutrAvidin-agarose (Pierce). Proteins were rinsed four times with radioimmune precipitation assay buffer, solubilized in Laemmli buffer, and resolved by SDS-PAGE.

Measurement of ERK Phosphorylation

Phosphorylated ERK was measured using cell-based ELISA according to a protocol described previously (29). Cells were grown and treated with agonists in 96-well plates. After incubation with agonist, cells were fixed in 4% formaldehyde/PBS for 20 min at room temperature. After three washes in 0.1% Triton X-100 in PBS (PBST) for membrane permeabilization, endogenous peroxidase activity was quenched by a 20-min incubation in PBS containing 0.5% H2O2 and 0.2% NaN3. After three more washes in PBST, cells were blocked with 2% BSA for 1 h and incubated overnight with primary antibody against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling Technology). Cells were then washed for 5 min three times in PBST and twice in PBS. An HRP-coupled goat anti-rabbit secondary antibody (Pierce) was incubated for 1 h at room temperature, and then cells were again washed five times. Cells were exposed to the colorimetric HRP substrate one-step ultra TMB (Pierce). After 10 min of developing, the reaction was stopped in 4 m H2SO4, and absorbance was read at 450 nm.

Assessment of Cell Viability

Viability of cells cultured on 96-well plates was measured by incubation for 1 h at 37 °C with 0.2 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, which was purchased from Invitrogen. The formation of the formazan product, proportional to the number of viable cells, was measured colorimetrically at 570 nm after extraction with 70 μl DMSO (30).

Measurements of PI Hydrolysis

Cells, cultured in 96-well plates, were incubated overnight with 0.625 μCi/well myo-[3H]inositol (PerkinElmer Life Sciences) to label the cell membrane phosphoinositides. After two washes with 0.1 ml of Locke's buffer (156 mm NaCl, 5.6 mm KCl, 3.6 mm NaHCO3, 1 mm MgCl2, 1.3 mm CaCl2, 5.6 mm glucose, and 20 mm Hepes, pH 7.4) incubations with receptor ligands were carried out for 45 min at 37 °C in Locke's buffer containing 20 mm LiCl to block inositol phosphate degradation. The reaction was terminated by aspiration of media and inositol phosphates were extracted with 60 μl formic acid (0.1 m) for 30 min. 40-μl samples were transferred to opaque-walled plates and incubated with 60 μl polylysine-coated yttrium scintillation proximity assay beads (PerkinElmer Life Sciences) and incubated at room temperature for 1 h with vigorous shaking. After 8 h of incubation with SPA beads, inositol phosphates were detected by scintillation counting.

Calculations and Statistics

For dose-response data, curves were fitted by nonlinear regression to data points using four-parameter logistic equation. Significance testing was performed using Student's t tests when comparing two groups or by Bonferroni-corrected t tests when comparing multiple groups. Statistical significance was deemed by p < 0.01.

RESULTS

Protective Effects of Glutamate

Our previous studies in primary cultures of cortical and cerebellar neurons have shown that glutamate, acting selectively at mGlu1a receptors, rescued these neurons from apoptotic cell death induced by conditions of trophic deprivation (1). This protective effect of glutamate was revealed in the presence of antagonists of ionotropic glutamate receptors, which suppressed the excitotoxic actions of glutamate. Moreover, in these cells, the protective effect was elicited by high concentrations of glutamate, ∼10 times higher than those needed to activate mGlu1-mediated PI hydrolysis.

To study the pharmacological and protective properties of mGlu1a without interference from other glutamate receptors, we used CHO cells with stably expressed mGlu1a receptors to assess the potency of mGlu1 agonists in producing the protective effect. Cells were transferred to serum-free culture medium to induce apoptosis (31), and their viability was monitored by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Under these conditions, after 3 days, cells exhibited ∼35–50% viability relative to controls grown in serum-containing medium. Cells exposed to 3 mm glutamate were fully protected from serum deprivation-induced apoptosis for up to 6 days, having similar viability to control cells grown in serum-containing growth media, whereas lower concentrations of glutamate (0.3 mm) produced a partial protection from serum deprivation (Fig. 1A). The protective effect of glutamate required the presence of mGlu1a as untransfected CHO cells were not protected from serum deprivation (Fig. 1B). Also, among PLC-coupled mGlu receptors only mGlu1, but not mGlu5, was effective in protecting from toxicity induced by serum deprivation (Fig. 1B).

