Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements
Ágnes Szabó 1, Gábor Horváth 1, János Szöllosi 1 and Peter Nagy 1*
1 University of Debrecen
* To whom correspondence should be addressed. E-mail: peter.v.nagy{at}gmail.com.
Submitted on March 13, 2008
Revised on April 7, 2008
Accepted on 1 May 2008
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Abstract |
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Association of receptor tyrosine kinases (RTK) is a key step in the initiation of growth factor-mediated signaling. Although ligand-induced dimerization of inactive, monomeric receptors was the central dogma of RTK activation for decades, the existence of larger oligomers is now accepted. Both homo- and heteroassociations are of extreme importance in the epidermal growth factor (EGF) receptor family leading to diverse and robust signaling. We present a statistically reliable, flow cytometric homo-FRET method for the quantitative characterization of large-scale receptor clusters. We assumed that a fraction of a certain protein species is monomeric, while the rest is present in homoclusters of N-mers. We measured fluorescence anisotropy as a function of the saturation of fluorescent antibody binding, and fitted the model to the anisotropy data yielding the fraction of monomers and the clusters size. We found that ErbB2 formed bigger homoclusters than ErbB1. Stimulation with EGF and heregulin lead to a decrease in ErbB2 homocluster size, whereas ErbB1 homoclusters got bigger after EGF stimulation. ErbB2 activation level was inversely proportional to its homocluster size. We conclude that homoclusters of ErbB1 and ErbB2 behave in a fundamentally different way; while huge ErbB2 clusters serve as a reservoir of inactive co-receptors and dissociate upon stimulation, small ErbB1 homoclusters form higher order oligomers after ligand binding.
Key Words:
ErbB proteins, anisotropy, flow cytometry, homo-FRET, protein clustering
