Native Metabotropic Glutamate Research Articles

Protein Networks Associated with Native Metabotropic Glutamate 1 Receptors (mGlu1) in the Mouse Cerebellum.

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.

Native metabotropic glutamate receptor 4 depresses synaptic transmission through an unusual Gαq transduction pathway

In cerebellar cortex, mGlu4 receptors located on parallel fibers play an essential role in normal motor function, but the molecular mechanisms involved are not yet completely understood. Using a strategy combining biochemical and electrophysiological approaches in the rodent cerebellum, we demonstrate that presynaptic mGlu4 receptors control synaptic transmission through an atypical activation of Gαq proteins. First, the Gαq subunit, PLC and PKC signaling proteins present in cerebellar extracts are retained on affinity chromatography columns grafted with different sequences of the cytoplasmic domain of mGlu4 receptor. The i2 loop and the C terminal domain were used as baits, two domains that are known to play a pivotal role in coupling selectivity and efficacy. Second, in situ proximity ligation assays show that native mGlu4 receptors and Gαq subunits are in close physical proximity in cerebellar cortical slices. Finally, electrophysiological experiments demonstrate that the molecular mechanisms underlying mGlu4 receptor-mediated inhibition of transmitter release at cerebellar Parallel Fiber (PF) – Molecular Layer Interneuron (MLI) synapses involves the Gαq-PLC signaling pathway.Taken together, our results provide compelling evidence that, in the rodent cerebellar cortex, mGlu4 receptors act by coupling to the Gαq protein and PLC effector system to reduce glutamate synaptic transmission.

A Study of the Spatial Protein Organization of the Postsynaptic Density Isolated from Porcine Cerebral Cortex and Cerebellum

Postsynaptic density (PSD) is a protein supramolecule lying underneath the postsynaptic membrane of excitatory synapses and has been implicated to play important roles in synaptic structure and function in mammalian central nervous system. Here, PSDs were isolated from two distinct regions of porcine brain, cerebral cortex and cerebellum. SDS-PAGE and Western blotting analyses indicated that cerebral and cerebellar PSDs consisted of a similar set of proteins with noticeable differences in the abundance of various proteins between these samples. Subsequently, protein localization in these PSDs was analyzed by using the Nano-Depth-Tagging method. This method involved the use of three synthetic reagents, as agarose beads whose surface was covalently linked with a fluorescent, photoactivable, and cleavable chemical crosslinker by spacers of varied lengths. After its application was verified by using a synthetic complex consisting of four layers of different proteins, the Nano-Depth-Tagging method was used here to yield information concerning the depth distribution of various proteins in the PSD. The results indicated that in both cerebral and cerebellar PSDs, glutamate receptors, actin, and actin binding proteins resided in the peripheral regions within ∼ 10 nm deep from the surface and that scaffold proteins, tubulin subunits, microtubule-binding proteins, and membrane cytoskeleton proteins found in mammalian erythrocytes resided in the interiors deeper than 10 nm from the surface in the PSD. Finally, by using the immunoabsorption method, binding partner proteins of two proteins residing in the interiors, PSD-95 and α-tubulin, and those of two proteins residing in the peripheral regions, elongation factor-1α and calcium, calmodulin-dependent protein kinase II α subunit, of cerebral and cerebellar PSDs were identified. Overall, the results indicate a striking similarity in protein organization between the PSDs isolated from porcine cerebral cortex and cerebellum. A model of the molecular structure of the PSD has also been proposed here.

Purification and Identification of G Protein-coupled Receptor Protein Complexes under Native Conditions

G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. The identification of GPCR-associated proteins is an important step toward a better understanding of these receptors. However, current methods are not satisfying as only isolated receptor domains (intracellular loops or carboxyl-terminal tails) can be used as "bait." We report here a method based on tandem affinity purification coupled to mass spectrometry that overcomes these limitations as the entire receptor is used to identify protein complexes formed in living mammalian cells. The human MT(1) and MT(2) melatonin receptors were chosen as model GPCRs. Both receptors were tagged with the tandem affinity purification tag at their carboxyl-terminal tails and expressed in human embryonic kidney 293 cells. Receptor solubilization and purification conditions were optimized. The method was validated by the co-purification of G(i) proteins, which are well known GPCR interaction partners but which are difficult to identify with current protein-protein interaction assays. Several new and functionally relevant MT(1)- and MT(2)-associated proteins were identified; some of them were common to both receptors, and others were specific for each subtype. Taken together, our protocol allowed for the first time the purification of GPCR-associated proteins under native conditions in quantities suitable for mass spectrometry analysis.

