title: Development of a beta-lactamase reporter gene assay for metabotropic glutamate receptor 1 by using coexpression of glutamate transporter.
abstract: mGluR1 antagonists have been postulated to be novel CNS drugs, including antipsychotics. Toward this end, the authors developed a beta-lactamase reporter assay to identify mGluR1 antagonists. beta-Lactamase has several interesting features for high-throughput screening, including very high sensitivity and less well-to-well variation than other reporter enzymes. mGluR1-expressing Chinese hamster ovary (CHO) cells with the beta-lactamase gene under control of the nuclear factor of activated T cells (NFAT) promoter (CHO-NFAT-bla-hmGluR1b) exhibited very high basal activity, resulting in an inadequate signal-to-basal (S/B) ratio. Coexpression of glutamate/aspartate transporter (GLAST) with mGluR1 in the cell line (CHO-NFAT-bla-hmGluR1b-GLAST) dramatically decreased basal activity and improved the S/B ratio (from 2- to 20-fold). The contribution of GLAST to lowering basal activity and increasing the S/B ratio was validated by the expression level of GLAST mRNA and by a GLAST inhibitor. Antagonistic activities of known mGluR1 antagonists in the beta-lactamase reporter assay were comparable with those in the conventional Ca(2+) mobilization assay. The Z' factor of the beta-lactamase reporter assay was 0.89 under optimized conditions. Taken together, the beta-lactamase reporter assay with CHO-NFAT-bla-hmGluR1b-GLAST could be a novel high-throughput assay for mGluR1 antagonist screening. This is the first description of a successful beta-lactamase reporter assay among all mGluR subtypes.
authors.affiliation: Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan.
authors.forename: Gentaroh
authors.initials: G
authors.lastname: Suzuki
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawamoto
authors.forename: Hisashi
authors.initials: H
authors.lastname: Ohta
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D005260:Female; D017930:Genes, Reporter; D006801:Humans; D008954:Models, Biological; D012333:RNA, Messenger / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
chemical_list: D027341:Excitatory Amino Acid Transporter 1; D012333:RNA, Messenger; D018094:Receptors, Metabotropic Glutamate; C104077:metabotropic glutamate receptor type 1; D001618:beta-Lactamases