Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), play crucial roles in cell signaling.
• Distinct ligands binding to the same receptor can elicit different cellular responses, a phenomenon not fully understood.
• Epidermal Growth Factor (EGF) and Transforming Growth Factor-alpha (TGF-α) are ligands that bind to EGFR but induce different downstream effects, namely receptor degradation and recycling, respectively.

Purpose of the Study:

• To decipher the functional selectivity of EGF and TGF-α in regulating EGFR trafficking and downstream cellular outcomes.
• To identify the molecular mechanisms and protein players responsible for the differential processing of EGFR upon stimulation by EGF versus TGF-α.
• To explore the potential for manipulating these mechanisms to switch cellular responses in EGFR-positive cancer cells.

Main Methods:

• Development and application of an integrated multilayered proteomics approach (IMPA).
• Quantitative mass spectrometry (MS) was employed to analyze dynamic changes in the EGFR interactome, ubiquitinome, phosphoproteome, and late proteome.
• Analysis was performed in human cells upon stimulation with EGF and TGF-α.

Main Results:

• Identified 67 proteins regulated at multiple levels in response to EGF and TGF-α.
• Discovered RAB7 phosphorylation and Regulator of Cytokine Production (RCP) recruitment to EGFR as critical switches determining the signaling output.
• Demonstrated that manipulating RCP levels or RAB7 phosphorylation could switch TGF-α-mediated responses to EGF-like responses and vice versa by rerouting EGFR trafficking.

Conclusions:

• EGF and TGF-α binding to EGFR initiate distinct signaling pathways through specific protein interactions and post-translational modifications.
• RAB7 phosphorylation and RCP recruitment are key determinants of EGFR trafficking fate (degradation vs. recycling), influencing signaling duration, proliferation, and migration.
• The integrated multilayered proteomics approach (IMPA) is a powerful strategy for uncovering fine-tuned regulatory mechanisms in complex cell signaling pathways, with implications for cancer research.