Agonist-dependent signaling by group I metabotropic glutamate receptors is regulated by association with lipid domains

Abstract

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, play critical functions in forms of activity-dependent synaptic plasticity and synapse remodeling in physiological and pathological states. Importantly, in animal models of fragile X syndrome, group I mGluR activity is abnormally enhanced, a dysfunction that may partly underlie cognitive deficits in the condition. Lipid rafts are cholesterol- and sphingolipid-enriched membrane domains that are thought to form transient signaling platforms for ligand-activated receptors. Many G protein-coupled receptors, including group I mGluRs, are present in lipid rafts, but the mechanisms underlying recruitment to these membrane domains remain incompletely understood. Here, we show that mGluR1 recruitment to lipid rafts is enhanced by agonist binding and is supported at least in part by an intact cholesterol recognition/interaction amino acid consensus (CRAC) motif in the receptor. Substitutions of critical residues in the motif reduce mGluR1 association with lipid rafts and agonist-induced, mGluR1-dependent activation of extracellular-signal-activated kinase1/2 MAP kinase (ERK-MAPK). We find that alteration of membrane cholesterol content or perturbation of lipid rafts regulates agonist-dependent activation of ERK-MAPK by group I mGluRs, suggesting a potential function for cholesterol as a positive allosteric modulator of receptor function(s). Together, these findings suggest that drugs that alter membrane cholesterol levels or directed to the receptor-cholesterol interface could be employed to modulate abnormal group I mGluR activity in neuropsychiatric conditions, including fragile X syndrome.

Keywords: CRAC Motif; Cholesterol; ERK; Glutamate Receptors Metabotropic; Lipid Raft; Protein Motifs.

FIGURE 1.

Group I mGluR association with lipid domains is enhanced by agonists. MGluR1 abundance in DRMs is increased by brief exposure to glutamate. A, representative immunoblots (IB) of extracts from transfected cells illustrating mGluR1 co-fractionation with DRMs in absence (basal) or presence of glutamate for different times; arrowheads indicate receptor monomers (∼135 kDa) and dimers (∼260 kDa). Numbers above gel lanes indicate gradient fractions (1, first fraction; 13, bottom gradient fraction): Input, total homogenate. B, quantification of mGluR1 abundance in DRMs in the absence (basal) or presence of glutamate for different times. MGluR1 abundance in DRMs (sum of band densities in fraction 2–5) is presented as the percentage of total receptor (sum of band densities in fractions 1–13) from images like those in A. Data are the means ± S.E.: basal, 4.58 ± 0.87% of total, n = 6; glutamate, 2 min, 8.68 ± 1.45%, n = 3; glutamate, 5 min, 20.73 ± 3.31%, n = 4; glutamate, 20 min, 10.48 ± 0.55%, n = 3. One-way ANOVA, Bonferroni post hoc; *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, glutamate does not affect localization to DRMs of Gα_q/11, caveolin-1, or transferrin receptor 1. Representative immunoblots from extracts of myc-mGluR1 transfected cells corresponding to those in A were probed with indicated antibodies. Endogenous Gα_q/11 and caveolin-1 associate with DRMs with similar efficiency in the absence or presence of glutamate. Estimated Gα_q/11 abundance in DRMs versus total: basal, ∼39%; glutamate, 5 min ∼37%; glutamate, 20 min ∼38%. D, MGluR5 abundance in neuronal DRMs is enhanced by exposure to DHPG. Rat cortical neurons were treated with GPT/pyruvate (1 h; basal) and stimulated with DHPG (50 μm, 5 min); representative immunoblots were probed with anti-mGluR5, anti-flotillin-1, and anti-transferrin-1 antibodies. MGluR5 abundance in DRMs was calculated as the percentage of total receptor and expressed as the ratio of DHPG versus basal; data are the means ± S.E.: 1.87 ± 0.115, n = 3, correlation coefficient r = 0.974 (ratio paired t test, significant, p > 0.05). Paired values in independent experiments were: control 7.09% versus DHPG 11.86%; control 49% versus DHPG 62.10%; control 3.7% versus DHPG 11.50%.

FIGURE 2.

