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Phostensin carries a novel Eps 15 homology domain-containing protein 1 (EHD1) and EHD4-binding motif to regulate endocytic recycling
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  • Hsien-Bin Huang,
  • Kuang-Yung Huang,
  • Hui-Chun Yu,
  • Ming-Chi Lu,
  • Hsien-Yu Huang,
  • Jyun-Jie Shen,
  • Chia-Ying Lin,
  • Pei-Rong Chung,
  • Hsiao-Kuei Tsai,
  • Ya-Ting Shen,
  • Si-Ru Zhou,
  • Chih-Wei Fan,
  • Chia-Lin Wang,
  • Ning-Sheng Lai,
  • Ta-Hsien Lin
Hsien-Bin Huang
National Chung Cheng University

Corresponding Author:[email protected]

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Kuang-Yung Huang
Dalin Tzu Chi Hospital
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Hui-Chun Yu
Dalin Tzu Chi Hospital Department of Medical Research
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Ming-Chi Lu
Dalin Tzu Chi Hospital
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Hsien-Yu Huang
Dalin Tzu Chi Hospital
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Jyun-Jie Shen
National Chung Cheng University
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Chia-Ying Lin
National Chung Cheng University
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Pei-Rong Chung
National Chung Cheng University
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Hsiao-Kuei Tsai
National Chung Cheng University
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Ya-Ting Shen
National Chung Cheng University
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Si-Ru Zhou
National Chung Cheng University
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Chih-Wei Fan
National Chung Cheng University
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Chia-Lin Wang
Taipei Veterans General Hospital Department of Medical Education
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Ning-Sheng Lai
Dalin Tzu Chi Hospital
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Ta-Hsien Lin
Taipei Veterans General Hospital Department of Medical Education
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Abstract

Phostensin (PTS) encoded by KIAA1949 is a scaffold protein. It can bind to protein phosphatase 1 (PP1), F-actin, Eps 15 homology domain-containing protein 1 (EHD1) and EHD4. PTS contains both PP1-binding and actin-binding motifs. Most EHD-binding proteins contain a consensus motif, Asn-Pro-Phe (NPF) motif, which interacts with the C-terminal EH domain of EHD proteins. Nevertheless, the NPF motif is absent in PTS. The binding motif for PTS to associate with EHD1 (or EHD4) remains unknown. Here, we used structure-function, far western blotting and GST pull-down assays to investigate this issue and identified that PTS-α binds to EHD1 (or EHD4) through the region of residues 60-80 which contains a consensus motif, 64ILV(X) 4(L/V)RL 74S. This novel motif was also found in the vacuolar protein sorting 35 (vps35) of retromer, an EHD1-associating complex and regulating retrograde trafficking, Replacement of 64ILV(X) 4(L/V)RL 74S with 64AAA(X) 4(L/V)RL 74S or with 64ILV(X) 4AEA 74A significantly reduces the binding efficiency of PTS-α to either EHD1 or EHD4. Overexpression of the PTS-β in 293T cells attenuated the endocytic trafficking of transferrin. However, this attenuation of transferrin in endocytic trafficking was disrupted when 293T cells overexpressed the mutant PTS-β with a defective EHD-binding motif, suggesting that PTS-β can regulate the endocytic recycling via associating with EHD1 or EHD4.