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Pharmacological properties of mGlu1a-mediated protection in CHO cells. A, time course of serum deprivation-induced toxicity and protection by glutamate in CHO cells expressing mGlu1a receptors. B, the effect of 1 mm glutamate on serum deprivation-induced toxicity for 3 days on CHO cells expressing mGlu1a, mGlu5a, or untransfected CHO cells. C, potency of glutamate to protect CHO cells expressing mGlu1a receptors from toxicity due to serum deprivation (EC50 = 154 ± 31 μm). D, the protective effect of glutamate blocked by three selective noncompetitive mGlu1 antagonists CPCCOEt (CP; 100 μm), YM-298198 (YM; 10 μm), and JNJ16259685 (JNJ; 30 μm)). E, dose-response curves of glutamate-mediated protection in the presence of varying concentrations of YM-298198. Cell viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide after 72 h of incubation with the agonists in serum-free conditions and is expressed as a percent of cells viable in parallel cultures grown in the presence of serum. All data points are means from at least three independent experiments performed in triplicate with error bars representing S.E. **, p < 0.001 as compared with untreated controls using Student's t test.

The addition of glutamate to the serum-free culture medium produced a dose-dependent increase in viability with an EC50 of 153 + 31 μm; however, quisqualate, the most potent mGlu1 agonist, was not effective in concentrations up to 3 mm (Fig. 1C). To further ascertain that the protective effect is in fact due to the activation of mGlu1, and not to a nonspecific effect involving glutamate transporters, glutamate-induced protection was tested in the presence of three distinct noncompetitive mGlu1-selective antagonists. Application of 100 μm CPCCOEt (32), 10 μm YM-298198 (33), or 30 μm JNJ16259685 (34) completely inhibited the activity of 1 mm glutamate (Fig. 1D). A more detailed analysis showed that YM-298198 inhibited glutamate-induced protection in a noncompetitive manner with increasing concentrations of the antagonist decreasing glutamate efficacy but having no effect on its potency as shown by similar glutamate EC50 values in the presence of different concentrations of antagonist (Fig. 1E and Table 1).

TABLE 1

Comparison of potency and efficacy of glutamate to induce protection and stimulate PI hydrolysis in the absence and presence of the noncompetitive antagonist YM-289198 in CHO cells expressing mGlu1a receptors

Potency (EC50) refers to the concentration of glutamate producing a half-maximal effect. Efficacy (Emax) represents the maximal effect of glutamate expressed as a percent of control cell viability in the presence of serum for toxicity experiments and as a percent of basal activity for measurements of PI hydrolysis. Data are means ± S.E. calculated from dose-response curves obtained in three to six experiments.

Protection from toxicity
PI hydrolysis
EC50EmaxEC50Emax
μm% controlμm% basal
YM-298198
    0 μm191 ± 2793 ± 1.728 ± 3.1259 ± 5.0
    0.03 μm180 ± 1174 ± 3.6a42 ± 4.8a233 ± 16
    0.3 μm160 ± 5663 ± 2.4a94 ± 6.0a198 ± 14a
    3 μm153 ± 2053 ± 0.9a171 ± 11a157 ± 12a

a p < 0.05 compared with values obtained in the absence of antagonist using Bonferroni-adjusted t test.

Pharmacological Properties of mGlu1

The observed pharmacological properties of mGlu1a in inducing the protective effect were inconsistent with its known properties in stimulating G protein-mediated PI hydrolysis (7). To study this discrepancy, we have determined the potency of agonists to stimulate PI hydrolysis in CHO cells expressing mGlu1a that were used in these experiments. As shown in Fig. 2A, both glutamate and quisqualate stimulated PI hydrolysis in a dose-dependent manner with quisqualate being more potent (EC50 = 0.63 ± 0.13 μm) than glutamate (EC50 = 16 ± 1.2 μm). Hence, the potency of glutamate to induce protection was ∼10 times lower than its potency to stimulate PI hydrolysis.

