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 of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal 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 constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were 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 EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.
Souriau
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Institut de Génétique Moléculaire, UMR CNRS 5535, BP5051, 1919 Route de Mende, 34033 Montpellier cedex 1, France.
Fort
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Roux
P
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Hartley
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O
Lefranc
M P
MP
Weill
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M
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D017930:Genes, Reporter; D005819:Genetic Markers; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000032:analysis*; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D008156:Luciferases; D066246:ErbB Receptors