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Journal of Biomolecular Screening
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Development of Generic Calcium Imaging Assay for Monitoring Gi-Coupled Receptors and G-Protein Interaction

Takashi Ueda

Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan, tueda{at}med.nagoya-cu.ac.jp

Shinya Ugawa

Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan

Yusuke Ishida

Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan

Aki Hondoh

Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan

Shoichi Shimada

Department of Neurobiology and Anatomy, Graduate School of Medical Science, Nagoya City University, Nagoya, Japan

G-protein-coupled receptors (GPCRs) are important therapeutic targets for many areas of drug research and development. Although chimeric G{alpha}16 proteins are valuable tools for detecting the activation of G{alpha}i/o-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both G{alpha}16 and G{alpha} i/o (G{alpha}16/o, G{alpha}16/i2, and G{alpha} 16/i3) into a well-established transient expression system to examine the ability of these chimeras to interact with D2 long-form (D 2L) dopamine and 5-HT1A serotonin receptors. The pEC 50 data obtained for known agonists were similar to results from previous studies that used other cell-based assays, thus indicating sufficient sensitivity for the assay. Moreover, quinpirole exhibited similar intrinsic activity to dopamine at the D2L receptor, whereas S-(—)-3-PPP displayed partial activity of dopamine and quinpirole in the presence of the G{alpha}16/o chimera. The potency of dopamine for D2L receptors was similar among G{alpha}16/o, G{alpha}16/i2, and G{alpha} 16/i3. In contrast, the 5-HT1A receptor exhibited a significantly preferential coupling for G{alpha}16/i3 compared with G{alpha} 16/i2 when serotonin was used as a ligand. This finding was in close agreement with the results of previous reports. The present system could therefore be used as a rapid functional assay for high-throughput screening and deorphanization. (Journal of Biomolecular Screening 2009:781-788)

Key Words: Gi-coupled receptors • D2L dopamine receptor • 5-HT1A serotonin receptor • G{alpha}16 • chimeric G{alpha} proteins

This version was published on August 1, 2009

Journal of Biomolecular Screening, Vol. 14, No. 7, 781-788 (2009)
DOI: 10.1177/1087057109335258
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