MGluR1 association with lipid domains is independent of G protein activation. DHPG enhances mGluR1 abundance in DRMs in the presence of the non-competitive antagonist CPCCOEt. A, representative immunoblots (IB) of extracts from transfected cells probed with anti-mGluR1, -flotillin-1 (Flot-1), and -TfR1 antibodies illustrating mGluR1 co-fractionation in DRMs under basal conditions or after application of DHPG (5 min) in the absence or presence of CPCCOEt. Input, total homogenate; arrowheads indicate receptor monomers (∼135 kDa) and dimers (∼260 kDa). B, quantification of myc-mGluR1 abundance in DRMs from images like those in A are presented as the percentage of total receptor; data are the means ± S.E., n = 3; *, p < 0.05. ns, not significant. C, representative immunoblots, probed with anti-phospho-ERK1/2(Thr-202/Tyr-204) and anti-ERK1/2 antibodies, of extracts from cells expressing myc-mGluR1 preincubated without or with CPCCOEt and stimulated with DHPG (5 min). D, quantification of ERK phosphorylation from experiments like those in C measured as the ratio of band densities for p-ERK2 versus ERK2; data are the means ± S.E., n = 4, one-way ANOVA with Bonferroni post test; *, p < 0.05; **, p < 0.01. E, representative immunoblot of extracts from transfected cells probed with anti-mGluR1 antibody illustrating co-fractionation in DRMs of the inactive mutant mGluR1^F781S. Estimated mGluR1^F781S abundance in DRMs versus total: basal, ∼6%; DHPG (5 min), ∼9%.

FIGURE 3.

MGluR1 association with lipid domains is independent of caveolin-1. Glutamate enhances the association with DRMs of a mutant receptor (myc-mGluR1mut-i1/i3) lacking functional caveolin-1 binding domains. A and B, mutation of caveolin-1 binding motifs does not affect receptor affinity for agonist. Saturation isotherms of [³H]quisqualate binding to myc-mGluR1 (A) and myc-mGluR1-i1/i3 (B) are shown. C, representative immunoblots of extracts from transfected cells probed with anti-mGluR1 antibody illustrating mGluR1mut-i1/i3 co-fractionation in DRMs in absence (basal) or presence of glutamate applied for 5 min. Input, total homogenate. D, quantification of myc-mGluR1mut-i1/i3 co-fractionation in DRMs from images like those in C expressed as -fold change in receptor abundance in DRMs in the presence of glutamate versus basal; data are the means ± S.E.: basal 1.0 ± 0.5; glutamate, 7.6 ± 1.4-fold basal, n = 3; *, p < 0.05. -Fold change of wild type mGluR1 abundance in DRMs was calculated from experiments in Fig. 1 and is presented here for comparison. E, native mGluR1 and mGluR5 are present in lipid domains in the absence of caveolin-1. Representative immunoblots (IB) of DRMs isolated from wild type and caveolin-1 knock-out (Cav1^−/−) mouse brain cortex probed with anti-mGluR1 and -mGluR5 antibodies are shown. Immunoblots with antibodies against flotillin-1 (Flot-1), a lipid raft marker, and TfR1 excluded from rafts illustrate raft integrity in the absence of caveolin-1. Input, total cortical homogenate.

FIGURE 4.

