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

• DOI: 10.1016/j.bpj.2022.11.003
• Publication Date: 2022-11-05T02:25:00Z
• Authors: Harikrushnan Bala...; Jagadish Sankaran; Shambhavi Pandey; Corinna Jie Hui Goh; Thorsten Wohland
• Keywords: ERBB3

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