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Persistent URL http://purl.org/net/epubs/work/41416
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Record Id 41416
Title Single molecule imaging and FLIM show different structures for high and low-affinity EGFRs in A431 cells
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Abstract Epidermal growth factor (EGF) receptor (EGFR) modulates mitosis and apoptosis through signalling by its high-affinity (HA) and low-affinity (LA) EGF-binding states. The prevailing model of EGFR activation - derived from X-ray crystallography - involves the transition from tethered ectodomain monomers to extended back-to-back dimers and cannot explain these EGFR affinities or their different functions. Here, we use single-molecule Förster resonant energy transfer analysis in combination with ensemble fluorescence lifetime imaging microscopy to investigate the 3D architecture of HA and LA EGFR-EGF complexes in cells by measuring the inter-EGF distances within discrete EGF pairs and the vertical distance from EGF to the plasma membrane. Our results show that EGFR ectodomains form interfaces resulting in two inter-EGF distances (~8 nm and <5.5 nm), different to the back-to-back EGFR ectodomain interface (~11nm). Distance measurements from EGF to the plasma membrane show that HA EGFR ectodomains are oriented flat on the membrane, while LA ectodomains stand proud from it. Their flat orientation confers HA EGFR ectodomains the exclusive ability to interact via asymmetric interfaces, head-to-head with respect to the EGF- binding site, while LA EGFRs must interact only side-by-side. Our results support a structural model in which asymmetric EGFR head-to-head interfaces may be relevant for HA EGFR oligomerisation.
Organisation CCLRC , SRS , CSE , CCP4
Keywords Biology , signalling , single molecule , FRET
Funding Information
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Language English (EN)
Type Details URI(s) Local file(s) Year
Journal Article Biophys J 94, no. 3 (2007): 803-819. doi:10.1529/biophysj.107.112623 BiophysFinal-1.pdf 2007
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