The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study.

Number and brightness (N&B) analysis is a fluorescence spectroscopy technique to quantify oligomerization of the mobile fraction of proteins. Accurate results, however, rely on a good knowledge of nonfluorescent states of the fluorescent labels, especially of fluorescent proteins, which are widely used in biology. Fluorescent proteins have been characterized for confocal, but not camera-based, N&B, which allows, in principle, faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment are still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor's oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization is not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, lead to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization dependent, involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation are the outcomes of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.