Cell-based high-throughput screening assay system for monitoring G protein-coupled receptor activation using beta-galactosidase enzyme complementation technology.
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 beta-arrestin and ligand-activated GPCR, is described. A protein-protein interaction technology, the InteraX trade mark system, uses a pair of inactive beta-galactosidase (beta-gal) deletion mutants as fusion partners to the protein targets of interest. To monitor GPCR activation, stable cell lines expressing both GPCR- and beta-arrestin-beta-gal fusion proteins are generated. Following ligand stimulation, beta-arrestin binds to the activated GPCR, and this interaction drives functional complementation of the beta-gal mutant fragments. GPCR activation is measured directly by quantitating restored beta-gal activity. The authors have validated this assay system with two functionally divergent GPCRs: the beta2-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 beta2-adrenergic receptor cell line was screened with the LOPAC trade mark 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.
Yan
Yu-Xin
YX
Applied Biosystems, Bedford, MA, USA. yansy@appliedbiosystems.com
Boldt-Houle
Deborah M
DM
Tillotson
Bonnie P
BP
Gee
Melissa A
MA
D'Eon
Brian J
BJ
Chang
Xiao-Jia
XJ
Olesen
Corinne E M
CE
Palmer
Michelle A J
MA
D019390:Arrestins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D018773:Genes, erbB-1; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000295:instrumentation / Q000379:methods*; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D023063:Receptors, Interleukin-8B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D019390:Arrestins; D010750:Phosphoproteins; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D023063:Receptors, Interleukin-8B; D011994:Recombinant Proteins; D000242:Cyclic AMP; D001616:beta-Galactosidase