Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis

Endocytic proteins such as epsin, AP180, and Hip 1R (Sla2p) share a conserved modular region termed the epsin NH₂-terminal homology (ENTH) domain, which plays a crucial role in clathrin-mediated endocytosis through an unknown target. Here, we demonstrate a strong affinity of the ENTH domain for phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂]. With nuclear magnetic resonance analysis of the epsin ENTH domain, we determined that a cleft formed with positively charged residues contributed to phosphoinositide binding. Overexpression of a mutant, epsin Lys⁷⁶→ Ala⁷⁶, with an ENTH domain defective in phosphoinositide binding, blocked epidermal growth factor internalization in COS-7 cells. Thus, interaction between the ENTH domain and PtdIns(4,5)P₂ is essential for endocytosis mediated by clathrin-coated pits.