Regulation of EGFR nanocluster formation by ionic protein-lipid interaction

Abstract

The abnormal activation of epidermal growth factor receptor (EGFR) is strongly associated with a variety of human cancers but the underlying molecular mechanism is not fully understood. By using direct stochastic optical reconstruction microscopy (dSTORM), we find that EGFR proteins form nanoclusters in the cell membrane of both normal lung epithelial cells and lung cancer cells, but the number and size of clusters significantly increase in lung cancer cells. The formation of EGFR clusters is mediated by the ionic interaction between the anionic lipid phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane and the juxtamembrane (JM) region of EGFR. Disruption of EGFR clustering by PIP2 depletion or JM region mutation impairs EGFR activation and downstream signaling. Furthermore, JM region mutation in constitutively active EGFR mutant attenuates its capability of cell transformation. Collectively, our findings highlight the key roles of anionic phospholipids in EGFR signaling and function, and reveal a novel mechanism to explain the aberrant activation of EGFR in cancers.

N -Glycosylation as determinant of epidermal growth factor receptor conformation in membranes

“…Unlike the other simulations, in simulation 1 of the glycosylated receptor, JM-A unfolds promptly and remains unfolded throughout the simulation, which correlates with the limited formation of the membrane interface. This finding, together with the previously described electrostatic membrane interactions with lipids, highlights the capacity of the intracellular JM-A domain to sense and interact with various lipid environments, possibly in an electrostatic switch-like mechanism (6,8,56,57). It should also be noted that the orientation of the TKD and the helicity of the JM-A segment do not seem to have an influence on the properties of the kinase active site of the TKD.…”

“…To prevent receptor activation and signaling in the absence of ligand, the structurally tethered ECD of monomeric EGFR blocks the intrinsic capacity of the TMD and the intracellular TKD to dimerize (2). Ligand binding is believed to release the self-inhibitory tether and facilitate receptor oligomerization and activation (3)(4)(5)(6). A detailed understanding of the structural regulation of the intact full-length receptors in their native membranes promises to reveal the molecular basis for receptor regulation (7); however, the methodological limitations associated with crystallizing transmembrane proteins, together with the high flexibility of the full-length receptor, have prevented high-resolution crystallographic analysis.…”

EGF and NRG induce phosphorylation of HER3/ERBB3 by EGFR using distinct oligomeric mechanisms

“…In some studies, such indications came from the experiments in which FRET was detected between two EGF ligand molecules; because of the structural constraints of their interaction, this transfer is most logically explained by receptor oligomerization (17)(18)(19)(20)(21). Other studies imaged fluorescently labeled EGFR itself using image correlation microscopy (ICS) (22,23), fluorescence correlation spectroscopy (FCS) (24,25), stochastic optical reconstruction microscopy (STORM) (26), FRET, fluorescence lifetime imaging microscopy (FLIM) (27,28), spatial mapping of the receptor by immuno-electron microscopy (29,30), or number and brightness analysis (31). This spectrum of experimental approaches all led to a unifying conclusion that EGFR is organized in larger clusters in response to ligand binding.…”

Development of nanoscale structure in LAT-based signaling complexes

“…Nanoclustering might be involved in both the activation and regulation of signaling pathways. Recent work has suggested a role for nanoclusters in both EGF receptor signaling (Wang et al, 2014b) and neural circuits (Broadhead et al, 2016). The nanoclustering of H-Ras has a direct effect on specific Raf effector recruitment (Guzman et al, 2014).…”