Development of a β-Lactamase Reporter Gene Assay for Metabotropic Glutamate Receptor 1 by Using Coexpression of Glutamate Transporter

Metabotropic glutamate receptors (mglurs) belong to a family of G-protein-coupled receptors and are thought to be involved in the modulation of neuronal excitability and neurotransmitter release. Eight subtypes of the receptor (mGluR1-mGluR8) have been identified to date and are classified into 3 groups based on sequence homology, pharmacological profile, and signal transduction pathway. mGluR1 and mGluR5 are classified into group I mGluRs and are coupled to phospholipase C and subsequent intracellular calcium mobilization via Gq-protein. Earlier efforts to identify an mGluR1-selective antagonist by chemical library screening using an orthosteric radioligand binding assay were unsuccessful. This was presumably due to the highly conserved amino acid sequences of the L-glutamatebinding site in mGluR1 and mGluR5. On the other hand, a functional assay based on the detection of intracellular Ca increases mediated via receptor activation has allowed us to identify mGluR1-selective allosteric antagonists. The success of this approach may stem from the fact that functional assays can identify compounds interacting with sites different from the L-glutamate binding site, such as the transmembrane domain. These sites are less conserved than the L-glutamate binding site. Several animal studies using these mGluR1selective allosteric antagonists indicate that blockage of mGluR1 could ameliorate CNS disorders, including neuropathic pain, neurodegeneration, and psychiatric diseases. However, it is not known whether mGluR1 is involved in human CNS disorders. In addition to the Ca mobilization assay, a reporter gene assay under the control of the NFAT promoter (which is activated by an increase in intracellular Ca) is another functional assay applicable to the identification of Gq-protein-coupled mGluR1 ligands. Of the known reporter gene assays, β-lactamase has several interesting features for high-throughput screening (HTS). For example, this reporter assay uses β-lactamase and its cell-permeable fluorogenic substrate, CCF2. Excitation of CCF2 produces green fluorescence Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan.