Flow cytometric FRET analysis of ErbB receptor tyrosine kinase interaction

The homologous and heterologous interaction of members of the epidermal growth factor (EGF)-related receptor tyrosine kinase (RTK) family (ErbB or HER family receptors) upon ligand binding is the initial key event in signal transduction by these receptors. In addition to the availability of their respective ligands, the relative expression level of the four ErbB receptors triggers receptor cross-activation, which determines signal diversification and the cells' biological response. However, the function of ErbB receptors and their ligands appears highly complex, and its impact on cell growth and proliferation of normal and tumor cells is incompletely understood. Flow cytometric fluorescence resonance energy transfer (FRET) measurements facilitate the quantitative analysis of receptor interaction. This unit delineates the cell-by-cell analysis of epidermal growth factor receptor (EGFR, ErbB1, HER1) and ErbB2 (HER2) receptor interaction in ErbB2-overexpressing BT474 and SK-BR-3 breast cancer cell lines, using a dual-laser flow cytometer. ReFlex software-based quantification of energy transfer efficiency (E) directly reflects the amount of receptor interaction.