Pharmacology and Signaling Properties of Epidermal Growth Factor Receptor Isoforms Studied by Bioluminescence Resonance Energy Transfer

2006 ◽  
Vol 71 (2) ◽  
pp. 508-518 ◽  
Author(s):  
Hans H. Schiffer ◽  
Esther C. Reding ◽  
Stephen R. Fuhs ◽  
Qing Lu ◽  
Fabrice Piu ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Resonance Energy Transfer ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Receptor Isoforms ◽  
Epidermal Growth

scholarly journals Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

2015 ◽  
Vol 26 (22) ◽  
pp. 4087-4099 ◽  
Author(s):  
Christopher C. Valley ◽  
Donna J. Arndt-Jovin ◽  
Narain Karedla ◽  
Mara P. Steinkamp ◽  
Alexey I. Chizhik ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Resonance Energy Transfer ◽  
Growth Factor Receptor ◽  
Resonance Energy ◽  
Epidermal Growth

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization.

scholarly journals Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation.

1995 ◽  
Vol 129 (6) ◽  
pp. 1543-1558 ◽  
Author(s):  
T W Gadella ◽  
T M Jovin
Keyword(s):  
Energy Transfer ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
A431 Cells ◽  
High Affinity ◽  
Fret Efficiency ◽  
Epidermal Growth

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.

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