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Pharmacological properties of mGlu1a-mediated signal transduction in CHO cells expressing mGlu1a receptors. A, PI hydrolysis is activated in a dose-dependent manner by glutamate (Glu; EC50 = 11 ± 1.2 μm) and quisqualate (Quis; EC50 = 0.63 ± 0.13 μm). B, stimulation of PI hydrolysis by fresh agonists and agonists conditioned in the presence of CHO cells for 3 days. EC50 for fresh and conditioned agonists was as follows: glutamate (19 ± 3.2 μm and 12 ± 2.6 μm) and quisqualate (0.23 ± 0.11 μm and 0.24 ± 0.13 μm). C, dose-response curves of glutamate-induced PI hydrolysis in the presence of varying concentrations of mGlu1-selective noncompetitive antagonist YM-298198. With increasing concentrations of YM, the EC50 for glutamate increases (see Table 1), indicating the presence of receptor reserve. All data points are means from at least three independent experiments performed in triplicate with error bars representing S.E.

One possible explanation for these differences would be that the potencies observed in protection reflect an artifactual decrease of agonist concentrations occurring during the 3-day incubation that would not appear in the 45-min incubation used when measuring PI hydrolysis. In control experiments testing agonist stability, glutamate and quisqualate were incubated with CHO cells for 3 days and then used to stimulate PI hydrolysis. As shown in Fig. 2B, both conditioned and freshly prepared agonists stimulated PI hydrolysis to the same extent, demonstrating that these compounds remain intact for the entire 3-day incubation and that the observed discrepancies do not result from a decrease in agonist concentration.

As expected, glutamate-stimulated PI hydrolysis was abolished by the selective noncompetitive mGlu1 antagonist YM-298198 (33), and the antagonism appeared noncompetitive as seen by the decrease of the maximal effect of glutamate with increasing concentrations of the antagonist (Fig. 2C). However, in contrast with the data obtained for glutamate protective signaling, the calculated potency of glutamate in PI hydrolysis experiments decreased with increasing antagonist concentrations (Table 1). Usually, such right shifts of agonist potency in the presence of a noncompetitive antagonist indicate the existence of receptor reserve (also known as spare receptors) and are frequently seen in heterologous expression systems. This allows us to conclude that the apparent difference in potency of glutamate to produce both effects results from the presence of a high receptor reserve for PI hydrolysis but no receptor reserve for protective signaling. The use of the highest concentration of YM-298198 in PI hydrolysis experiments increases the EC50 of glutamate to the levels observed in protection experiments. Hence, once this receptor reserve is abolished by the use of a noncompetitive antagonist, the potency of glutamate to produce both effects is the same.

Although our pharmacological results confirm that the protective signaling and PI hydrolysis are mediated by the same receptor, they also indicate the existence of ligand bias in producing the two effects. Ligand bias is defined as the ability of some agonists, such as quisqualate, to activate only selected signal transduction mechanisms, whereas other agonists, such as glutamate, may activate all signaling associated with this receptor (25). Our data also suggest that the protective effect mediated by mGlu1a receptor is not related to its ability to stimulate PI hydrolysis but, rather, is mediated by a different signal transduction mechanism.

Signal Transduction of Protective Signaling through mGlu1

To identify the mechanism through which mGlu1a elicits protective signaling, we used a selective inhibitor of signaling, which occurs downstream of G protein activation. Because mGlu1 typically couples to PLC through Gq/11 (7, 8), transfected CHO cells were treated with the PLC inhibitor U73122 (35). In conditions of serum deprivation for 3 days, U73122 (30 μm) failed to block the protective effect of glutamate (Fig. 3A). In control experiments, 30 μm culture-conditioned U73122 effectively blocked glutamate-induced PI hydrolysis (Fig. 3B), indicating that this compound is stable in these culture conditions and is effective in blocking PI hydrolysis in this model. These data, therefore, indicate that the protective effect is not mediated by the coupling of mGlu1a receptors to PLC.