MGluR1 association with lipid domains is dependent on membrane cholesterol content. MGluR1 association with DRMs is abolished by cholesterol depletion and restored after cholesterol replenishment. A, representative immunoblots (IB) of DRMs prepared from transfected cells in which cholesterol was acutely removed from membranes by incubation with mβCD (depletion) or added back after depletion (replenishment); control cells were left untreated. Immunoblots were probed with antibodies against mGluR1, the lipid raft-associated protein Gα_q/11, or TfR1, which is excluded from lipid rafts; Input, total homogenate. B, quantification of myc-mGluR1 co-fractionation in DRMs from images like those in A. Abundance in DRMs is presented as the percentage of total receptor; data are the means ± S.E.: control, 9.1 ± 1.4% of total, n = 4; cholesterol depletion, 1.0 ± 0.6% of total, n = 3; cholesterol replenishment, 29.6 ± 6.7% of total, n = 4; *, p < 0.05. Estimated Gα_q/11 abundance in DRMs from images like those in A, means ± S.E.: control, 10.5 ± 4.4% of total; cholesterol depletion, none detected; cholesterol replenishment, 13.5 ± 3.2% of total. C, cholesterol depletion reduces but does not abolish association of endogenous caveolin-1 with DRMs prepared from cells as those in A; representative immunoblots were probed with anti-caveolin-1 antibody. Estimated caveolin-1 in DRMs was expressed as the percentage of total: control, 41%; cholesterol depletion, 23%; cholesterol replenishment, 48%. D, DRMs are rich in cholesterol and relatively depleted of proteins (protein content in DRM 4.6 ± 1% of total, n = 3); cholesterol can be rapidly and effectively removed from and restored to membranes by incubation with mβCD or a mβCD-cholesterol complex, respectively. Cholesterol concentration is in μg/ml in DRMs (fraction 3); data are the means ± S.E., n = 5 from two independent experiments.

FIGURE 5.

A putative CRAC motif in mGluR1 participates in promoting association with lipid domains. A, MGluR1 contains a putative CRAC motif spanning fifth transmembrane helix (TM5) and N-terminal portion of third intracellular loop (i3). Critical residues in the predicted CRAC consensus are in bold or in color in the mGluR1 sequence; X, any amino acid. B, sequence alignment of mGluR family members in the corresponding TM5 to i3 region where a putative CRAC motif is predicted in mGluR1; the bold font indicates potential critical residues in putative CRAC motif(s). C, the group II receptor mGluR2 associates with DRMs in the brain cortex. Representative immunoblots (IB) were probed with anti-mGluR2, anti-flotillin-1, and anti-transferrin receptor 1 antibodies. Input, total homogenate. D–G, representative immunoblots of extracts from transfected cells illustrating co-fractionation in DRMs of mutant receptors with amino acid substitutions at critical residues in the CRAC motif in the absence (basal) or presence of agonists applied for 5 min (DHPG for mGluR1^L763S, mGluR1^Y769A/Y770A, mGluR1^R775G, or glutamate for mGluR1^R775W). Input, total homogenate. Immunoblots were sequentially probed with anti-mGluR1, anti-flotillin-1 (Flot-1) and anti-TfR1 antibodies. Estimated abundance in DRMs of mutant receptors (percentage of total): mGluR1^L763S basal, 0.45 ± 0.15% of total, n = 4; agonist, 0.75 ± 0.25%, n = 2; mGluR1^Y769A/Y770A basal, 3.46 ± 1.75%, n = 3; agonist, 0.29%; mGluR1^R775G basal, 3.92 ± 1.33%, n = 4; agonist, 1 ± 0.6%, n = 2; mGluR1^R775W basal, 0.6 ± 0.4%, n = 3; agonist, 0.6 ± 0.2%, n = 3. H and I, mutations introduced in a putative CRAC motif in mGluR1 do not significantly affect expression at the cell surface. H, representative immunoblots of biotin-labeled (surface) and total (lysate) proteins from extracts of cells expressing wild type mGluR1 or mutant receptors with substitutions in the putative CRAC motif or an inactive mGluR1 mutant (mGluR1^F781S). Immunoblots were probed with anti-mGluR1 and anti-γ-tubulin or anti-actin antibodies for loading and surface specificity control. I, quantification of receptor surface expression at steady state from immunoblots like those in H. Surface expression is calculated as the ratio of biotinylated versus total receptor and expressed as percentage of wild type mGluR1. Data are the means ± S.E.: mGluR1, n = 5; mGluR1^L763S, n = 5; mGluR1^Y769A/Y770A, n = 5; mGluR1^R775G, n = 5; mGluR1^R775W, n = 10; mGluR1^F781S, n = 11; one-way ANOVA with Tukey's post test, p = 0.8364.

FIGURE 6.

