Development of generic calcium imaging assay for monitoring gi-coupled receptors and g-protein interaction

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 Gαi/o-coupled receptors, the details of the activation process remain unclear. The authors introduce a series of chimeras that combine both Gα16 and Gαi/o (Gα16/o, Gα16/i2, and Gα16/i3) into a well-established transient expression system to examine the ability of these chimeras to interact with D2 long-form (D2L) dopamine and 5-HT1A 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 D2L receptor, whereas S-(-)-3)-3-PPP displayed partial activity of dopamine and quinpirole in the presence of the Gα16/o chimera. The potency of dopamine for D2L receptors was similar among Gα16/o, Gα16/i2, and Gα16/i3. In contrast, the 5-HT1A receptor exhibited a significantly preferential coupling for Gα16/i3 compared with Gα16/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. © 2009 Society for Biomolecular Sciences.