Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor.

Metabotropic glutamate receptors (mGluRs) are G-protein-coupled glutamate receptors that subserve a number of diverse functions in the central nervous system. The large extracellular amino-terminal domains (ATDs) of mGluRs are homologous to the periplasmic binding proteins in bacteria. In this study, a region in the ATD of the mGluR4 subtype of mGluR postulated to contain the ligand-binding pocket was explored by site-directed mutagenesis using a molecular model of the tertiary structure of the ATD as a guiding tool. Although the conversion of Arg(78), Ser(159), or Thr(182) to Ala did not affect the level of protein expression or cell-surface expression, all three mutations severely impaired the ability of the receptor to bind the agonist L-[(3)H]amino-4-phosphonobutyric acid. Mutation of other residues within or in close proximity to the proposed binding pocket produced either no effect (Ser(157) and Ser(160)) or a relatively modest effect (Ser(181)) on ligand affinity compared with the Arg(78), Ser(159), and Thr(182) mutations. Based on these experimental findings, together with information obtained from the model in which the glutamate analog L-serine O-phosphate (L-SOP) was "docked" into the binding pocket, we suggest that the hydroxyl groups on the side chains of Ser(159) and Thr(182) of mGluR4 form hydrogen bonds with the alpha-carboxyl and alpha-amino groups on L-SOP, respectively, whereas Arg(78) forms an electrostatic interaction with the acidic side chains of L-SOP or glutamate. The conservation of Arg(78), Ser(159), and Thr(182) in all members of the mGluR family indicates that these amino acids may be fundamental recognition motifs for the binding of agonists to this class of receptors.

Cloning and characterization of a metabotropic glutamate receptor, mGluR4b.

An alternative spliced variant of metabotropic glutamate receptor subtype mGluR4a, termed mGluR4b was isolated from a rat cDNA library. Subtype mGluR4b was identical to the previously described mGluR4a, except for the last 64 amino acids in the C-terminal region in which were replaced by 135 new amino acids in mGluR4b. Recombinant baculoviruses coding for mGluR4a and mGluR4b were expressed in Spodoptera frugiperda, Sf-9, insect cells and characterized pharmacologically by measuring [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding and second messenger formation. [3H]-L-AP4 binding to membranes prepared from Sf-9 cells expressing mGluR4a and mGluR4b revealed respective affinities (Kd) of 480 and 360 nM and maximal binding densities (Bmax) of 4.2 and 0.8 pmol/mg protein. The ligand selectivity of mGluR4a and mGluR4b was similar: L-AP4 > L-serine-O-phosphate > L-glutamate > L-2-amino 2-methyl-4-phosphonobutyrate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > or = quisqualate. A decrease in the affinity of [3H]-L-AP4 was observed in the presence of 0.1 mM guanosine 5'-O-(3-thio)trisphosphate-gamma-S, indicating that mGluR4a and mGluR4b were functionally coupled to G-proteins in Sf-9 cells. Agonists of mGluR4 caused a minor decrease in forskolin-induced cAMP formation in Sf-9 cells expressing either mGluR4a or mGluR4b, suggesting that both receptors are coupled to adenylate cyclase in an inhibitory manner. Thus, mGluR4a and mGluR4b share a common signal transduction pathway and pharmacology when expressed in Sf-9 insect cells.

Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor.

The application of the glutamate analog L-2-amino-4-phosphonobutyric acid (L-AP4) to neurons produces a suppression of synaptic transmission. Although L-AP4 is a selective ligand at a subset of metabotropic glutamate receptors (mGluRs), the precise physiological role of the L-AP4-activated mGluRs remains primarily unknown. To provide a better understanding of the function of L-AP4 receptors, we have generated and studied knockout (KO) mice lacking the mGluR4 subtype of mGluR that displays high affinity for L-AP4. The mGluR4 mutant mice displayed normal spontaneous motor activity and were unimpaired on the bar cross test, indicating that disruption of the mGluR4 gene did not cause gross motor abnormalities, impairments of novelty-induced exploratory behaviors, or alterations in fine motor coordination. However, the mutant mice were deficient on the rotating rod motor-learning test, suggesting that mGluR4 KO mice may have an impaired ability to learn complex motor tasks. Patch-clamp and extracellular field recordings from Purkinje cells in cerebellar slices demonstrated that L-AP4 had no effect on synaptic responses in the mutant mice, whereas in the wild-type mice 100 microM L-AP4 produced a 23% depression of synaptic responses with an EC50 of 2.5 microM. An analysis of presynaptic short-term synaptic plasticity at the parallel fiber-->Purkinje cell synapse demonstrated that paired-pulse facilitation and post-tetanic potentiation were impaired in the mutant mice. In contrast, long-term depression (LTD) was not impaired. These results indicate that an important function of mGluR4 is to provide a presynaptic mechanism for maintaining synaptic efficacy during repetitive activation. The data also suggest that the presence of mGluR4 at the parallel fiber-->Purkinje cell synapse is required for maintaining normal motor function.

Expression profiling of mRNA obtained from single identified crustacean motor neurons: determination of specificity of hybridization.

The purpose of this study was to determine if the technique of expression profiling would allow us to determine the changes in the abundance of certain mRNAs in identifiable, single neurons as a result of heightened electrical activity. In doing so we developed an approach to test the specificity of hybridization in expression profiling. Messenger RNA from single identified crayfish motor neurons was amplified by ligation-mediated reverse transcription PCR and hybridized by dot-blotting to 45 target cDNAs from different species. As a test of specificity, the hybridization was repeated using unlabelled cDNAs, the dots were excised, and the hybridized nucleic acids were re-amplified, cloned, and sequenced to confirm their identity. By cloning and sequencing the re-amplified product for each cDNA examined, one can determine the degree of background hybridization as compared to homologous hybridization. False positive results were also observed when a species-specific cDNA and highly stringent hybridization conditions were used. Our results demonstrate that ligation-mediated PCR is a useful technique for checking the specificity of expression profiling. This approach can easily be adapted to any situation when confirmation of the specificity of nucleic acid hybridization is required. During this study, part of a novel crayfish neuronal actin cDNA was cloned and sequenced.

Differential effects of lectins on recombinant glutamate receptors.

The effects of the lectins concanavalin A, succinyl concanavalin A, wheat-germ agglutinin and soybean agglutinin were studied at recombinant ionotropic glutamate receptors expressed in Xenopus oocytes. Homomeric and heteromeric receptors from each of the three major classes of ionotropic glutamate receptors (N-methyl-D-asparate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate) were studied. The lectins potentiated homomeric configurations of kainate, AMPA and NMDA receptors to a greater degree than the corresponding heteromeric configurations although the rank order of the lectin potentiating effects was the same for both homomeric and heteromeric receptors within a given glutamate receptor class. The most profound effects of the lectins were observed with the kainate receptors; the rank order of potentiating effects of the lectins at the homo- and heteromeric kainate receptors (Glu6 and Glu6/KA-2) was concanavalin A > succinyl concanavalin A > wheat-germ agglutinin > soybean agglutinin. At the recombinant Glu3 and Glu2/3 AMPA receptor complexes, wheat-germ agglutinin and concanavalin A produced the largest enhancements of the glutamare-activated currents followed by succinyl concanavalin A; soybean agglutinin had no significant potentiating effect. Agonistevoked currents recorded from oocytes expressing the homo- and heteromeric NMDA receptors were only slightly enhanced by concanavalin A and succinyl concanavalin A but not by wheat-germ agglutinin or soybean agglutinin. These results demonstrate that kainate. AMPA and NMDA receptors display dramatic differences in their responses to lectins, and suggest that the receptor-bound oligosaccharide side chains may play different roles in the functional responses mediated by the three major classes of ionotropic glutamate receptors.