A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation

Abstract

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight into EGFR activation mechanisms in living cells, we develop a Förster resonance energy transfer (FRET)-based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant reveals that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

Organization at criticality enables processing of time‐varying signals by receptor networks

“…The EGFR activity dynamics is regulated by a four‐component spatially distributed network, where the interactions of EGFR (R) with three specific membrane‐associated enzymes—protein tyrosine phosphatases via a double negative ( P DNF , PTPRG), a negative feedback ( P NF , PTPN2) and a negative regulation ( P NR , PTPRJ), are coupled via the vesicular trafficking of the receptor (Fig A). Ligand‐bound EGF receptors ( LR ) promote autocatalytic activation of ligandless receptors (red arrows; Reynolds et al , ; Tischer & Bastiaens, ; Baumdick et al , , ) and thereby transfer information about the extracellular environment before they are internalized and degraded ( LR E ; Schlessinger, ; Baumdick et al , ). The vesicular recycling, on the other hand, brings the internalized and deactivated ligandless EGFR ( R E ) back to the plasma membrane, establishing the EGF signal processing network.…”

Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation

“…Phosphorylation of EGFR tyrosine 845 (Y845) is also important for EGFR TK activity [57,58]. TGF- β stimulates the phosphorylation of EGFR Y845, via Src activation, as part of a ligand-independent pathway [24].…”

A Genetic Code Expansion‐Derived Molecular Beacon for the Detection of Intracellular Amyloid‐β Peptide Generation

“…The functional handle is then chemically derivatized with a probe of choice. With its single‐amino acid label, genetic code expansion can be used to precisely place a biophysical tag within small proteins or peptides with minimal functional perturbation, and has been used to construct conformational sensors [5] and to provide insight into functional dynamics of microproteins [6] . Despite the ability to precisely place the biophysical label, genetic code expansion by itself does not allow selective visualization of the proteolytic proteoforms in the presence of excess of their precursors, the latter of which also carry the same label.…”