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Protective signaling through mGlu1a is independent of PI hydrolysis but requires the phosphorylation of ERK. A, CHO cells expressing mGlu1a were subjected to serum deprivation for 72 h in the presence of glutamate and inhibitors U73122 (30 μm), PD98059 (10 μm), U0126 (10 μm), or dynasore (100 μm). Phospholipase C inhibitor U73122 fails to block protection by glutamate, whereas protection is blocked by MEK1 inhibitor PD98059, MEK 1/2 inhibitor U0126, and dynamin inhibitor, dynasore. B, inhibition of glutamate-induced PI hydrolysis in mGlu1a-expressing CHO cells by U73122 (30 μm) that is freshly prepared (U73) or preconditioned (U73-C) by exposure to CHO cells for 3 days. C, inhibition of glutamate-induced internalization of mGlu1a receptors by dynasore (100 μm). Cells were labeled with cleavable biotin and stimulated with glutamate for 1 h. Extracellular biotin was removed and internalized mGlu1a receptors were pulled down in NeutrAvidin. P, pellet (internalized during incubation); S, supernatant (remaining receptors); Con, control. D, quantification of Western blot from Fig. 2C. Values are means from at least three independent experiments performed in triplicate with error bars representing S.E. **, p < 0.001 as compared with serum-deprived controls using Bonferroni-adjusted t test.

Because mGlu1 also has been reported to activate the MAP kinase pathway (23, 36), we investigated the involvement of this pathway in the protective effect of glutamate. Indeed, treatment of mGlu1a-expressing CHO cells with MEK1 inhibitor PD98059 (37) or with the MEK1/2 inhibitor U0126 (38) abolished the efficacy of glutamate to cause protective signaling (Fig. 3A). Furthermore, dynasore, an inhibitor of dynamin, a protein in the endocytotic pathway for mGlu1 (21, 39), also abolished glutamate-induced protection. In control experiments, monitoring receptor trafficking, dynasore (100 μm) blocked glutamate-induced internalization of mGlu1 receptors (Fig. 3, C and D). These data suggest that the protective effect of glutamate is not mediated by the classical G protein-mediated mechanism, but instead, a G protein-independent activation of MAPK pathway, most probably involving receptor internalization and the phosphorylation of ERK.

Time Course and Pharmacology of mGlu1-mediated ERK Phosphorylation

Previous reports have demonstrated that the stimulation of group I mGlu receptors causes transient ERK phosphorylation that occurs due to G protein activation and subsides within 30 min of agonist application (10). As shown by Western blot (Fig. 4, A and B), in CHO cells expressing mGlu1, glutamate and quisqualate increased ERK phosphorylation at 5 min after agonist application. In addition, when agonists were added for 24 h, glutamate, but not quisqualate, produced a sustained ERK phosphorylation. As shown in Fig. 4A, both isoforms of ERK were equally phosphorylated.

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Effect of mGlu1 agonists on ERK phosphorylation in CHO cells. A, representative Western blot of phosphorylated ERK (pERK) and total ERK (ERK) at 5 min and 24 h after the application of glutamate or quisqualate. B, quantification of Western blot from A, expressed as the ratio of measured intensities for the sum of pERK bands divided by total ERK. ELISA-based quantification of ERK phosphorylation in CHO cells expressing mGlu1a (C) or mGlu5a (D) receptors treated with agonists for 5 min, 30 min, and 24 h. Agonist profiles of ERK phosphorylation in CHO cells expressing mGlu1a receptors after 5 min (E) and 24 h (F) of treatment. Glu, glutamate; Quis, quisqualate; YM, YM-298198 (10 μm). All values are means from at least three experiments performed in triplicate with error bars representing S.E. and are expressed as the percent of ERK phosphorylation measured in cells not treated with agonists. *, p < 0.01 and **, p < 0.001 as compared with untreated controls using Bonferroni-adjusted t test.

These effects were further quantified using an ELISA-based assay to compare the levels of ERK phosphorylation in CHO cells expressing either mGlu1a or mGlu5a receptors. In cells expressing mGlu5a, both glutamate and quisqualate elicited a transient ERK phosphorylation, which was elevated at 5 min after the addition of agonists and subsided after 30 min (Fig. 4D). However, in cells expressing mGlu1a, while both agonists induced a transient increase in ERK phosphorylation, after the addition of glutamate ERK phosphorylation was elevated up to 24 h (Fig. 4C). A closer look at the pharmacology of ERK phosphorylation revealed that the transient, 5-min phosphorylation was induced by both glutamate and quisqualate (Fig. 4E), with a pharmacological profile and agonist potencies similar to those observed for PI hydrolysis (Fig. 2A). Also, like PI hydrolysis, agonist-induced ERK phosphorylation was abolished by the mGlu1-selective noncompetitive antagonist YM-298198 (Fig. 4E). In contrast, the sustained 24-h ERK phosphorylation was only induced by glutamate, but not by quisqualate, and was blocked by YM-298198 (Fig. 4F). The observed agonist potencies were lower than those for PI hydrolysis and transient ERK phosphorylation, but approximated agonist potencies observed for the protective signaling through mGlu1a (Fig. 1C).

