A Review of Molecular Imaging of Glutamate Receptors.

Molecular imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT) is a well-established and important in vivo technique to evaluate fundamental biological processes and unravel the role of neurotransmitter receptors in various neuropsychiatric disorders. Specific ligands are available for PET/SPECT studies of dopamine, serotonin, and opiate receptors, but corresponding development of radiotracers for receptors of glutamate, the main excitatory neurotransmitter in mammalian brain, has lagged behind. This state of affairs has persisted despite the central importance of glutamate neurotransmission in brain physiology and in disorders such as stroke, epilepsy, schizophrenia, and neurodegenerative diseases. Recent years have seen extensive efforts to develop useful ligands for molecular imaging of subtypes of the ionotropic (N-methyl-D-aspartate (NMDA), kainate, and AMPA/quisqualate receptors) and metabotropic glutamate receptors (types I, II, and III mGluRs). We now review the state of development of radioligands for glutamate receptor imaging, placing main emphasis on the suitability of available ligands for reliable in vivo applications. We give a brief account of the radiosynthetic approach for selected molecules. In general, with the exception of ligands for the GluN2B subunit of NMDA receptors, there has been little success in developing radiotracers for imaging ionotropic glutamate receptors; failure of ligands for the PCP/MK801 binding site in vivo doubtless relates their dependence on the open, unblocked state of the ion channel. Many AMPA and kainite receptor ligands with good binding properties in vitro have failed to give measurable specific binding in the living brain. This may reflect the challenge of developing brain-penetrating ligands for amino acid receptors, compounded by conformational differences in vivo. The situation is better with respect to mGluR imaging, particularly for the mGluR5 subtype. Several successful PET ligands serve for investigations of mGluRs in conditions such as schizophrenia, depression, substance abuse and aging. Considering the centrality and diversity of glutamatergic signaling in brain function, we have relatively few selective and sensitive tools for molecular imaging of ionotropic and metabotropic glutamate receptors. Further radiopharmaceutical research targeting specific subtypes and subunits of the glutamate receptors may yet open up new investigational vistas with broad applications in basic and clinical research.

Identification and mRNA expression profile of glutamate receptor-like gene in quinclorac-resistant and susceptible Echinochloa crus-galli.

Animal ionotropic glutamate receptors (iGluRs) function as Ca(2+) ion channels during excitatory neurotransmission in nerve cells. Here, a glutamate receptor-like gene (GLR) was identified and characterized from a plant - Echinochloa crus-galli. The GLR gene was designated EcGLR1 with GenBank no: JX518597. It has a 2,793 bp open reading frame predicted to encode a 101.7 kDa protein. Sequence alignment showed that EcGLR1 is a GLR homologue. Its expression in response to quinclorac treatment was assessed by real-time PCR in near-isogenic lines of quinclorac-resistant (R) and susceptible (S) biotypes of E. crus-galli. The expression of EcGLR1 in the seedling leaf and root at least increased 5 times in the S plants and 22 times in the R plants after exposure to quinclorac. In the adult plant leaves, roots and stems, its expression increased 11-14 times in the S plants and 23-25 times in the R plants after quinclorac stimulation. In the seed, its expression was 4 times less in the S plants than that in the R plants, but after treatment, the levels all increased by about 24 times in the two biotypes. EcGLR1 expression was 1-4 times greater in the R plants than in that in the S plants, and after treatment by quinclorac, the difference increased to a ratio of 4 to 9. Its expression was higher in all tissues tested of R biotypes than in that of S plants before or after quinclorac treatment. The results of this study provide basic information for the further research of function of the EcGLR1 in resistance to quinclorac in E. crus-galli.

The origin of GPCRs: identification of mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in fungi.

G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily.

Cloning and characterization of glutamate receptors in Californian sea lions (Zalophus californianus).

Domoic acid produced by marine algae has been shown to cause acute and chronic neurologic sequelae in Californian sea lions following acute or low-dose exposure. Histological findings in affected animals included a degenerative cardiomyopathy that was hypothesized to be caused by over-excitation of the glutamate receptors (GluRs) speculated to be present in the sea lion heart. Thus tissues from five sea lions without lesions associated with domoic acid toxicity and one animal with domoic acid-induced chronic neurologic sequelae and degenerative cardiomyopathy were examined for the presence of GluRs. Immunohistochemistry localized mGluR 2/3, mGluR 5, GluR 2/3 and NMDAR 1 in structures of the conducting system and blood vessels. NMDAR 1 and GluR 2/3 were the most widespread as immunoreactivity was observed within sea lion conducting system structures. PCR analysis, cloning and subsequent sequencing of the seal lion GluRs showed only 80% homology to those from rats, but more than 95% homologous to those from dogs. The cellular distribution and expression of subtypes of GluRs in the sea lion hearts suggests that exposure to domoic acid may induce cardiac damage and functional disturbances.

