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Abstract

G-protein-coupled receptors (GPCRs) are important therapeutic targets for many areas of drug research and development. Although chimeric Gα₁₆ proteins are valuable tools for detecting the activation of Gα_i/o-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both Gα₁₆ and Gα_i/o (Gα_16/o, Gα_16/i2, and Gα_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-HT_1A serotonin receptors. The pEC₅₀ 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 D_2L receptor, whereas S-(—)-3-PPP displayed partial activity of dopamine and quinpirole in the presence of the Gα_16/o chimera. The potency of dopamine for D_2L receptors was similar among Gα_16/o, Gα_16/i2, and Gα_16/i3. In contrast, the 5-HT_1A receptor exhibited a significantly preferential coupling for Gα_16/i3 compared with Gα_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)

Keywords

Gi-coupled receptors D2L dopamine receptor 5-HT1A serotonin receptor Gα16 chimeric Gα proteins

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

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