Effects of N361 Glycosylation on Epidermal Growth Factor Receptor Biological Function

Abstract

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently modified by glycosylation post-translationally. In cancer, EGFR amplifications and hotspot mutations such as L858R that promote proliferation have been detected in a significant fraction of non-small cell lung carcinomas and breast adenocarcinomas. Molecular dynamic simulations suggested that glycosylation at asparagine residue 361 (N361) promotes dimerization and ligand binding. We stably expressed glycosylation-deficient mutant EGFR N361A, with or without the oncogenic mutation L858R. Immunofluorescence and flow cytometry demonstrated that the mutants were each well expressed at the cell membrane. N361A decreased proliferation relative to wild-type EGFR as well as decreased sensitivity to ligands. Proximity ligation assays measuring co-localization of EGFR with its binding partner HER2 in cells revealed that N361A mutations increased co-localization. N361A, located near the binding interface for the EGFR inhibitor necitumumab, desensitized cells expressing the oncogenic EGFR L858R to antibody-based inhibition. These findings underline the critical relevance of post-translational modifications on oncogene function. SIGNIFICANCEEGFR transduces signals from growth factors into cell proliferation and is frequently hyperactivated in tumors. Glycosylation of EGFR at N361 regulates EGFR dimerization, growth factor stimulation of proliferative signaling, and susceptibility to targeted inhibition. Insights into EGFR glycosylation may expand therapeutic opportunities to benefit cancer patients.