EGFR activation monitored by SW-FCCS in live cells

Abstract

Single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) has been applied to the quantitative determination of molecular interactions at equilibrium for different molecular systems in vitro and in vivo, including living cells and organisms. Here we report for the first time the measurement of an activation and time dependent interaction between a cytosolic and a membrane bound protein by SW-FCCS in live cells. On the example of the epidermal growth factor (EGF) receptor (EGFR) we confirm the existence of pre-formed dimers in the absence of stimulation and demonstrate that the activation of the receptor can be detected by the phosphorylation dependent binding of a phosphotyrosine binding (PTB) domain. SWFCCS results indicate that in CHO cells there is low specific interaction between PTB and EGFR, possibly indicating a low level of EGFR phosphorylation even in the absence of EGF stimulation. After EGF stimulation the interaction between PTB and EGFR increases significantly in a time dependent manner.