T1R receptors mediate mammalian sweet and umami taste.

The T1R family of taste receptors mediates 2 taste qualities: T1R2/T1R3 for sweet taste and T1R1/T1R3 for umami taste. Functional expression in heterologous system and gene knockout studies has shown their functions as taste receptors. Structure-function relation studies on T1R2/T1R3 showed multiple ligand binding sites on both subunits. The umami taste of l-glutamate can be drastically enhanced by 5' ribonucleotides, and the synergy is a hallmark of this taste quality. On the basis of chimeric T1R receptors, site-directed mutagenesis, and molecular modeling data, we recently proposed a cooperative ligand binding model that involved the Venus flytrap domain of T1R1 in which l-glutamate binds close to the hinge region and 5' ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This novel mechanism may apply to other class C, G protein-coupled receptors.

Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse.

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKgamma, are major unrecognized components of this synapse type. We demonstrate that MRCKgamma can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.

Identification of a neurosteroid binding site contained within the GluR2-S1S2 domain.

Glutamate receptors play a major role in neural cell plasticity, growth, and maturation. The degree to which ionotropic glutamate receptors (iGluR) conduct current is dependent on binding of extracellular ligands, of which glutamate is the native agonist. Although the glutamate binding site of the GluR2 class of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) iGluR has been structurally characterized, the allosteric sites attributed to neurosteroid binding have yet to be localized. Here, using intrinsic tryptophan fluorescence spectroscopy, we show that the extracellular glutamate binding core of the GluR2 class of AMPA receptors also binds to two neurosteroids, pregnenolone sulfate (PS) and 3alpha-hydroxy-5beta-pregnan-20-one sulfate, both of which negatively modulate its activity. Interest in these sulfated neurosteroids stems from their differential modulation of other members of the iGluR family and their potential use as endogeneous agents for stroke therapy. In particular, whereas PS inhibits AMPA and other non-N-methyl-D-aspartate (NMDA) family members, it activates the NMDA receptor. In addition to providing evidence for binding of these neurosteroids to the glutamate binding core of the GluR2 class of AMPA receptors, our data suggests that both neurosteroids bind in a similar manner, consistent with their modulation of activity of this class of iGluR. Interestingly, the conformational change induced upon binding of these neurosteroids is distinct from that induced upon glutamate binding.

Channel properties of the purified glutamate receptor from rat brain reconstituted in planar lipid bilayer membrane.

The properties of the purified rat brain glutamate receptor (GluR), reconstituted in planar lipid bilayer (BLM) were characterised. The single channel currents activated by glutamate and aspartate were similar. The different kinetics of current fluctuation were observed. Paroxysms of channel activity seems to be resulted from the transit of GluR through its active conformation from which it can open several times before desensitising. The effect of concanovaline A (Con A) as an agent blocking desensitisation of glutamatergic synapses was investigated. It was shown that Con A evokes high levels of conductivity and prolonged opening events of channels. Another agent, which stabilises glutamate activated conductivity, dithiothreitol (DTT), evokes "chronic" channel activity. This study demonstrates that purified GluR reconstituted in planar lipid bilayers exhibits the ion-conductivity properties that are associated with the postsynaptic membrane.

[3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts.

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.

[Identification of vestibular compensation-associated molecules by means of differential display].

The differential display method was used to identify gene expression which is altered in the cerebellar flocculus after unilateral labyrinthectomy (UL). Total RNA from flocculi of sham-operated and labyrinthectomized rats was isolated, amplified by PCR using arbitrary primer sets and separated by electrophoresis on a polyacrylamide gel. PCR products, whose amounts were significantly different in samples from labyrinthectomized animals and those from controls, were cut out of the gel and sequenced. One of the up-regulated products was the rat protein phosphatase 2A beta catalytic subunit mRNA and one of the down-regulated products was the rat glutamate receptor delta-2 subunit mRNA. Histochemical examination of in situ hybridization showed that those molecules were intensively localized in the Purkinje cell layer. In labyrinthectomized rats, UL-induced nystagmus gradually disappeared within 3 days after UL. These findings suggest that changes in expression of those molecules in the floccular Purkinje cells after UL is involved in vestibular compensation. So far various kinds of neural plasticity-associated molecules have been investigated, mainly by slice-in vitro studies. This study indicates that differential display is a feasible molecular biological in vivo method for investigation of the mechanism of neural plasticity.

Protein half-lives of two subunits of an NMDA receptor-like complex, the 71-kDa glutamate-binding and the 80-kDa CPP-binding protein.

We determined the half-lives for two subunits of a complex that functions as a glutamate and N-methyl-D-aspartate (NMDA) receptor-ion channel in synaptic membranes. These two proteins are a 71 kDa glutamate-binding protein (GBP) and an 80 kDa CPP-binding protein (CBP). Seven month-old Fischer 344 rats were injected with L-[14C] leucine. The radioactivity in the two proteins was determined in a crude synaptosomal membrane fraction obtained from the brains of rats sacrificed from 4 hours to 13 days after the injection. The previously reported data on time-dependent appearance and loss of L-[14C] leucine radioactivity in the serum (Ferrington et al., 1997, Biochem. Biophys. Res. Commun. 237, 163-165) was used in the present study to estimate the half-lives of GBP and CBP. Theoretical curves best fit the experimental data obtained for the two proteins assuming apparent half-lives of 14 (+/- 2.4) and 18 (+/- 1.2) hours for CBP and GBP, respectively.

A unitary non-NMDA receptor short subunit from Xenopus: DNA cloning and expression.

A high-affinity homomeric, non-NMDA glutamate receptor was previously purified from the amphibian Xenopus laevis. We have obtained nine peptide sequences from its subunit, applied in cDNA cloning. The cDNA encodes a subunit (XenU1) containing all nine sequences. The 51,600-dalton mature subunit has four hydrophobic domains homologous to the four in the C-terminal half of mammalian non-NMDA receptor subunits. Transient expression in COS cells showed 1:1 binding (at Bmax) of [3H] kainate (KD = 9.1 nM) and of [3H] AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid; KD = 62 nM). The competitive binding series domoate > kainate > AMPA > NBQX > glutamate was established (where NBQX is 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo (f) quinoxaline). Each agonist shows the same KI value against [3H] kainate and [3H] AMPA binding, suggesting a common agonist site, but two conformations thereof are distinguishable by their different affinities for the antagonist NBQX and by the allosteric effect of thiocyanate anion (greatly potentiating AMPA binding, inert with kainate). XenU1 is exceptional among non-NMDA receptor subunits because it lacks most of the large N-terminal domain found in those of mammals and it has high affinity for both kainate and AMPA. It differs from the similarly-short "kainate-binding proteins" (KBPs), in binding AMPA and in forming glutamate receptor channels when the native protein is reconstituted. Moreover, whereas a full-length kainate receptor of mammals, GluR6, is shown here (from a partial cDNA sequence) to exist also in Xenopus, with approximately 97% sequence identity to rat GluR6, XenU1 is much less homologous to any rat kainate or AMPA receptor and also to the KBPs, even from another amphibian, Rana. Another difference is that a potential concensus sequence ("EF hand") for Ca2+ binding is present in the N-terminal domain of XenU1, but not in the chicken (glial) KBP. XenU1 is deduced to be in a new family of non-NMDA receptors.

Purification of recombinant GluR-D glutamate receptor produced in Sf21 insect cells.

GluR-D glutamate receptors carrying FLAG and polyHis affinity tags at the N-terminus and C-terminus, respectively, were expressed in recombinant baculovirus-infected Spodoptera frugiperda Sf21 cells. Affinity-tagged receptors displayed ligand-binding affinity (Kd = 40 nM) and an expression level (Bmax 10-30 pmol/mg protein) similar to that of insect-cell-expressed wild-type GluR-D, as determined by [3H]-alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid ([3H]AMPA) binding. The receptor was solubilized in Triton X-100, and purified using a two-step protocol consisting of immobilized metal-chelation affinity chromatography followed by immunoaffinity chromatography. The purified receptor preparation contained over 2000 pmol high-affinity [3H]AMPA-binding sites/mg protein, and migrated as a single 110-kDa species in SDS/PAGE. Peptide:N-glycosidase F treatment reduced the size of GluR-D from 110 kDa to 100 kDa, indicating the presence of N-linked glycans. Up to 100 micrograms purified GluR-D was obtained from 1 l Sf21 suspension culture (2-3 x 10(6) cells/ml). High-level expression of affinity-tagged GluRs in insect cells should be an efficient strategy to produce GluR subtypes for biochemical and structural studies.

Partial purification of [3H]glutamate-associating-proteins with sensitivity to displacement by N-methyl-D-aspartate from rat brain.