Qualitative and Quantitative Analyses of Protein Phosphorylation in Naive and Stimulated Mouse Synaptosomal Preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

Opposite effects of Zn on the in vitro binding of [3H]LY354740 to recombinant and native metabotropic glutamate 2 and 3 receptors

We investigated the effect of Zn on agonist binding to both recombinant and native mGlu2 and mGlu3 receptors. Zn had a biphasic inhibitory effect on recombinant mGlu2 with IC(50) values for the high- and low-affinity components of 60 +/- 10 microM and 2 +/- 0.7 mM, respectively. Zn induced a complex biphasic effect of inhibition and enhancement of [(3)H]LY354740 binding to mGlu3. Observations with a series of chimeric mGlu2/3 receptors suggest that the Zn effect resides in the N-terminal domain of mGlu2 and mGlu3. We observed that the His56 of mGlu2, which corresponds to Asp63 in mGlu3 was largely accountable for the second phase of the Zn effect. As revealed by quantitative receptor radioautography, the addition of up to 100 microm Zn to brain sections of wild-type mice resulted in significant decreases in binding density in most brain regions. In particular, the mid-molecular layer of the dentate gyrus (DGmol) and the CA1 lacunosum moleculare of hippocampus (CA1-LMol) showed reductions of 62 and 67%, respectively. In contrast, the addition of 300 microM Zn to brain sections of mGlu2(-/-) mice caused large increases in binding density of 289 and 242% in DGmol and CA1-LMol, respectively. Therefore, Zn might play a role as a physiological modulator of group II mGlu receptor function.

Proteomic analysis of native metabotropic glutamate receptor 5 protein complexes reveals novel molecular constituents.

We used a proteomic approach to identify novel proteins that may regulate metabotropic glutamate receptor 5 (mGluR5) responses by direct or indirect protein interactions. This approach does not rely on the heterologous expression of proteins and offers the advantage of identifying protein interactions in a native environment. The mGluR5 protein was immunoprecipitated from rat brain lysates; co-immunoprecipitating proteins were analyzed by mass spectrometry and identified peptides were matched to protein databases to determine the correlating parent proteins. This proteomic approach revealed the interaction of mGluR5 with known regulatory proteins, as well as novel proteins that reflect previously unidentified molecular constituents of the mGluR5-signaling complex. Immunoblot analysis confirmed the interaction of high confidence proteins, such as phosphofurin acidic cluster sorting protein 1, microtubule-associated protein 2a and dynamin 1, as mGluR5-interacting proteins. These studies show that a proteomic approach can be used to identify candidate interacting proteins. This approach may be particularly useful for neurobiology applications where distinct protein interactions within a signaling complex can dramatically alter the outcome of the response to neurotransmitter release, or the disruption of normal protein interactions can lead to severe neurological and psychiatric disorders.

A novel selective allosteric modulator potentiates the activity of native metabotropic glutamate receptor subtype 5 in rat forebrain.

We found that N-[4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl]-2-hydroxybenzamide (CPPHA), is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). CPPHA alone had no agonist activity and acted as a selective positive allosteric modulator of human and rat mGluR5. CPPHA potentiated threshold responses to glutamate in fluorometric Ca(2+) assays 7- to 8-fold with EC(50) values in the 400 to 800 nM range, and at 10 microM shifted mGluR5 agonist concentration-response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine (DHPG) 4- to 7-fold to the left. The only effect of CPPHA on other mGluRs was weak inhibition of mGluR4 and 8. Neither CPPHA nor the previously described 3,3'-difluorobenzaldazine (DFB) affected [(3)H]quisqualate binding to mGluR5, but although DFB partially competed for [(3)H]3-methoxy-5-(2-pyridinylethynyl)pyridine binding, CPPHA had no effect on the binding of this 2-methyl-6-(phenylethynyl)-pyridine analog to mGluR5. Although the binding sites for the two classes of allosteric modulators seem to be different, these different allosteric sites can modulate functionally and mechanistically similar allosteric effects. In electrophysiological studies of brain slice preparations, it had been previously shown that activation of mGluR5 receptors by agonists increased N-methyl-D-aspartate (NMDA) receptor currents in the CA1 region of hippocampal slices. We found that CPPHA (10 microM) potentiated NMDA receptor currents in hippocampal slices induced by threshold levels of DHPG, whereas having no effect on these currents by itself. Similarly, 10 microM CPPHA also potentiated mGluR5-mediated DHPG-induced depolarization of rat subthalamic nucleus neurons. These results demonstrate that allosteric potentiation of mGluR5 increases the effect of threshold agonist concentrations in native systems.

