Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements.
The association of receptor tyrosine kinases is a key step in the initiation of growth factor-mediated signaling. Although the ligand-induced dimerization of inactive, monomeric receptors was the central dogma of receptor tyrosine kinase activation for decades, the existence of larger oligomers is now accepted. Both homoassociations 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-fluorescence resonance energy transfer method for the quantitative characterization of large-scale receptor clusters. We assumed that a fraction of a certain protein species is monomeric, whereas the rest are 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 cluster size. We found that ErbB2 formed larger homoclusters than ErbB1. Stimulation with EGF and heregulin led to a decrease in ErbB2 homocluster size, whereas ErbB1 homoclusters became larger after EGF stimulation. The activation level of ErbB2 was inversely proportional to its homocluster size. We conclude that homoclusters of ErbB1 and ErbB2 behave in a fundamentally different way. Whereas huge ErbB2 clusters serve as a reservoir of inactive coreceptors and dissociate upon stimulation, small ErbB1 homoclusters form higher-order oligomers after ligand binding.
Szabó
Agnes
A
Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, University of Debrecen, Debrecen, Hungary.
Horváth
Gábor
G
Szöllosi
János
J
Nagy
Peter
P
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods
D048868:Adaptor Proteins, Signal Transducing; C413364:ERBIN protein, human; D066246:ErbB Receptors