Biophysical characterization of recurrent ErbB2 missense mutations reveals alterations in receptor organization and membrane dynamics

• Mutation-dependent ErbB2 activation leads to accelerated ErbB2 diffusion. • EGF/EGFR-induced ErbB2 activation results in restricted lateral diffusion of ErbB2. • Cancer-associated ErbB2 mutations lead to dimerization without phosphorylation. The purpose of this study was to characterize three recurrent, cancer-associated missense mutations in ErbB2, R143Q, R678Q, and V842I, located in the extracellular, juxtamembrane and kinase domains, respectively, to determine how single amino acid substitutions affect receptor organization and dynamics. Using confocal microscopy, Förster resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP), we assessed homo- and heteroassociation, lateral diffusion and tyrosine phosphorylation of ErbB2 cancer-associated variants either alone or co-expressed with EGFR. The only individual biophysical property that differentiated the mutation-activated ErbB2 variants from the wild-type was an accelerated diffusion in the absence of EGFR coexpression. Correlative analysis of the biophysical properties of ErbB2 revealed that ErbB2-activating mutations, including the cancer-associated R143Q, R678Q and V842I mutations, may promote a pre-dimerized receptor state associated with accelerated lateral mobility, but without full-scale activation implied by the lack of growth factor-independent tyrosine phosphorylation. The faster mobility of mutation-activated ErbB2 contrasted with the EGF-induced slowing down of its lateral diffusion. In summary, single amino acid substitutions across ErbB2 domains may modulate receptor dynamics, organization, and responsiveness.