TY - JOUR T1 - Binding of single-mutant epidermal growth factor (EGF) ligands alter the stability of the EGF receptor dimer and promote growth signaling JF - bioRxiv DO - 10.1101/677393 SP - 677393 AU - Stefano Pascarelli AU - Dalmira Merzhakupova AU - Gen-Ichiro Uechi AU - Paola Laurino Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/05/28/677393.abstract N2 - The Epidermal Growth Factor Receptor (EGFR) is a membrane-anchored tyrosine kinase that is able to selectively respond to multiple extra-cellular stimuli. Previous studies have indicated that the modularity of this system is affected by ligand-induced differences in the stability of the dimerized receptor in a process known as “Biased signaling”. However, this hypothesis has not been explored using single-mutant ligands thus far. Herein, we developed a new approach to identify residues responsible for functional divergence combining the conservation and co-evolution information of ortholog and paralog genes encoding the epidermal growth factor (EGF) ligand. Then, we mutated these residues and assessed the mutants’ effects on the receptor by employing a combination of molecular dynamics (MD) and biochemical techniques. Although the EGF mutants had comparable binding affinities to the wild type ligand for EGFR, the EGF mutants induced a different phosphorylation and cell growth pattern in multiple cell lines. The MD simulations of the EGF mutants show a long-range effect on the receptor dimer interface. For the first time in this study, a single mutation in EGF is shown to be enough to alter the activation of the pathway at the cellular level. These results also support the theory of biased signaling in the tyrosine kinase receptor system and demonstrate a promising new way to study ligand-receptor interactions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsEGFEpidermal Growth Factor;EGFREpidermal Growth Factor Receptor;HBEGFHeparin-Binding Epidermal Growth Factor;EPGNEpigen;BTCBetacellulin;EREGEpiregulin;AREGAmphiregulin;PPIProtein-Protein Interaction;MSAMultiple Sequence Alignment;MSTAMultiple STructural Alignment;ITCIsothermal Titration Calorimetry;MSTMicroScale thermophoresis;DMEMDulbecco’s Modified Eagle Medium;ECDExtra Cellular Domain;WTWild Type;CDcircular dichroism ER -