title: A novel binding assay for metabotropic glutamate receptors using [3H] L-quisqualic acid and recombinant receptors.
abstract: We established a methodology to analyze radioligand binding to the recombinant type la metabotropic glutamate receptor (mGluRla). A full-length cDNA encoding mGluR1a, which was isolated from a lambda gt 11 cDNA library of human cerebellar origin, was expressed in a baculovirus/Sf9 insect cell system. Membrane fractions with recombinant receptor expression were analyzed for the binding of [3H]L-quisqualic acid (L-QA), which is known to be a potent agonist of mGluRla. Efficient binding of the radioligand to the human receptor was observed in a saturable manner, giving an apparent Kd= 0.091 microM. [3H]L-QA bound to the human mGluR1a was displaced by known ligands such as L-QA, L-Glu, t-ACPD ((+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid) with IC50s = 0.056, 0.97 and 4.0 microM, respectively. MCPG (alpha-methyl-4-carboxyphenylglycine) displaced the radioligand binding with lower potency. Using this binding protocol, we then evaluated the ligand ability of synthetic dipeptides. Among peptides tested, only Glu-containing dipeptides inhibited the radioligand binding, e.g. IC50 of L-Met-L-Glu was 4.3 microM. When phosphatidyl inositol turnover was assayed in mGluR1a-expressing CHO cells, L-Met-L-Glu was partially agonistic. We further expanded this [3H]L-QA binding protocol to type 5a mGluR, another member of group I mGluRs, as well as to AMPA receptor, a member of ionotropic glutamate receptors, since L-QA is also known to be a potent ligand for these receptors. Data shown here will provide a novel system not only to search for ligands for the glutamate receptors, but also to biochemically analyze the interaction modes between glutamate receptors and their ligands.
authors.affiliation: Biomedical Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co, Ltd, Ibaraki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ohashi
authors.forename: Takaharu
authors.initials: T
authors.lastname: Maruyama
authors.forename: Hidemi
authors.initials: H
authors.lastname: Higashi-Matsumoto
authors.forename: Takashi
authors.initials: T
authors.lastname: Nomoto
authors.forename: Susumu
authors.initials: S
authors.lastname: Nishimura
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Takeuchi
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D016318:Quisqualic Acid / Q000493:pharmacokinetics*; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014316:Tritium
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007295:Inositol Phosphates; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; C104077:metabotropic glutamate receptor type 1; D014316:Tritium; D016318:Quisqualic Acid