Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis.
Endocytic proteins such as epsin, AP180, and Hip1R (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.
Itoh
T
T
Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Koshiba
S
S
Kigawa
T
T
Kikuchi
A
A
Yokoyama
S
S
Takenawa
T
T
D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008958:Models, Molecular; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D033921:Vesicular Transport Proteins; D016335:Zinc Fingers
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D004268:DNA-Binding Proteins; D007295:Inositol Phosphates; D008081:Liposomes; D009479:Neuropeptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; C114154:epsin; D004815:Epidermal Growth Factor