title: Unligated epidermal growth factor receptor forms higher order oligomers within microclusters on A431 cells that are sensitive to tyrosine kinase inhibitor binding.
abstract: Characterization of the association states of the unligated epidermal growth factor receptor (EGFR) is important in understanding the mechanism of EGFR tyrosine kinase activation in a tumor cell environment. We analyzed, in detail, the association states of unligated, immunotagged EGFR on the surface of intact epidermoid carcinoma A431 cells, using AlexaFluor488 and AlexaFluor546 anti-EGFR antibody, mAb528, as probes. Image correlation microscopy revealed the presence of unligated EGFR in submicron scale clusters containing an average of 10-30 receptors (mean cluster density = 32 +/- 9 clusters per square micron). Lifetime-based Förster resonance energy transfer (FRET) techniques as a function of acceptor:donor labeling ratio disclosed a clustering of the unligated EGFR in clusters containing an average of four receptors on the nanometer (<10 nm) scale. The relationship between the nanoscale and submicron scale associations was determined using a new analysis that combines nanoscale information from lifetime-detected FRET imaging with submicron scale information obtained with image correlation microscopy. This analysis revealed the presence of monomers (or small oligomers) and larger clusters containing 15-30 receptors that were partially associated on the sub-10 nm scale. Pretreatment of the cells with the tyrosine kinase inhibitor AG1478 caused a partial dispersal of the submicron clusters (mean cluster density = 85 +/- 15 clusters per square micron; mean degree of association = 4-10 receptors per cluster) and reduced the level of FRET down to our limit of detection. These results are consistent with a higher order nanoscale receptor organization of the unligated receptor population that is partially controlled by the kinase domains. The ramifications of the results to mechanisms of EGFR activation in a tumor cell environment are discussed.
authors: The Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, P.O. Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia. andrew.clayton@ludwig.edu.au
authors: Andrew H A
authors: AH
authors: Clayton
authors: Maria L
authors: ML
authors: Tavarnesi
authors: Terrance G
authors: TG
authors: Johns
mesh_terms: D045744:Cell Line, Tumor; D019281:Dimerization; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011806:Quinolinium Compounds / Q000737:chemistry
chemical_list: C417561:Alexa 488 hydrazide; C481052:Alexa fluor 546; D005456:Fluorescent Dyes; D006834:Hydrazines; D047428:Protein Kinase Inhibitors; D011806:Quinolinium Compounds; D066246:ErbB Receptors