IMR Press / FBE / Volume 3 / Issue 1 / DOI: 10.2741/E216

Frontiers in Bioscience-Elite (FBE) is published by IMR Press from Volume 13 Issue 2 (2021). Previous articles were published by another publisher on a subscription basis, and they are hosted by IMR Press on imrpress.com as a courtesy and upon agreement with Frontiers in Bioscience.

Open Access Article
EGFR activation monitored by SW-FCCS in live cells
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1 Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543
2 Institute of Medical Biology, 8A Biomedical Grove, Singapore 138665

*Author to whom correspondence should be addressed.

Academic Editor: Vicente Andres

Front. Biosci. (Elite Ed) 2011, 3(1), 22–32; https://doi.org/10.2741/E216
Published: 1 January 2011
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. SW-FCCS 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.

Keywords
Epidermal Growth Factor Receptor
Tyrosine Kinase
Phosphotyrosine Binding Domain
FCS
FCCS
SW-FCCS
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