Decoding EGFR ligand bias through an endocytic organelle platform

Abstract How growth factor receptors decode ligand identity into distinct cellular responses remains a fundamental question in cell signaling. Here, we identify a receptor-proximal mechanism that links ligand-specific EGFR activation to distinct endocytic and biological outputs. We show that EGF, but not TGFα, selectively engages a RAC1-PLCγ2-IP3R signaling axis that supports EGFR non-clathrin endocytosis (NCE). PLCγ2, but not PLCγ1, localizes to RTN3-dependent PM-ER contact sites, where it generates localized Ca²⁺ signals required for completion of NCE, mitochondrial activation and cell motility. This specificity requires the RAC-binding interface of PLCγ2 and is associated with RAC1-dependent formation of CTxB-positive PM regions, indicating that spatial organization contributes to signaling specificity. TGFα fails to efficiently assemble the EGFR-associated organelle platform and instead favors clathrin-dependent EGFR uptake, prolonged proliferative signaling, greater organoid yield, and reduced migration compared with EGF. Together, our findings identify the RAC1-PLCγ2 axis as the key determinant that decodes EGFR ligand bias by coupling receptor trafficking to the metabolic program that supports cell migration.