Clathrin-independent endocytosis of ubiquitinated cargos

Plasma membrane receptors can be endocytosed through clathrindependentand clathrin-independent pathways. Here, we showthat the epidermal growth factor (EGF) receptor (EGFR), whenstimulated with low doses of EGF, is internalized almost exclusivelythrough the clathrin pathway, and it is not ubiquitinated. At higherconcentrations of ligand, however, a substantial fraction of thereceptor is endocytosed through a clathrin-independent, lipidraft-dependent route, as the receptor becomes ubiquitinated. Anubiquitination-impaired EGFR mutant was internalized throughthe clathrin pathway, whereas an EGFRubiquitin chimera, thatcan signal solely through its ubiquitin (Ub) moiety, was internalizedexclusively by the non-clathrin pathway. Non-clathrin internalizationof ubiquitinated EGFR depends on its interaction withproteins harboring the Ub-interacting motif, as shown through theablation of three Ub-interacting motif-containing proteins, eps15,eps15R, and epsin. Thus, eps15s and epsin perform an importantfunction in coupling ubiquitinated cargo to clathrin-independentinternalization.