AbstractThe extracellular part of ErbB‐2 is formed by 4 domains, specifically, L1, L2 that adopt a β‐helical structure and S1, S2 that consist of several cysteine‐rich, EGF‐fold modules. These ectodomains mediate ErbB‐2 dimerisation with itself or with other members of the epidermal growth factor receptor (EGFR) family, events essential to both ErbB‐2 signaling and the development of certain malignancies. The anti‐ErbB‐2 monoclonal antibodies N12, N28 and L87 bind to the polypeptides C531‐A586, T216‐C235 and C220‐C235 respectively. In this study, glycine walking and random mutagenesis were used to further delineate the critical residues involved in antibody binding. A molecular model of ErbB‐2 ectodomains was then constructed based on the recently published coordinates of the EGFR (EGFR) model. This model rationalized successfully many features of our epitope mapping, including their location in modules within the S1 and S2 domains and the importance of Arg545, Gln548 and Leu561 for N12 binding. Further investigation of the functional effects of the anti‐ErbB‐2 monoclonal antibodies demonstrated that N28 strongly stimulated ErbB‐2 phosphorylation and MAPK activation whereas N12 had no effect. As bivalency is required for the action of these antibodies we propose that at least 2 different kinds of ErbB‐2 homodimers can be formed as relative rotational isomers and that the S1 and S2 domains are instrumental in determining the relative orientations of the ErbB‐2 homodimers, such that different signaling effects are induced. © 2003 Wiley‐Liss, Inc.