Affiliation:
1. Biomedical Research Laboratories
2. Clinical Development Institute, Banyu Pharmaceutical Co., Ltd., AIG Kabutocho Building, 5D1 Nihombashi-kabutocho, Chuo-ku, Tokyo 103D 0026, Japan
3. Drug Discovery Research Laboratories, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., 300Ð0312 Ibaraki, Japan
Abstract
We established a methodology to analyze radioligand binding to the recombinant type 1a metabotropic glutamate receptor (mGluR1a). A full-length ᴄDNA encoding mGluR1a, which was isolated from a λ gt 11 ᴄDNA 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 (ʟ-QA), which is known to be a potent agonist of mGluR1a. Efficient binding of the radioligand to the human receptor was observed in a saturable manner, giving an apparent Kd= 0.091 μᴍ. [3H]ʟ-QA bound to the human mGluR1a was displaced by known ligands such as ʟ-QA, ʟ-Glu, t-ACPD(( ±)-1- aminocyclopentane-trans-1,3-dicarboxylic acid) with IC50s = 0.056, 0.97 and 4.0 μᴍ, respectively. MCPG (α-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 ʟ-Met-ʟ-Glu was 4.3 μᴍ. When phosphatidyl inositol turnover was assayed in mGluR1a-expressing CHO cells, ʟ-Met-ʟ-Glu was partially agonistic. We further expanded this [3H]ʟ-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 ʟ-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
Subject
General Biochemistry, Genetics and Molecular Biology
Cited by
22 articles.
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