Abstract
The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.
Highlights
The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is a member of the ErbB family of signaling receptors
In cells treated with clathrin siRNA and these ligands, surface EGFR levels were only increased to approximately 70% of surface levels in unstimulated cells. 3.22 nM AR or EPI did not induce significant EGFR internalization
To further study the clathrin-dependency of EGFR endocytosis, HeLa cells treated with siRNA against clathrin heavy chain (CHC) were stimulated with 10 nM ligand for 15 minutes, as we have previously shown that maximum internalization is reached after 15 minutes stimulation with all ligands [7]
Summary
The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase (RTK), is a member of the ErbB family of signaling receptors. It is involved in regulating growth and differentiation as well as in the pathogenesis of a number of cancers. The EGFR is activated upon ligand binding. Activation leads to internalization of the receptor and trafficking to the early endosomal compartment of the cell. From there the receptor can either be degraded via lysosomes or recycled to the cell surface depending on the ligand bound Endocytic downregulation of the EGFR is an important mechanism of signal attenuation
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