Effects of Recurrent Missense Mutations on ErbB2 Receptor Organization and Membrane Dynamics

Abstract

This research focused on elucidating the biophysical and functional impact of three clinically relevant somatic mutations in the ErbB2 receptor: R143Q in the extracellular domain, R678Q in the transmembrane domain, and V842I in the kinase domain, on its organization and dynamics of the plasma membrane. To ensure precise measurements, the study first evaluated the methodological use of the fluorophore-conjugated monoclonal antibody trastuzumab. It was determined that maintaining a labeling degree of approximately 1 is key to preserving the structural integrity of the antibody and the affinity of its epitope, avoiding steric hindrance or artificial homodimerization that could alter the basal dynamic measurement. The membrane evaluation revealed that, when expressed in isolation, the cancer associated ErbB2 variants exhibit higher lateral diffusion and a lower immobile fraction than WT. This increased mobility suggests that the alteration in the autoinhibitory conformation allows the mutant receptor to escape from cytoskeletal constraints and lipid nanodomain clusters. In a more physiological context, when expressed with its main dimerization partner (EGFR), the mutant variants exhibited dimerization, indicating pre-activation. However, upon EGF stimulation, the mutant variants showed an allosteric decoupling. The mutations stabilize the EGFR-ErbB2 heterodimer in a rigid, asymmetrical architecture. In this state, the ErbB2 receptor permanently assumes the role of active kinase, while EGFR functions solely as an activator without catalytic capability, with EGFR being unable to phosphorylate key tyrosine residues in its C-terminal tail. The absence of this phosphorylation prevents the recruitment of degradation proteins. As a result, the heterodimers remain anchored to the plasma membrane in static clusters, sending constitutive signals for cellular survival and proliferation. In summary, this work shows that single-amino-acid substitutions in ErbB2 can shape receptor membrane dynamics. These mutations not only alter signaling but also seem to induce an active, rigid conformation, conferring the oncogenic advantage described as a hallmark of cancer. Therefore, the study identifies receptor conformational dynamics as a promising new target for designing future targeted therapies to restore cellular homeostasis.