Role of the ENTH Domain in Phosphatidylinositol-4,5-Bisphosphate Binding and Endocytosis
10.1126/science.291.5506.1047
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Endocytic proteins such as epsin, AP180, and Hip1R (Sla2p) share a conserved modular region termed the epsin NH 2 -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)P 2 ]. 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 Lys 76 → Ala 76 , 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)P 2 is essential for endocytosis mediated by clathrin-coated pits.

Bibliography

Itoh, T., Koshiba, S., Kigawa, T., Kikuchi, A., Yokoyama, S., & Takenawa, T. (2001). Role of the ENTH Domain in Phosphatidylinositol-4,5-Bisphosphate Binding and Endocytosis. Science, 291(5506), 1047–1051.

Authors 6
  1. Toshiki Itoh (first)
  2. Seizo Koshiba (additional)
  3. Takanori Kigawa (additional)
  4. Akira Kikuchi (additional)
  5. Shigeyuki Yokoyama (additional)
  6. Tadaomi Takenawa (additional)
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  37. An EGF internalization assay was carried out 48 hours after transfection. COS-7 cells were incubated with EGF (0.1 μg/ml) [biotinylated complexed to Texas Red–streptoavidin (Molecular Probes Eugene OR)] in binding buffer [20 mM Hepes–NaOH (pH 7.5) 130 mM NaCl and 0.1% bovine serum albumin] at 4°C for 60 min. Internalization of EGF was allowed by incubation in Dulbecco's modified Eagle's medium at 37°C for 10 min then excess EGF was removed with 0.2 M AcOH (pH 2.5) and 0.5 M NaCl at 4°C for 5 min. Cells were fixed in 3.7% formaldehyde permeabilized with 0.2% Triton X-100 and immunostained with a polyclonal antibody to myc (Santa Cruz Biotechnology Santa Cruz CA) and fluorescein isothiocyanate–conjugated antibody to rabbit (Organon Teknika Boxtel Netherlands). Internalization of EGF was observed by confocal microscopy (Bio-Rad).
  38. We thank Y. Watanabe (Ehime University Japan) for providing us with various synthetic phosphoinositides.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:47 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:30 a.m.)
Indexed 2 days, 4 hours ago (Aug. 30, 2025, 1:06 p.m.)
Issued 24 years, 6 months ago (Feb. 9, 2001)
Published 24 years, 6 months ago (Feb. 9, 2001)
Published Print 24 years, 6 months ago (Feb. 9, 2001)
Funders 0

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@article{Itoh_2001, title={Role of the ENTH Domain in Phosphatidylinositol-4,5-Bisphosphate Binding and Endocytosis}, volume={291}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.291.5506.1047}, DOI={10.1126/science.291.5506.1047}, number={5506}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Itoh, Toshiki and Koshiba, Seizo and Kigawa, Takanori and Kikuchi, Akira and Yokoyama, Shigeyuki and Takenawa, Tadaomi}, year={2001}, month=feb, pages={1047–1051} }