Clathrin-independent endocytosis of ubiquitinated cargos

TLDR

Plasma membrane receptors are internalized via clathrin‑dependent and clathrin‑independent pathways. Ubiquitination of EGFR drives its clathrin‑independent, lipid‑raft–dependent internalization via Ub‑interacting motif proteins eps15, eps15R, and epsin, while a ubiquitination‑impaired EGFR mutant is taken up through the clathrin pathway. At low EGF concentrations EGFR is internalized by clathrin without ubiquitination, but at higher concentrations a significant portion is ubiquitinated and internalized via a clathrin‑independent, lipid‑raft–dependent pathway that requires eps15, eps15R, and epsin.

Abstract

Plasma membrane receptors can be endocytosed through clathrin-dependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR/ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.