Human Epidermal Growth Factor Receptor (EGFR) Aligned on the Plasma Membrane Adopts Key Features of Drosophila EGFR Asymmetry

Abstract

The ability of epidermal growth factor receptor (EGFR) to control cell fate is defined by its affinity for ligand. Current models suggest that ligand-binding heterogeneity arises from negative cooperativity in signaling receptor dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the isolated extracellular region of the human EGFR. Human EGFR also differs from the Drosophila EGFR in that negative cooperativity is found only in full-length receptors in cells. To gain structural insights into the human EGFR in situ, we developed an approach based on quantitative Förster resonance energy transfer (FRET) imaging, combined with Monte Carlo and molecular dynamics simulations, to probe receptor conformation in epithelial cells. We experimentally demonstrate a high-affinity ligand-binding human EGFR conformation consistent with the extracellular region aligned flat on the plasma membrane. We explored the relevance of this conformation to ligand-binding heterogeneity and found that the asymmetry of this structure shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative cooperativity is conserved from invertebrates to humans but that in human EGFR the extracellular region asymmetry requires interactions with the plasma membrane.The human epidermal growth factor receptor (hEGFR) is a classic receptor tyrosine kinase (26). The hEGFR was originally identified in A431 epithelial carcinoma cells (47) and consists of an extracellular growth factor-binding region, a single-pass transmembrane region, and a cytoplasmic domain that has tyrosine kinase activity (42).The extracellular domain of hEGFR is comprised of four subdomains (I to IV). The unliganded receptor monomer is held in a closed conformation by an intramolecular tether formed by loops in subdomains I and III (17). In ligand-occupied receptor dimers, the intramolecular tether is broken, and the receptor is opened into an extended conformation which interacts with another monomer, forming a back-to-back dimer (19, 36). Ligand-induced receptor dimerization is thought to be the key stimulatory step, leading to allosteric transactivation of the two associated intracellular hEGFR kinases (42).The EGFR family has diversified during evolution from one ligand/one receptor in Caenorhabditis elegans, to multiple ligands and one receptor in flies, and to a family of multiple ligands and four closely related receptors that form homo-and heterodimers in humans (10). The EGFR itself is highly conserved across species. Wild-type hEGFR and Drosophila EGFR (dEGFR) show extensive conservation, with 55% homology in the kinase domain and 41% homology in the ligandbinding portion of the extracellular domain (39).An important unresolved question regarding the mechanism of hEGFR activation is the structural basis for the concave-up Scatchard plots exhibited by epidermal growth factor (EGF) (31, 43). These plots were initially interpreted ...

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“…This reflects a difference of binding affinity, albeit small, that may be correlated with asymmetrical conformations. A recent fluorescence resonance energy transfer study agrees with this interpretation [38].…”

“…This reflects a difference of binding affinity, albeit small, that may be correlated with asymmetrical conformations. A recent fluorescence resonance energy transfer study agrees with this interpretation [38].In addition to ligand binding, we were also interested in determining the energies of symmetric and asymmetric dimerization. If dimerization of a ligated sEGFR and an unligated untethered sEGFR is more favorable than two ligated monomers, this should be reflected in differences in binding energy.…”

A single ligand is sufficient to activate EGFR dimers

“…The fact that intracellular regions are required for negative cooperativity likely reflects the importance of these regions for stabilizing receptor dimers in the absence of ligand (17). A theoretical study recently suggested interactions with the cell membrane may also induce a Drosophila EGFR-like dimer in human EGFR (18). (11) complexes has been superposed on domains I, II, and III of the yellow sErbB4 subunit.…”

Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy

“…Using a combination of FRET microscopy and Monte Carlo and molecular dynamics simulations, Tynan et al [49] showed recently a high-affinity ligand-binding human EGFR conformation consistent with the extracellular region aligned flat on the plasma membrane ( Figure 3). Interestingly, the asymmetry of this structure shares key features with that of dEGFR [48], suggesting that the structural basis for negative co-operativity is conserved from invertebrates to humans, but that, in human EGFR, the extracellular region asymmetry requires interactions with the plasma membrane.…”