A SIMPLE LUCIFERASE ASSAY FOR SIGNAL-TRANSDUCTION ACTIVITY DETECTION OF EPIDERMAL GROWTH-FACTOR DISPLAYED ON PHAGE

Studies on receptor-ligand interactions are important for the design o f agonists or antagonists of natural ligands. We developed a luciferas e reporter assay to screen epidermal growth factor receptor (EGFR) bin ding molecules rapidly for their ability to stimulate or inhibit signa l transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR , for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructe d in which the aspartic acid residue at position 46 or the arginine re sidue at position 41 were randomised. EGF mutants displayed on phage w ere screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGF R and their transducing activity corresponded perfectly with their bin ding. For the more crucial 41 position, only the wild-type EGF was abl e to bind the EGFR. Our approach allowed a simple determination of cru cial positions and paved the way for identification of agonists with a ltered transduction activity.