Characterization of the Dimerization of Metabotropic Glutamate Receptors Using an N-Terminal Truncation of mGluR1α

The metabotropic glutamate receptor mGluR1alpha in membranes isolated both from rat brain and from cell lines transfected with cDNA coding for the receptor migrates as a disulphide-bonded dimer on sodium dodecyl sulphate-polyacrylamide gels. Dimerization of mGluR1alpha takes place in the endoplasmic reticulum because it is not prevented by exposing transfected human embryonic kidney (HEK) 293 cells to the drug brefeldin A, a drug that prevents egress of proteins from the endoplasmic reticulum. Dimerization was also not dependent on protein glycosylation as it was not prevented by treatment of the cells with tunicamycin. Using a mammalian expression vector containing the N-terminal domain of mGluR1alpha, truncated just before the first transmembrane domain (NT-mGluR1alpha), we show that the N-terminal domain is secreted as a soluble disulphide-bonded dimeric protein. In addition, the truncated N-terminal domain can form heterodimers with mGluR1alpha when both proteins are cotransfected into HEK 293 cells. However, mGluR1alpha and its splice variant mGluR1beta did not form heterodimers in doubly transfected HEK 293 cells. These results show that although the N-terminal domain of mGluR1alpha is sufficient for dimer formation, other domains in the molecule must regulate the process.     

The C-Terminal Domain of the mGluR1 Metabotropic Glutamate Receptor Affects Sensitivity to Agonists

Abstract: The metabotropic glutamate receptor (mGluR) subtype 1 exists as at least three variants (−1a, −1b, and −1c) generated by alternative splicing at the C-terminal domain. Fluorometric Ca²⁺ measurements were used to compare the concentration dependency of agonist-induced rises in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in human embryonic HEK 293 cells transiently expressing rat mGluR1a, mGluR1b, or mGluR1c. The rank order of agonist potencies was quisqualate ≫ (2 S, 1' S, 2' S )-2-(carboxycyclopropyl)glycine (L-CCG-I) > (1 S, 3 R )-1-aminocyclopentane-1,3-dicarboxylic acid [(1 S, 3 R )-ACPD] and did not differ among the splice variants. However, agonists were consistently more potent at mGluR1a than at mGluR1c and mGluR1b. In the same system, we characterized the agonist pharmacology of two chimeric rat mGluR3/1 receptors where the first and/or the second intracellular loop(s) and the C-terminal domain were exchanged with the corresponding mGluR1a or mGluR1c sequences and that were previously shown to mediate elevations in [Ca²⁺]_i in response to agonists. The potency of agonists was higher at the chimera having the C-terminus of mGluR1a as compared with those having the mGluR1c C-terminus. Both chimeric mGluR3/1 receptors had the same rank order of agonist potencies: L-CCG-I ≫ (1 S, 3 R )-ACPD ∼ quisqualate. These data support the hypothesis that the C-terminal domain of mGluRs plays a role in determining the potency of agonists for inducing mGluR-mediated functional responses.     

(S)-Carboxy-3-Hydroxyphenylglycine, an Antagonist of Metabotropic Glutamate Receptor (mGluR)1a and an Agonist of mGluR2, Protects Against Audiogenic Seizures in DBA/2 Mice

Abstract: The in vivo anticonvulsant effects and in vitro metabo-tropic glutamate receptor selectivity of ( S )-4-carboxy-3-hydroxy-phenylglycine [(S)-4C3HPG] were examined. Intracerebroventricular injection of (S)-4C3HPG dose-dependently antagonized audiogenic-induced clonic and tonic convulsions in DBA/2 mice with ED₆₀ values of 76 and 110-nmol per mouse, respectively. (S)-4C3HPG dose-dependently inhibited the spontaneously evoked epileptic spikes in a cingulate cortex-corpus callosum slice preparation. (SJ-4C3HPG displaced the binding of [³H]glutamate in membranes prepared from baby hamster kidney (BHK) cells expressing the metabotropic glutamate receptor mGluR1a with an EC₅₀ of 5 β 1 u M. ( S )-4C3HPG dose-dependently antagonized glutamate-stimulated phosphoinositide hydrolysis in BHK cells expressing mGluR 1a with an IC₅₀ of 15 β 3 μ M. ( S )-4C3HPG was, however, an agonist at mGluR2 with an EC₆₀ of 21 β 4 μ M for inhibition of forskolin-stimulated cyclic AMP formation in BHK cells expressing the mGluR2. ( S )-4C3HPG had no effects at mGluR4a. These data suggest that the anticonvulsant action of ( S )-4C3HPG is mediated by combined antagonism of mGluRIa and agonism of mGluR2. These results suggest the importance of mGluR1a and/or mGluR2 in the control of epileptic activity.     

Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

1. The serotonin_1A(5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.     

Developmental Changes in the Modulation of Cyclic AMP Formation by the Metabotropic Glutamate Receptor Agonist 1S,3R-Aminocyclopentane-1,3-Dicarboxylic Acid in Brain Slices

Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.     

Improved Visualization and Specific Binding for Metabotropic Glutamate Receptor Subtype 1 (mGluR1) Using [11C]ITMM with Ultra-High Specific Activity in Small-Animal PET

Metabotropic glutamate receptor subtype 1 (mGluR1) is a crucial target in the development of new medications to treat central nervous system (CNS) disorders. Recently, we developed N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-4-[¹¹C]methoxy-N-methyl-benzamide ([¹¹C]ITMM) as a useful positron emission tomography (PET) probe for mGluR1 in clinical studies. Here, we aimed to improve visualization and threshold of specific binding for mGluR1 using [¹¹C]ITMM with ultra-high specific activity (SA) of > 3,500 GBq/μmol in rat brains. A two-tissue compartment model indicated large differences between the two SAs in the constants k₃ and k₄, representing binding ability for mGluR1, while constants K ₁ and k₂ showed no differences. The total distribution volume (V_T) values of conventional and ultra-high SA were 9.1 and 11.2 in the thalamus, 7.7 and 9.7 in the striatum, and 6.4 and 8.5 mL/cm³ in the substantia nigra, respectively. The specific binding of [¹¹C]ITMM with ultra-high SA was significantly higher than the conventional SA, especially in the basal ganglia. Parametric PET images scaled with V_T of the ultra-high SA clearly identified regional differences in the rat brain. In conclusion, PET studies using [¹¹C]ITMM with ultra-high SA could sufficiently improve visualization and specific binding for mGluR1, which could help further understanding for mGluR1 functions in CNS disorders.     

Metabotropic Glutamate Receptor (mGluR)-Mediated Potentiation of Cyclic AMP Responses Does Not Require Phosphoinositide Hydrolysis: Mediation by a Group II-Like mGluR

Abstract: Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (1 S ,3 R -ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to G_s. It has been suggested that mGluR-mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR-mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1 S ,3 R -ACPD-stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1 S ,3 R -ACPD induced a cAMP response that was greater than additive. Finally, ( RS )-3,5-dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2 S ,1' R ,2' R ,3' R )-2-(2,3-dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR-mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.     

Preparative resolution of 1-aminoindan-1,5-dicarboxylic acid (AIDA) by chiral ligand-exchange chromatography

Chiral ligand-exchange chromatography has been shown to be effective in the resolution and semipreparative separation of 1-aminoindan-1,5-dicarboxylic acid (AIDA) enantiomers. In functional activity experiments, only (S)-AIDA was a potent and mGluR1 subtype selective antagonist.     

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

Interaction Between A1 Adenosine and Class II Metabotropic Glutamate Receptors in the Regulation of Purine and Glutamate Release from Rat Hippocampal Slices

Abstract: Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time-dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [¹⁴C]glutamate and [³H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8-cyclopentyl-1,3-dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)-α-methyl-4-carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2-chloro-N⁶-cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)-(2,3-dicarboxycyclopropyl)glycine (DCG-IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG-IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG-IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.     

Protein Kinase C-Mediated Suppression of the Presynaptic Adenosine A1 Receptor by a Facilitatory Metabotropic Glutamate Receptor

Abstract: KCl-evoked glutamate exocytosis from cerebrocortical synaptosomes can be inhibited by the adenosine A1 receptor agonist cyclohexyladenosine (CHA). Inhibition is associated with a decreased KCl-evoked Ca²⁺ level elevation, and the effect of the agonist is occluded by prior incubation with the Agelenopsis aperta neurotoxin ω-agatoxin-IVA at 250 n M . The inhibition is suppressed in the presence of 3 n M phorbol dibutyrate (PDBu) or by activation of the protein kinase C (PKC)-coupled metabotropic glutamate receptor by 100 µ M (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )ACPD]. A tonic inhibition of release by leaked exogenous adenosine can be reversed by adenosine deaminase or by PDBu addition. The CHA-induced inhibition can be enhanced by the PKC inhibitor Ro 31-8220. The mechanism for the suppression of the adenosine A1 receptor-mediated inhibition is distinct from that previously described for the (1 S ,3 R )ACPD-evoked, PKC-mediated, facilitatory pathway, which enhances phosphorylation of the MARCKS protein, 4-aminopyridine-induced action potentials, and release of glutamate because the latter requires at least 100 n M PDBu [or the combination of (1 S ,3 R )ACPD and arachidonic acid] and is not seen following KCl depolarization. Both PKC-mediated pathways may be involved in the presynaptic events associated with the establishment of synaptic plasticity.     

