Characterization of CHO Cells Stably Expressing a Gα 16/z Chimera for High Throughput Screening of GPCRs

Abstract

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Gα protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G_i/o- and G_s-coupled receptors to mobilize intracellular Ca²⁺ upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca²⁺ mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.