Together, these data indicate that transient (5 min) ERK phosphorylation is activated by both mGlu1a and mGlu5a receptors and has a pharmacological profile similar to the activation of PI hydrolysis, hence is mediated by a G protein-mediated mechanism. In contrast, the sustained ERK phosphorylation, lasting up to 24 h, only is activated by mGlu1a, and only glutamate is able to produce protective mGlu1a signaling. These results further support the existence of a ligand bias, which extends to ERK phosphorylation and reveals the existence of two distinct signal transduction mechanisms. The classical mechanism, activated by both glutamate and quisqualate, mGlu1 agonists with an apparent high potency, involves a G protein-mediated PI hydrolysis and transient ERK phosphorylation. The second mechanism, which correlates well with protective mGlu1a signaling, is activated with an apparent lower potency by glutamate and is mediated by a G protein-independent mechanism, which leads to prolonged activation of MAPK pathway and sustained ERK phosphorylation.

The Role of β-Arrestin-1 in Sustained ERK Phosphorylation and the Protective Signaling of mGlu1a

Based on similarities with other GPCRs (40), we hypothesized that the mGlu1a-induced phosphorylation of ERK may involve a β-arrestin-1-dependent internalization of mGlu1a receptors and formation of a signaling complex mediating this protective signaling. To investigate this hypothesis, the expression of β-arrestin-1 in mGlu1a-expressing CHO cells was silenced using shRNA targeted against rat β-arrestin-1. Transfection with a plasmid encoding shRNA caused a substantial decrease in protein expression levels of β-arrestin-1 as monitored by Western blots, whereas a scrambled shRNA used in control experiments had no effect (Fig. 5, A and B). In rescue experiments, cells expressing targeted shRNA were transfected with cDNA for human β-arrestin-1, which is refractory to the silencing effects of rat shRNA due to having mismatches of several bases. Expression of human β-arrestin-1 caused an approximate 3-fold increase of β-arrestin-1 protein levels relative to untransfected controls (Fig. 5, A and B). Additional control experiments showed that shRNA silencing of β-arrestin-1 failed to decrease agonist-induced PI hydrolysis in CHO cells expressing mGlu1a receptors (Fig. 5D). In contrast, β-arrestin-1 shRNA effectively abolished the protective effect of glutamate against serum deprivation (Fig. 5C). Protection by glutamate was restored after transfection of human β-arrestin-1 (Fig. 5C).

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Effect of shRNA silencing of β-arrestin-1 on mGlu1a signal transduction in CHO cells. A, protein levels of β-arrestin-1 in mGlu1a-expressing CHO cells. Samples in lane 3 were transfected with plasmids encoding shRNA targeted against rat β-arrestin-1, which reduces expression to 24% of untransfected controls (lane 1). Scrambled control shRNA does not affect protein levels of β-arrestin-1 (lane 2). Knockdown due to shRNA is reversed by transfection of human β-arrestin-1 (lane 4). Knockdown due to shRNA is reversed by transfection of human β-arrestin-1 (lane 4). Transfection with human β-arrestin-1 causes expression levels of 314% relative to untransfected controls. B, quantification of the Western blot expressed as a ratio of band densities measured for β-arrestin-1 and β-actin. C, protective signaling through mGlu1a is abolished by shRNA silencing of β-arrestin-1. In shRNA-expressing cells, transfection of human β-arrestin-1 restores protective signaling by glutamate. D, lack of effect of β-arrestin-1 silencing on agonist-induced PI hydrolysis in CHO cells expressing mGlu1a. E and F, glutamate (Glu)-induced internalization of mGlu1a is attenuated by shRNA silencing of β-arrestin-1. Cells were labeled with cleavable biotin and stimulated with glutamate for 1 h. Extracellular biotin was removed and internalized mGlu 1a receptors were pulled down in NeutrAvidin. P, pellet (internalized during incubation); S, supernatant (remaining receptors). Effect of shRNA silencing of β-arrestin1 on ERK phosphorylation induced by agonists after 5 min (G) and 24 h (H). Transient ERK phosphorylation is sensitive to the inhibition of PLC by U73122 (30 μm) (G). Sustained ERK phosphorylation is blocked by shRNA silencing of β-arrestin-1 but is not affected by the inhibition of PLC (H). ERK phosphorylation was measured using ELISA, and the data are expressed as a percent of ERK phosphorylated in the absence of agonists. All values are means from at least three experiments performed in triplicate with error bars representing S.E. Double asterisks represent significantly different (p < 0.001) as compared with untreated controls using Bonferroni-adjusted t test. Quis, quisqualate.

