IMPROVED SYSTEM FOR SENSITIVE DETECTION OF G-PROTEIN COUPLED RECEPTOR AND ORPHAN RECEPTOR FUNCTION USING REPORTER ENZYME MUTANT COMPLEMENTATION

摘要

PROBLEM TO BE SOLVED: To figure out methods for assaying G-protein coupled receptor (GPCR) activity, methods for screening for GPCR ligands, and G-protein-coupled receptor kinase (GRK) activity.SOLUTION: Methods for expanding ICAST technologies for assaying GPCR activity are provided. These methods include: engineering serotonin/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the state that G-protein coupled receptor kinases is limited; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs. More specific methods are included for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead compound discovery and ligand and function discovery for orphan GPCRs.