# Effects of N361 Glycosylation on Epidermal Growth Factor Receptor Biological Function

## TL;DR

This study shows that glycosylation at a specific site on the EGFR protein affects its function and response to cancer treatments.

## Contribution

The study reveals that N361 glycosylation is critical for EGFR function and therapeutic response in cancer.

## Key findings

- Glycosylation-deficient EGFR increases membrane localization and co-localizes with HER2.
- Mutant EGFR reduces cell proliferation and ligand responsiveness.
- Loss of N361 glycosylation decreases the effectiveness of the antibody necitumumab.

## Abstract

Activating genetic changes in the gene that encodes epidermal growth factor receptor (EGFR) frequently promote uncontrolled proliferation in lung cancer and breast cancer. Glycosylation modifications added to asparagines after protein translation can impact protein function. This study focuses on the impacts of a site on EGFR that is frequently N-glycosylated. We found that glycosylation-deficient mutation of this site promotes increased membrane localization and non-productive co-localization of EGFR with its binding partner HER2. Proliferation was reduced in mutant cells. The mutant EGFR was deficient at binding and responding to growth factor ligands that promote cell proliferation. Loss of this glycosylation site reduced the efficacy of the cancer therapeutic antibody necitumumab, which binds nearby. These findings underline the critical relevance of post-translational glycosylation modifications on EGFR function, cell biology, and responses to therapeutic agents.

Background: Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that is frequently post-translationally modified by glycosylation. 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. Analyses of disruptions to glycosylation pathways in cancer cells identified EGFR glycosylation at residue N361 as one of the most heavily impacted sites. Methods: We stably expressed a glycosylation-deficient mutant EGFR, N361A, with or without the oncogenic EGFR mutation L858R in cells. Proximity ligation assays were employed to study the effects of the glycosylation mutant on the co-localization of EGFR and HER2. The effects of the glycosylation-deficient mutant on the efficacy of agonists, such as EGF and amphiregulin, or antagonists, such as osimertinib and necitumumab, were defined using cell viability assays and immunoblots. Results: N361A increased the membrane localization and co-localization of EGFR with its binding partner HER2. The glycosylation-deficient mutation decreased cell proliferation, including proliferative responses to EGFR ligands. The mutant cells demonstrated reduced sensitivity to inhibition using the antibody inhibitor necitumumab, which inhibits EGFR by binding the extracellular domain. Conclusions: Disruption of glycosylation at N361, located near the ligand binding and dimerization regions, created a dominant negative form of EGFR, which non-productively co-localized with HER2, resulting in a blockage in proliferation. These findings underline the critical relevance of post-translational glycosylation modifications on EGFR function.