Binding Interaction of the Heregulinβ egf Domain with ErbB3 and ErbB4 Receptors Assessed by Alanine Scanning Mutagenesis

Abstract

Individual residues of the heregulinβ (HRG)<i>egf</i> domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGβ <i>egf</i> domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGβ <i>egf</i> domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the<i>egf</i> domain; including the NH₂ terminus (His² and Leu³), the two β-turns (Val¹⁵–Gly¹⁸ and Gly⁴²–Gln⁴⁶), and some discontinuous residues (including Leu³, Val⁴, Phe¹³, Val²³, and Leu³³) that form a patch on the major β-sheet and the COOH-terminal region (Tyr⁴⁸ and Met⁵⁰–Phe⁵³). Binding affinity was least changed by mutations throughout the Ω-loop and the second strand of the major β-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor α binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of <i>egf</i>-like analogs with broad or narrow specificity.