An attempt was made to solubilize and isolate [3H]L-glutamic acid (Glu) binding sensitive to displacement by N-methyl-D-aspartic acid (NMDA) from rat brain. Brain synaptic membranes were solubilized by deoxycholic acid, followed by gel filtration with Sephadex G-25. In these turbid supernatants, significant but fragile binding was detected with a variety of radioligands related to ionotropic subclasses of receptors for excitatory amino acids. These included [3H]5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine (MK-801), [3H]glycine, [3H]spermidine, [3H]Glu, [3H]DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic and [3H]kainic acids. Re-solubilization of turbid supernatants by Triton X-100 resulted in detection of [3H]Glu binding which was only stable for 24 h, with [3H]MK-801 binding being entirely lost. In these clear preparations after re-solubilization, Glu was exclusively effective in completely displacing [3H]Glu binding with other ligands being partially active. Furthermore, [3H]Glu binding displaceable by NMDA was eluted with 0.5 M KCl together with [3H]Glu binding insensitive to NMDA on DEAE-Toyopearl column chromatography, while fractions eluted with 0.2 M KCl had NMDA-insensitive [3H]Glu binding only. Chromatography on chelate (Zn)-Toyopearl resin resulted in elution of both NMDA-sensitive and NMDA-insensitive [3H]Glu binding with 10 mM EDTA. High performance liquid chromatography revealed that NMDA-sensitive [3H]Glu binding was detected at retention times of 10-20 min when eluted from an Asahipak ES-502N column with NaCl at linearly graded concentrations up to 0.5 M. In order to detect NMDA-sensitive [3H]Glu binding, however, the whole procedures needed to be completed within 24 h after re-solubilization. Accordingly, the identity of the NMDA-sensitive [3H]Glu binding partially purified here is still unclear at present. The NMDA recognition domain could be more stable than the NMDA channel domain on the NMDA receptor ionophore complex under aqueous conditions.

Differential assembly of coexpressed glutamate receptor subunits in neurons of rat cerebral cortex.

In the rat, subunits of the glutamate receptor family fall into three pharmacologically distinct groups: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring receptors (Glu R1-4), kainate preferring receptors (Glu R5-7, KA 1, KA 2), and N-methyl-D-aspartate preferring receptors (NMDA R1, NMDA R2A-2D). In the present study, we demonstrate immunocytochemically that the majority of neurons in rat cerebral cortex coexpress members of all three groups of glutamate receptor subunits, Glu R2/3, Glu R5/6/7, and NMDA R1. Using immunoaffinity purified or immunoprecipitated alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid, kainate and N-methyl-D-aspartate receptors, we show that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors containing Glu R1-4, kainate receptors containing Glu R6, Glu R7, and KA 2 and N-methyl-D-aspartate receptors containing NMDA R1 each form distinct protein complexes that do not share subunits. Our data indicate that a mechanism exists which allows for the specific assembly of selected glutamate receptor subunits into functionally and structurally distinct heteromeric receptors.

Expression of ionotropic glutamate receptor genes by P19 embryonal carcinoma cells.

P19 embryonal carcinoma cells can be induced to differentiate into neuron-like cells by retinoic acid. P19 neurons were recently shown to express both NMDA and non-NMDA type glutamate receptor-mediated currents and be susceptible to glutamate excitotoxicity. In this study, we used RT-PCR to survey differentiated P19 cultures for glutamate receptor transcript expression. The following transcripts were detected: at least one member of the GluR1-4 family, GluR5, GluR6, GluR7, KA1, KA2, NMDAR1, and NMDAR2B. Nuclease protection assays revealed a large quantitative induction of GluR6 transcripts following retinoic acid treatment. Inotropic glutamate receptors are a fundamental and major feature of CNS neurons which are not expressed by the cell lines commonly used as experimental models for mammalian neurons. The present results show that P19 cells express multiple genes involved in glutamate receptor biology. Since the stem cells can be manipulated genetically, the system has the basic requirements for analyzing mechanisms involved in glutamate receptor gene expression.

Selective expression of the glutamate receptor channel delta 2 subunit in cerebellar Purkinje cells.

The primary structure of a putative subunit of the mouse glutamate receptor channel, designated as the delta 2 subunit, has been deduced by cloning and sequencing the cDNA. The delta 2 subunit has four putative transmembrane segments characteristic for neurotransmitter-gated ion channels, and shares 56% amino acid sequence identity with the delta 1 subunit of the mouse glutamate receptor channel and 14-24% identity with the subunits of the AMPA-, kainate- or NMDA-selective glutamate receptor channel. RNA blot and in situ hybridization analyses show that the delta 2 subunit mRNA is localized in cerebellar Purkinje cells. Furthermore, immunoblot and immunohistochemical analyses suggest that the delta 2 subunit protein is actually expressed in vivo in Purkinje neurons. The selective localization of the delta 2 subunit in Purkinje cells may imply a role of the delta 2 subunit in Purkinje cell-specific function such as the cerebellar LTD.