Using internalization assays, we found that a 30-min pulse of 1 mm glutamate caused 50% of mGlu1a receptors to become internalized in contrast to control cells, where ∼18% of mGlu1a receptors were internalized during the 30-min incubation (Fig. 5, E and F). Transfection with shRNA targeted toward β-arrestin-1 caused internalization in the presence of 1 mm glutamate to be equivalent with the constitutive internalization seen in cells not exposed to glutamate (Fig. 5, E and F).

Further experiments showed that β-arrestin-1 silencing differently affected the transient and sustained ERK phosphorylation mediated by mGlu1a receptors. The transient ERK phosphorylation induced by glutamate and quisqualate was reduced by the PLC inhibitor U73122 but not by β-arrestin-1 silencing (Fig. 5G). In contrast, the sustained ERK phosphorylation, measured 24 h after the application of agonist, was activated only by glutamate and was abolished by β-arrestin-1 silencing, but not by the PLC inhibitor U73122 (Fig. 5H).

These results show that the classical mGlu1a signaling through PI hydrolysis, which also includes transient ERK phosphorylation, does not depend on the presence of β-arrestin-1. In fact, Fig. 5B suggests that PI hydrolysis after silencing of β-arrestin-1 was slightly elevated, which corresponds well to the reported ability of β-arrestin to inhibit G protein-mediated signal transduction (41). In contrast, our results indicate that the presence of β-arrestin-1 is required for both sustained ERK phosphorylation and protective mGlu1a signaling.

DISCUSSION

In a previous report, we have shown that glutamate, acting selectively at mGlu1 receptors, protects neurons from apoptotic death induced by trophic deprivation (1). In this study, we show the existence of a novel signal transduction mechanism that allows mGlu1 receptors to mediate this protective signaling. This mechanism is distinct from the well described classical G protein-mediated signal transduction of group I mGlu receptors (7, 8) and is not shared by mGlu5 receptors. We now show that in CHO cells expressing mGlu1a receptors, glutamate caused protection from toxicity due to serum deprivation, and this protection was blocked not only by noncompetitive antagonists of mGlu1, but also by the inhibition of receptor internalization by silencing β-arrestin-1 expression and inhibition of dynamin. Furthermore, protective signaling was blocked by inhibitors of MEK1/2. The pathway through which mGlu1 acts appears to be unrelated to classical G protein signaling, as protection was not attenuated by inhibition of phospholipase C. Instead, our data reveal the existence of a previously unreported signaling mechanism associated with mGlu1a receptors, namely a G protein-independent protective signaling pathway that requires β-arrestin-1 and activation of MEK followed by ERK phosphorylation.

Antiapoptotic signaling through β-arrestin has been demonstrated for numerous GPCRs (42). As signaling molecules, β-arrestins have been shown to mediate the stimulation of phosphorylation of several protein kinases, including ERK (43). Additionally, β-arrestin-mediated signaling has been shown to be independent of G protein signaling for multiple receptors including the angiotensin II type 1a receptor (44), the D3 dopamine receptor, (45) the β2-adrenergic receptor (46), and the μ opioid receptor (47). Consistent with previous reports that β-arrestin-1 is required for agonist-induced internalization of mGlu1 (21), our findings suggest that the protective signaling of mGlu1a may be initiated by agonist-dependent receptor internalization, followed by a nonclassical, G protein-independent mechanism of signal transduction. Our results also indicate that, similarly to several other GPCRs (25), mGlu1a shows a ligand bias toward the different signaling mechanisms, manifested by the ability of glutamate to activate both signaling pathways, whereas quisqualate activates only G protein-mediated PI hydrolysis.

