EGF binding to the Epidermal Growth Factor Receptor (EGFR) promotes its homodimerization and transphosphorylation. Formation of the resulting phosphorylated dimer is considered the committed step in signal transduction through the Ras/MAPK pathway because it is necessary and sufficient to recruit signaling adapters such as Shc, Grb2, and the Ras-GEF SOS to the membrane. In addition to initiating the recruitment of these signaling adapters, EGF binding also contributes to the concomitant spatial rearrangement of the receptor into larger aggregates. Whether these nano and microscale aggregates are passive intermediates in the process of clathrin mediates endocytosis, or whether they play a more active role in the propagation of downstream signaling, remains unclear. The study of these species has been challenging because receptor phosphorylation and spatial rearrangement are rapid, asynchronous spatially across a cell, and intimately linked to EGF binding. To investigate the signaling potential of EGFR dimers and higher oligomers, we decoupled EGFR activation from spatial reorganization by preparing a pure population of phosphorylated and full-length EGFR “trapped-dimers” using a chemical genetic strategy. We expressed full length EGFR bearing an N-terminal SNAP-tag at physiological levels and in multiple cell types, and formed trapped dimers upon the addition of PEG or DNA polymers modified at their ends with benzyl-guanine. We found that trapped dimers of EGFR were phosphorylated at both kinase proximal and distal sites, but did not further oligomerize to form nanoscale clusters, traffic to clathrin-coated pits, or become internalized by endocytosis. Despite a lack of oligomerization and spatial reorganization, phosphorylated trapped dimers recruited key signaling adapters at equivalent levels to phosphorylated EGF-activated receptors, including Shc, Grb2 and SOS1. Surprisingly, however, trapped dimers did not activate Ras or Erk. Loss of Erk activation was not a consequence of a lack of endocytosis or colocalization with clathrin coated pits, suggesting that when stimulated by EGF, maximal signaling through Ras occurred through an oligomeric species. Our results suggest that receptors dimerization, phosphorylation, and adapter recruitment are not the committed steps in signaling through the MAPK cascade, and implicate a role for higher order oligomers in efficient Ras activation.

Covalent trapping reveal that phosphorylated EGFR oligomers activate Ras more potently than dimers

253rd National Meeting (2017)

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