Calcium dependence of native metabotropic glutamate receptor signaling in central neurons.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that are distributed throughout the brain and play important roles in regulation of synaptic efficacy. Some studies report that mGluRs heterologously expressed in nonneuronal cells are sensitive not only to glutamate but also to extracellular Ca2+ (Ca2+o). We studied the Ca2+o-sensitivity of native mGluRs in mammalian central neurons. In cerebellar Purkinje cells that naturally express type-1 mGluR (mGluR1), physiological levels of Ca2+o (around 2 mM) activate mGluR1-mediated intracellular Ca2+ mobilization. The activation of the native mGluR1 response to Ca2+o appears to be slower than that to glutamate. Ca2+o (2 mM) also augments glutamate analog-evoked, native mGluR1-mediated inward cation current and intracellular Ca2+o mobilization. Detailed analysis of this effect suggests that Ca2+o modulates the glutamate responsiveness of native and heterologously expressed mGluR1s in different manners. These findings suggest that Ca2+o may enhance the basal level and glutamate responsiveness of neuronal mGluR signaling in vivo.

Pharmacological properties of native metabotropic glutamate receptors in freshly dissociated Golgi cells of the rat cerebellum

We have examined the pharmacological properties of native metabotropic glutamate (mGlu) receptors in freshly isolated rat cerebellar Golgi cells using the whole-cell configuration of the patch-clamp technique. Group II mGlu receptor agonists inhibited voltage-gated Ca 2+ channels (VGCC) currents in a reversible and concentration-dependent manner with a rank order of potency being LY354740> DCG-IV > L-CCG-I > glutamate >>1S,3R-ACPD > NAAG. The maximum degree of inhibition obtained was similar for all drugs tested, saturating at about 33–41%, except for NAAG that had a non saturating effect of 50% at 1mM. Two novel group II mGlu receptor antagonists, LY341495 and Ro 65-3479, reversed VGCC current inhibition by LY354740 with pK B values of 7.0 and 6.3, respectively. In a subpopulation of Golgi cells, the antagonistic effect of LY341495 was only partial, suggesting a remaining effect of group I mGlu receptors. This was confirmed by experiments with S-DHPG, a selective group I mGlu receptor agonist. These experiments suggest that Golgi cells of the cerebellum express group II mGlu receptors that couple to the inhibition of VGCCs. Therefore, inhibition of VGCCs in cerebellar Golgi cells is a useful model system to evaluate novel group II mGlu receptor ligands.

Selective Blockade of P/Q-Type Calcium Channels by the Metabotropic Glutamate Receptor Type 7 Involves a Phospholipase C Pathway in Neurons

Although presynaptic localization of mGluR7 is well established, the mechanism by which the receptor may control Ca(2+) channels in neurons is still unknown. We show here that cultured cerebellar granule cells express native metabotropic glutamate receptor type 7 (mGluR7) in neuritic processes, whereas transfected mGluR7 was also expressed in cell bodies. This allowed us to study the effect of the transfected receptor on somatic Ca(2+) channels. In transfected neurons, mGuR7 selectively inhibited P/Q-type Ca(2+) channels. The effect was mimicked by GTPgammaS and blocked by pertussis toxin (PTX) or a selective antibody raised against the G-protein alphao subunit, indicating the involvement of a G(o)-like protein. The mGuR7 effect did not display the characteristics of a direct interaction between G-protein betagamma subunits and the alpha1A Ca(2+) channel subunit, but was abolished by quenching betagamma subunits with specific intracellular peptides. Intracellular dialysis of G-protein betagamma subunits did not mimic the action of mGluR7, suggesting that both G-protein betagamma and alphao subunits were required to mediate the effect. Inhibition of phospholipase C (PLC) blocked the inhibitory action of mGluR7, suggesting that a coincident activation of PLC by the G-protein betagamma with alphao subunits was required. The Ca(2+) chelator BAPTA, as well as inhibition of either the inositol trisphosphate (IP(3)) receptor or protein kinase C (PKC) abolished the mGluR7 effect. Moreover, activation of native mGluR7 induced a PTX-dependent IP(3) formation. These results indicated that IP(3)-mediated intracellular Ca(2+) release was required for PKC-dependent inhibition of the Ca(2+) channels. Possible control of synaptic transmission by the present mechanisms is discussed.

Characterization ok the pharmacological profile of (RS)-1-aminoindan-1,5-dicarboxylic acid (UPF523) on native metabotropic glutamate receptors (mGluRs) in the rat

Characterization ok the pharmacological profile of (RS)-1-aminoindan-1,5-dicarboxylic acid (UPF523) on native metabotropic glutamate receptors (mGluRs) in the rat