Disruption of a putative CRAC motif impairs mGluR1 signaling to ERK-MAPK. A, representative immunoblots (IB) of extracts from transfected cells expressing YFP (mock), wild type, or mutant mGluR1 with substitutions at critical residues in the presumptive CRAC motif (mGluR1^L763S, mGluR1^Y769A/Y770A, mGluR1^R775G, mGluR1^R775W) were probed with anti-phospho-ERK1/2(Thr-202/Tyr-204) and anti-ERK1/2 antibodies. Cells were preincubated with vehicle or the inverse agonist BAY 36-7620 (BAY) or exposed to DHPG for 5 min. B, quantification of receptor constitutive activity as determined by incubation with BAY 36-7620 from experiments like those in A, ERK phosphorylation is measured as the ratio of band densities for p-ERK2 and ERK2. Data are the means ± S.E.: YFP, n = 8; mGluR1, n = 7; mGluR1^L763S, n = 6; mGluR1^Y769A/Y770A, n = 6; mGluR1^R775G, n = 5; mGluR1^R775W, n = 7. Similar changes in phosphorylation were observed for ERK1 but not quantified, as p-ERK1 was generally below detection in controls. C, quantification of agonist-induced ERK phosphorylation expressed as -fold increase versus basal from experiments like those in A; data are the means ± S.E.: mGluR1, n = 6; mGluR1^L763S, n = 5; mGluR1^Y769A/Y770A, n = 6; mGluR1^R775G, n = 5, mGluR1^R775W n = 6. One-way ANOVA, Bonferroni post test: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

FIGURE 7.

Membrane cholesterol content regulates agonist-dependent, mGluR1-mediated activation of ERK-MAPK. A, representative immunoblots of extracts from transfected cells expressing mGluR1 incubated without (vehicle) or with mβCD and stimulated with DHPG (5 min) were probed with anti-phospho-ERK1/2(Thr-202/Tyr-204), anti-ERK1/2, and anti-actin antibodies. B and C, quantification of ERK phosphorylation from experiments like those in A measured as the ratio of band densities for p-ERK1 versus ERK1 (B) and p-ERK2 versus ERK2 (C); data are the means ± S.E.: n = 7, two-way ANOVA with Bonferroni post test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ns, not significant. D, representative immunoblots probed with anti-phospho-ERK1/2(Thr-202/Tyr-204), anti-ERK1/2, and anti-GAPDH antibodies of extracts from cells expressing mGluR1 incubated without (control) or with supplemental cholesterol and stimulated with DHPG (2.5, 5.0, or 50 μm) for 5 min. E, quantification of ERK phosphorylation from experiments like those in D measured as the ratio of band densities for p-ERK2 versus ERK2 normalized to basal (no DHPG); data are the means ± S.E.: n = 6; *, p < 0.05; **, p < 0.01; two-way ANOVA, Bonferroni post test. F, representative immunoblots probed with anti-phospho-ERK1/2(Thr-202/Tyr-204), anti-ERK1/2, and anti-GAPDH antibodies of extracts from cells expressing mGluR1^R775W with or without (control) supplemental cholesterol and stimulated with 50 μm DHPG applied for 5 min. G, quantification of ERK phosphorylation from experiments like those in F, measured as the ratio of band densities for p-ERK2 versus ERK2 normalized to basal (no DHPG); data are the means ± S.E., n = 4; p > 0.05.

FIGURE 8.

Treatment with HMG-CoA reductase inhibitors impairs agonist-dependent, mGluR1-mediated activation of ERK-MAPK in neurons. A, representative immunoblots (IB) probed with anti-phospho-ERK1/2(Thr-202/Tyr-204), anti-ERK1/2, and anti-α-tubulin of extracts from cortical neurons incubated with vehicle or the HMG-CoA reductase inhibitors lovastatin or simvastatin and stimulated with DHPG for indicated times. B, quantification of ERK2 phosphorylation from experiments like those in A; data are the mean ± S.E.; n = 6; *, p < 0.05, one-way ANOVA. C, representative immunoblots probed with anti-phospho-ERK1/2, anti-ERK1/2, and anti-γ-tubulin antibodies of extracts from FMR1 knock-out neurons incubated with vehicle, lovastatin, or simvastatin and stimulated with DHPG. D, quantification of ERK2 phosphorylation from experiments like those in C; data are the means ± S.E.; *, p < 0.05, one-way ANOVA.