Binding interaction of the heregulinbeta egf domain with ErbB3 and ErbB4 receptors assessed by alanine scanning mutagenesis.

Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf 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 HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-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 alpha 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 egf-like analogs with broad or narrow specificity.