Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

PLoS One. 2013;8(3):e58148. doi: 10.1371/journal.pone.0058148. Epub 2013 Mar 5.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amiloride / pharmacology
  • Animals
  • Betacellulin
  • Caveolin 1 / metabolism
  • Clathrin / genetics
  • Clathrin / metabolism*
  • Dynamins / metabolism
  • Endocytosis
  • Epidermal Growth Factor / metabolism*
  • ErbB Receptors / metabolism*
  • HeLa Cells
  • Heparin-binding EGF-like Growth Factor
  • Humans
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Ligands
  • Mice
  • NIH 3T3 Cells
  • Pinocytosis
  • RNA, Small Interfering / metabolism
  • Transforming Growth Factor alpha / metabolism*

Substances

  • BTC protein, human
  • Betacellulin
  • Btc protein, mouse
  • Caveolin 1
  • Clathrin
  • HBEGF protein, human
  • Hbegf protein, mouse
  • Heparin-binding EGF-like Growth Factor
  • Intercellular Signaling Peptides and Proteins
  • Ligands
  • RNA, Small Interfering
  • Transforming Growth Factor alpha
  • Epidermal Growth Factor
  • Amiloride
  • ErbB Receptors
  • Dynamins

Grant support

This work is funded by Eva og Henry Frænkels Mindefond (grant number 2011-1-14-89; http://www.fraenkelsmindefond.org), The Danish Cancer Society (grant number DP08104; http://www.cancer.dk/), The Lundbeck Foundation (grant number R67-A6299; http://www.lundbeckfonden.com/), The Danish Medical Research Council (grant number 271-06-0505; http://en.fi.dk/councils-commissions/the-danish-council-for-independent-research/scientific-research-councils/medical-sciences), The Danish Cancer Research Foundation (http://www.dansk-kraeftforsknings-fond.dk), The Novo Nordic Foundation (http://www.novonordiskfonden.dk), The John and Birthe Meyer Foundation, The Arvid Nilssons Foundation, and The Vera & Carl Johan Michaelsens grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.