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

Area of Science:

• Cellular Biology
• Biochemistry
• Molecular Signaling

Background:

• Epidermal growth factor receptor (EGFR) activation is crucial for cell growth and is initiated by growth factors (GFs).
• EGFR activation involves receptor dimerization, allosteric kinase activation, and tyrosine trans-phosphorylation.
• Previous studies identified EGF-induced allosteric activation, but cellular imaging and simulations suggested additional catalytic mechanisms.

Purpose of the Study:

• To investigate EGFR activation mechanisms in living cells.
• To develop a Förster resonance energy transfer (FRET)-based indicator for EGFR conformational changes.
• To elucidate the role of Y845 phosphorylation in EGFR activation.

Main Methods:

• Development of a FRET-based conformational EGFR indicator (CONEGI) using genetic code expansion.
• Comparison of conformational transitions, self-association, and auto-phosphorylation of CONEGI and its Y845F mutant.
• Analysis of EGFR kinase activity and C-terminal auto-phosphorylation.

Main Results:

• Y845 phosphorylation induces a catalytically active conformation in EGFR monomers.
• This conformational transition is dependent on EGFR kinase activity and C-terminal auto-phosphorylation.
• A looped causality leading to autocatalytic amplification of EGFR phosphorylation at low EGF doses was identified.

Conclusions:

• EGFR activation involves a novel autocatalytic amplification mechanism.
• Y845 phosphorylation plays a key role in inducing a catalytically active monomeric EGFR conformation.
• This mechanism contributes to efficient EGFR signaling, particularly at low growth factor concentrations.