Activation of Preformed EGFR Dimers by Binding of Single EGF Molecules: Negative Cooperativity

Abstract

Epidermal growth factor receptor (EGFR) plays roles in cell proliferation, inhibition of apoptosis and angiogenesis among others. It has long been thought that binding of the ligand induces the dimer formation of monomeric EGFR (the “ligand-induced dimerization model”). Upon ligand binding, tyrosine residues in the C-terminal tail of EGFR are phosphorylated, and recruit effector proteins such as Shc1 and Grb2, which trigger activation of downstream signaling cascades. However, a pletora of recent studies indicate that EGFR exists in dimeric form (a preformed dimer), which is incompatible with the ligand-induced dimerization model. To understand the molecular mechanism of activation of EGFR, we directly observed interactions among EGFR, EGF labeled with two different fluorescent compounds, and GFP-tagged Shc1 by multi-color TIRF microscopy. Within 30 seconds of application of the dye-conjugated EGF to cultured cells expressing EGFR, three different fluorescent spots of EGF and Shc1 appeared on the cell surface. EGFR mutant that have no kinase activity could bind EGF but not recruit Shc1. Two different fluorescent colors derived from EGF molecules do not co-localize, and binding of a single EGF molecule induces Shc1 recruitment to EGFR. These results suggest that binding of a single EGF molecule can activate preformed EGFR dimer, consistently with negative cooperativity in EGF binding to EGFR.