Abstract
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.
MeSH terms
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Adaptor Proteins, Vesicular Transport
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Amino Acid Motifs
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Amino Acid Substitution
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Animals
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COS Cells
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism*
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Chlorocebus aethiops
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Clathrin / metabolism
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Coated Pits, Cell-Membrane / metabolism
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DNA-Binding Proteins / metabolism
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Endocytosis*
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Epidermal Growth Factor / metabolism
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Inositol Phosphates / metabolism
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Liposomes / metabolism
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Models, Molecular
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Neuropeptides / chemistry*
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Neuropeptides / metabolism*
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Nuclear Magnetic Resonance, Biomolecular
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Phosphatidylinositol 4,5-Diphosphate / metabolism*
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Protein Conformation
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Transcription Factors / metabolism
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Vesicular Transport Proteins*
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Zinc Fingers
Substances
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Adaptor Proteins, Vesicular Transport
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Carrier Proteins
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Clathrin
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DNA-Binding Proteins
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Inositol Phosphates
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Liposomes
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Neuropeptides
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Phosphatidylinositol 4,5-Diphosphate
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Recombinant Fusion Proteins
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Transcription Factors
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Vesicular Transport Proteins
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epsin
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Epidermal Growth Factor