PMID- 7790353 OWN - NLM STAT- MEDLINE DCOM- 19950725 LR - 20201222 IS - 0021-9525 (Print) IS - 1540-8140 (Electronic) IS - 0021-9525 (Linking) VI - 129 IP - 6 DP - 1995 Jun TI - Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation. PG - 1543-58 AB - 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 energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence 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 resolved 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 degrees 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 degrees C and 31% at 37 degrees 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 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody 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 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass 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 relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains. FAU - Gadella, T W Jr AU - Gadella TW Jr AD - Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Goettingen, Federal Republic of Germany. FAU - Jovin, T M AU - Jovin TM LA - eng PT - Journal Article PL - United States TA - J Cell Biol JT - The Journal of cell biology JID - 0375356 RN - 0 (Fluorescent Dyes) RN - 0 (Macromolecular Substances) RN - 62229-50-9 (Epidermal Growth Factor) RN - EC 2.7.10.1 (ErbB Receptors) SB - IM MH - Binding, Competitive MH - Carcinoma, Squamous Cell MH - Cell Line MH - Cell Membrane/metabolism MH - Energy Transfer MH - Enzyme Activation MH - Epidermal Growth Factor/metabolism MH - ErbB Receptors/chemistry/*metabolism MH - Fluorescent Dyes MH - Humans MH - Kinetics MH - Macromolecular Substances MH - Microscopy, Fluorescence/methods MH - Models, Structural MH - Models, Theoretical MH - Spectrometry, Fluorescence/methods MH - Time Factors MH - Tumor Cells, Cultured PMC - PMC2291181 EDAT- 1995/06/01 00:00 MHDA- 2001/03/28 10:01 PMCR- 1995/12/02 CRDT- 1995/06/01 00:00 PHST- 1995/06/01 00:00 [pubmed] PHST- 2001/03/28 10:01 [medline] PHST- 1995/06/01 00:00 [entrez] PHST- 1995/12/02 00:00 [pmc-release] AID - 95310335 [pii] AID - 10.1083/jcb.129.6.1543 [doi] PST - ppublish SO - J Cell Biol. 1995 Jun;129(6):1543-58. doi: 10.1083/jcb.129.6.1543.