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

Nature communications (2018-09-23)
Martin Baumdick, Márton Gelléri, Chayasith Uttamapinant, Václav Beránek, Jason W Chin, Philippe I H Bastiaens
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.

MATERIALS
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Brand
Product Description

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Phosphatase Inhibitor Cocktail 2, aqueous solution (dark coloration may develop upon storage, which does not affect the activity)
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Hydrocortisone, BioReagent, suitable for cell culture
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Atto 590 NHS ester, BioReagent, suitable for fluorescence, ≥60% (coupling to amines)

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