Flow Cytometric In Situ Proximity Ligation Analyses of Protein Interactions and Post-translational Modification of the Epidermal Growth Factor Receptor Family

摘要

Interactions between members of the epidermal growth factor receptor (EGFR) familymediates cellular responses to ligand stimulation. Measurement of these interactionscould provide important information and may prove useful as prognostic markers inmalignancy. Therefore, to develop methods to study these interactions in geneticallyunmodified cells, such as clinical samples, in a sensitive and selective way, with goodstatistical accuracy, is important. The in situ proximity ligation assay (in situ PLA) wasused to quantify homo- and heteromeric interactions between EGFR and HER2 in cul-tured cells, using flow cytometry as the readout method. Cells were monitored forchanges in dimerization patterns and phosphorylation status upon stimulation. Thedifferent cell lines displayed varying amounts of interactions between EGFR and HER2,but the amount of dimerization was not found to be affected significantly upon stimu-lation by EGF. Activation of EGFR could be visualized by in situ PLA, but not by immu--nofluorescence staining. In situ PLA was successfully used to study receptor dimeriza-tion and activation of the EGF-receptor family with high selectivity and sensitivity. Thecombination of in situ PLA and flow cytometry provided a statistically powerful way ofanalyzing protein-protein interactions and post-translational modifications on a single- cell basis.