Oncogenic EGFR Mutants Differentially Alter Dimerization, Adaptor Protein Engagement, and Clathrin-Mediated...

Area of Science:

• Cell Biology
• Molecular Oncology
• Biophysics

Background:

• Epidermal growth factor receptor (EGFR) signaling is crucial in non-small cell lung cancer (NSCLC).
• Activating EGFR mutations drive oncogenesis, but their effects on receptor clustering and endocytosis are unclear.
• Understanding these early events is key to targeting sustained signaling in RTK-driven cancers.

Purpose of the Study:

• To investigate how common oncogenic EGFR mutations (exon 19 deletions and L858R) affect receptor clustering and clathrin-mediated endocytosis (CME) at the plasma membrane.
• To elucidate the mechanistic link between EGFR mutation-specific dynamics and downstream signaling perturbation.

Main Methods:

• Utilized pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) to quantify EGFR dimerization and clustering.
• Employed quantitative live-cell total internal reflection fluorescence microscopy (TIRFM) to track EGFR and AP-2 dynamics during early endocytosis.
• Analyzed ligand-induced receptor multimerization, adaptor protein (AP-2) recruitment, and internalization kinetics.

Main Results:

• EGFR^WT showed ligand-induced multimerization; EGFR^L858R displayed ligand-independent dimerization and ligand-driven clustering; exon 19 deletion variants exhibited weaker ligand-dependent dimerization.
• EGFR^E746-A750del showed impaired AP-2 recruitment, EGFR^L747-A750>P demonstrated altered clathrin assembly with sustained AP-2 membrane association.
• EGFR^L858R had ligand-dependent AP-2 recruitment similar to wild type but with reduced net internalization.

Conclusions:

• Oncogenic EGFR mutations induce distinct receptor clustering and adaptor engagement patterns, differentially affecting early CME steps.
• Altered receptor dynamics at the plasma membrane contribute to sustained signaling in EGFR-mutated NSCLC.
• These findings offer mechanistic insights into RTK-driven cancer signaling and potential therapeutic targets.