Comparative Effects of Chronic Exposure to Ethanol and Calcium Channel Antagonists on Calcium Channel Antagonist Receptors in Cultured Neural (PC12) Cells

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.     

(R,S)-α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Receptors Mediate a Calcium-Dependent Inhibition of the Metabotropic Glutamate Receptor-Stimulated Formation of Inositol 1,4,5-Trisphosphate

Abstract: l -Glutamate (3-1,000 μ M ) and (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid (1S.3R-ACPD; 10-1,000 μ M ), a selective agonist for the metabotropic glutamate receptor, stimulated the formation of inositol 1,4,5-trisphosphate in a concentration-dependent manner. l -Glutamate was half as efficacious as 1S,3R-ACPD. N -methyl- d -aspartate (nMDA; 1 n M to 1 m M ) did not significantly influence the response to a maximally effective concentration of 1S,3R-ACPD (100 μ M ). On the other hand, coapplication of (R,S)-α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid (AMPA; 1-300 n M ) produced a concentration- and time-dependent inhibition of the 1S,3R-ACPD effect, with a maximal inhibition (97%) at 100 n M . Ten micromolar 6-cyano-7-nitroquinoxaline-2,3-dione. an antagonist of the AMPA receptor, blocked the inhibitory effect of AMPA. Reduced extracellular calcium concentration, as well as 10 μ M nimodipine, an l -type calcium channel antagonist, inhibited the AMPA influence on the 1S,3R-ACPD response. W-7, a calcium/calmodulin antagonist, prevented the inhibition by AMPA. whereas H-7. an inhibitor of protein kinase C, had no effect. These data suggest that activation of AMPA receptors has an inhibitory influence on inositol 1,4,5-trisphosphate formation mediated by stimulation of the metabotropic glutamate receptor. The mechanism of action involves calcium influx through l -type calcium channels and possible activation of calcium/calmodulin-dependent enzymes.     

Inhibition of the Vesicular Glutamate Transporter (VGLUT) with Congo Red Analogs: New Binding Insights

Neurochemical Research - The vesicular glutamate transporter (VGLUT) facilitates the uptake of glutamate (Glu) into neuronal vesicles. VGLUT has not yet been fully characterized pharmacologically...     

Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [³H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites ( B _max = 139 ± 9 fmol/mg of protein) displaying a high affinity ( K _D = 0.76 ± 0.09 n M ). The two cannabinoid receptor agonists, CP 55940 and WIN 55212-2, inhibited in a concentration-dependent manner cyclic AMP production induced by either 1 µ M forskolin or isoproterenol with EC₅₀ values in the nanomolar range (4.6 and 65 n M with forskolin and 1.0 and 5.1 n M with isoproterenol for CP 55940 and WIN 55212-2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µ M forskolin with a potency similar to that observed in cortical neurons (EC₅₀ values of 3.5 and 1.9 n M in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940- and WIN 55212-2-induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor-specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen-activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212-2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.     

Mechanism of Cation Binding to the Glutamate Transporter EAAC1 Probed with Mutation of the Conserved Amino Acid Residue Thr101

The glutamate transporter excitatory amino acid carrier 1 (EAAC1) catalyzes the co-transport of three Na⁺ ions, one H⁺ ion, and one glutamate molecule into the cell, in exchange for one K⁺ ion. Na⁺ binding to the glutamate-free form of the transporter generates a high affinity binding site for glutamate and is thus required for transport. Moreover, sodium binding to the transporters induces a basal anion conductance, which is further activated by glutamate. Here, we used the [Na⁺] dependence of this conductance as a read-out of Na⁺ binding to the substrate-free transporter to study the impact of a highly conserved amino acid residue, Thr¹⁰¹, in transmembrane domain 3. The apparent affinity of substrate-free EAAC1 for Na⁺ was dramatically decreased by the T101A but not by the T101S mutation. Interestingly, in further contrast to EAAC1_WT, in the T101A mutant this [Na⁺] dependence was biphasic. This behavior can be explained by assuming that the binding of two Na⁺ ions prior to glutamate binding is required to generate a high affinity substrate binding site. In contrast to the dramatic effect of the T101A mutation on Na⁺ binding, other properties of the transporter, such as its ability to transport glutamate, were impaired but not eliminated. Our results are consistent with the existence of a cation binding site deeply buried in the membrane and involving interactions with the side chain oxygens of Thr¹⁰¹ and Asp³⁶⁷. A theoretical valence screening approach confirms that the predicted site of cation interaction has the potential to be a novel, so far undetected sodium binding site.