Proteins and mechanisms involved in endosomal sorting of the epidermal growth factor receptor

Abstract

Signaling from growth factor receptors is tightly regulated. Failure of this regulation can lead to overstimulation of growth, proliferation and differentiation, and is often observed in cancer. The epidermal growth factor receptor (EGFR) is desensitized by internalization and degradation in lysosomes. Internalization takes place rapidly upon ligand induced activation, whereas degradation depends on retained ligand binding and subsequent sorting to inner vesicles on multivesicular bodies (MVBs). Using electron-microscopy, confocal immunofluorescence microscopy and standard molecular biology techniques, we have investigated endocytosis and endosomal sorting of EGFR. Endocytosis of the activated EGFR is believed to depend on ubiquitination of the receptor. Ubiquitination depends on the ubiquitin ligase Cbl, and we show that the direct interaction between EGFR and Cbl at the phosphorylated tyrosine residue 1045 (pY1045) of the EGFR is sufficient for endocytosis of the EGFR, but not sufficient for sorting to MVBs and degradation. On endosomes, different membrane domains are involved in sorting of the endocytosed EGFR, and we found that two different electron dense coats are involved, one coat containing clathrin and Hrs, and another coat devoid of clathrin and Hrs. Hrs is involved in sorting of ubiquitinated receptors for degradation, and we further found that the ubiquitination induced by direct interaction between EGFR and Cbl at the pY1045 of the EGFR is necessary for co-localization between the EGFR and Hrs. Finally, we found the non-receptor tyrosine kinase Ack1, which is often over-expressed in cancer, to be involved in EGFR endocytosis, most likely at the level of endosomal sorting. Ack1 colocalized with the EGFR on early endosomes and overexpression of Ack1 retained the EGFR on the limiting membrane of early endosomes and inhibited sorting of the EGFR to inner vesicles of MVBs.