- Oligomerization of epidermal growth factor receptors (EGFR) on A431 cells studied by time-resolved fluorescence imaging microscopy: a stereochemical model for tyrosine kinase receptor activation
- Journal of Cell Biology
- Volume | Issue number
- 129 | 6
- Pages (from-to)
- Number of pages
- Document type
- Faculty of Science (FNWI)
- Swammerdam Institute for Life Sciences (SILS)
The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance en- ergy transfer: donor photobleaching fluorescence reso- nance energy transfer (pbFRET) microscopy and fluo- rescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially re- solved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5 % was determined after a low temperature (4°C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20°C and 31% at 37°C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37°C, respectively. A431 cells were pretreated with the monoclonal anti- body mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4°C, indicative of a minimal receptor dimeriza- tion of 65% for the subpopulation of high affinity re- ceptors. These results are in accordance with prior stud- ies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional sub- class of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted rela- tive rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.
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