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Cell-Based High-Throughput Screening Assay System for Monitoring G Protein-Coupled Receptor Activation Using ß-Galactosidase Enzyme Complementation Technology

Applied Biosystems, Bedford, MA

Deborah M. Boldt-Houle

Applied Biosystems, Bedford, MA

Bonnie P. Tillotson

Applied Biosystems, Bedford, MA

Melissa A. Gee

Applied Biosystems, Bedford, MA

Brian J. D'Eon

Applied Biosystems, Bedford, MA

Xiao-Jia Chang

Applied Biosystems, Bedford, MA

Corinne E. M. Olesen

Applied Biosystems, Bedford, MA

Michelle A. J. Palmer

Applied Biosystems, Bedford, MA

A novel cell-based functional assay to directly monitor G protein-coupled receptor (GPCR) activation in a high-throughput format, based on a common GPCR regulation mechanism, the interaction between ß-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX^TM system, uses a pair of inactive ß-galactosidase (ß-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and ß-arrestin-ß-gal fusion proteins are generated. Following ligand stimulation, ß-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the ß-gal mutant fragments. GPCR activation is measured directly by quantitating restored ß-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the ß2-adrenergic amine receptor and the CXCR2 chemokine-binding receptor. Both receptors are activated or blocked with known agonists and antagonists in a dose-dependent manner. The ß2-adrenergic receptor cell line was screened with the LOPAC^TM compound library to identify both agonists and antagonists, validating this system for high-throughput screening performance in a 96-well microplate format. Hit specificity was confirmed by quantitating the level of cAMP. This assay system has also been performed in a high-density (384-well) microplate format. This system provides a specific, sensitive, and robust methodology for studying and screening GPCR-mediated signaling pathways.

Journal of Biomolecular Screening, Vol. 7, No. 5, 451-459 (2002)
DOI: 10.1177/108705702237677


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