The protective mGlu1a signaling appears to be mediated by a different mechanism of ERK phosphorylation than that described previously. Several reports show that agonists acting at mGlu1 induce a transient increase of ERK phosphorylation that peaks at 5 min and subsides within 30 min (23, 36). Consistent with these reports, in CHO cells expressing mGlu1, at 5 min, agonists induced a transient ERK phosphorylation, which had a pharmacological profile similar to that observed for PI hydrolysis, was blocked by the inhibition of PLC activity, and was not dependent on β-arrestin expression. At higher glutamate concentrations, β-arrestin silencing slightly reduced ERK phosphorylation also at 5 min, which is consistent with a previous report showing that a portion of mGlu1-mediated transient MAPK phosphorylation is β-arrestin-dependent (23). Together, these data suggest that transient ERK phosphorylation is mediated by the classical mechanism of G protein-mediated PI hydrolysis.

In addition, we have observed that glutamate, acting at mGlu1, activates also a long lasting phase of ERK phosphorylation, which was maintained in the presence of glutamate for 24 h. To our knowledge, this is the first report of a sustained phosphorylation of ERK due to stimulation of a metabotropic glutamate receptor. Similar to the protective effect of glutamate, the sustained phase of ERK phosphorylation was insensitive to PLC inhibition and was abolished completely by shRNA silencing of β-arrestin-1 expression. In contrast to transient ERK phosphorylation, the sustained phase was induced by much higher glutamate concentrations and was not elicited by quisqualate. As demonstrated, this difference in glutamate potency reflected the presence in transfected CHO cells of receptor reserve for PI hydrolysis. The pharmacological profile of the sustained ERK phosphorylation resembled that of protective mGlu1a signaling, but not that of PI hydrolysis and transient ERK phosphorylation. These data strongly suggest that protective mGlu1a signaling is mediated by glutamate-induced sustained ERK phosphorylation. Although the specific downstream targets of phosphorylated ERK have not been identified, phosphorylated ERK, acting at the cell nucleus, was shown to cause protective cellular responses (24).

In conclusion, we have demonstrated the existence of a new signal transduction mechanism of mGlu1a receptors. In contrast to the classical G protein-mediated PI hydrolysis, this mechanism appears to involve agonist-mediated, β-arrestin-1-dependent receptor internalization, followed by the activation of MEK cascade and a long lasting, sustained phase of ERK phosphorylation. This mechanism appears to be responsible for the protective action of glutamate mediated by mGlu1a receptors. We have proposed previously that mGlu1a may function as a dependence receptor causing apoptosis in the absence of glutamate, possibly due to the cleavage of its C-terminal domain (1). Now, we propose that the protective, positive signaling of mGlu1, which occurs in the presence of glutamate, is mediated by this new, G protein-independent, mechanism of signal transduction. This hypothesis needs now to be validated in systems expressing native mGlu1a receptors and in vivo. Further studies also are needed to address the mechanism and establish the conditions in which glutamate, classically viewed as an excitotoxin (12, 48, 49), also may produce a protective effect when acting at mGlu1. Acting as a dependence receptor, mGlu1a may serve as a sensor of extracellular glutamate, promoting neuronal survival in the presence of glutamate and inducing apoptosis in its absence. Such a mechanism could play an important role in brain physiology by allowing glutamate to act as a trophic factor contributing to neuronal development and neuronal selection during synaptogenesis and, possibly, by participating in the restructuring of damaged brain tissue.

*This work was supported in part by National Institutes of Health Grant NS37436.

3The abbreviations used are:

mGlu
metabotropic glutamate
GPCR
G protein-coupled receptor
PI
phosphoinositide(s)
YM-298198
6-amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide hydrochloride
CPCCOEt
7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester
JNJ16259685
(3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-methoxycyclohexyl) methanone
PLC
phospholipase C
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

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