Nanoscale Imaging of Epidermal Growth Factor Receptor Clustering

Abstract

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.The plasma membrane of the cell is a complex, carefully regulated, dynamic structure that is compartmentalized into cell surface domains such as lipid rafts, caveolae, and clathrincoated pits (1). Lipid rafts are postulated to be dynamic nanodomains ranging in size from 10 to 200 nm that are enriched in cholesterol, sphingolipids, and certain proteins and that play an important role in assembling signaling complexes (2-4). Caveolae are invaginated lipid raft domains (50 -150 nm) whose stability at the plasma membrane is attributable to the formation of stable oligomers of their coat protein, caveolin-1 (5). Caveolae are thought to serve as concentrators of various signal transduction machineries. Clathrin-coated pits (100 -150 nm) internalize rapidly upon formation at the plasma membrane, and their lateral cell surface mobility is enhanced by actin cytoskeleton depolymerization (6).The epidermal growth factor receptor (EGFR), 3 a 170-kDa transmembrane glycoprotein, is one of four members of the ErbB family of receptor tyrosine kinases involved in oncogene signaling. The initial step in receptor activation involves binding of the EGF peptide to the extracellular domain leading to dimerization or activation of pre-existing dimers (7,8). Following ligand binding, the EGFR is auto-phosphorylated in several tyrosine residues of the intracellular domain, creating high affinity sites for various adaptor molecules that transmit the mitogenic signal to the Ras/MAPK signal transduction pathway (9). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2, which regulate transcription of molecules that are linked to cell proliferation, survival, a...

The Epidermal Growth Factor Receptor Forms Location-Dependent Complexes in Resting Cells

“…The amount of EGFR complexes also increases upon EGF stimulation at different doses, leading to the formation of larger oligomers, which are then endocytosed at different rates and possibly with different fates. As shown earlier, the low-dose EGF stimulation (10 ng/mL) leads to the recycling of the receptors while high-dose EGF (100 ng/mL) to receptor degradation (33,74).…”

Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

“…Previous study using near-field scanning optical microscopy (NSOM) suggested that EGFR might be organized in aggregates with distinct diameters in the membrane of paraformaldehyde-fixed Hela cells [18]. However, the detailed information of EGFR distribution on cell surface, especially on living cell surface, still remains largely elusive.…”

A novel biotinylated lipid raft reporter for electron microscopic imaging of plasma membrane microdomains

“…6D and 7B ), the EGFR showed clustering over a broader size range (25-150 nm). Such submicron-scale clustering of the EGFR in cellular plasma membranes has been previously characterized by immuno-EM ( 11,39 ) and single molecule-resolution fl uorescence microscopy (40), although the factors responsible for the establishment of these microdomains are the subject of continuing investigation ( 7,(39)(40)(41). By analyzing the co-clustering of the EGFR and Lck-BAP-GFP reporter with the bivariate Ripley's K function statistic ( Fig.…”