Molecular characterization of angiomotin/JEAP family proteins: interaction with MUPP1/Patj and their endogenous properties

Authors

  • Yuko Sugihara-Mizuno,

    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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  • Makoto Adachi,

    Corresponding author
    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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  • Yuka Kobayashi,

    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
    2. Division of Molecular and Cell Biology, Institute for Medical Science, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu-machi, Tochigi 321-0293, Japan
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  • Yoko Hamazaki,

    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
    2. Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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  • Miyuki Nishimura,

    1. KAN Research Institute, Inc., Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
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  • Toshio Imai,

    1. KAN Research Institute, Inc., Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
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  • Mikio Furuse,

    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
    2. Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
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  • Shoichiro Tsukita

    1. Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
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  • Communicated by: Eisuke Nishida

* Correspondence: E-mail: ada@mfour.med.kyoto-u.ac.jp

Abstract

We have previously shown that MUPP1, which has an MRE domain and 13 PDZ domains, is expressed in epithelial cells and localize at tight junctions (TJs) and apical membranes. Using yeast two-hybrid screening, we found here that MUPP1 interacts with angiomotin (Amot), JEAP/Amot-like 1 and MASCOT/Amot-like 2, which we refer to as Amot/JEAP family proteins. PDZ2 and -3 were responsible for MUPP1's interaction with Amot and MASCOT, whereas only PDZ3 was responsible for its interaction with JEAP. All the Amot/JEAP family proteins also interacted with Patj, a close relative of MUPP1. The C-terminal PDZ-binding motives of the Amot/JEAP family were required for these interactions. We successfully generated specific antibodies for these proteins and analyzed the endogenous molecular properties of the family in parallel. Immunofluorescence microscopy of cultured epithelial cells showed that in subcellular distribution, the Amot/JEAP family proteins were indistinguishable; they were apparent at TJs as well as apical membranes, and mostly co-localized with MUPP1. They were also located at TJs in several mouse tissues, but each protein showed a distinct tissue distribution. In biochemical fractionation assays, the Amot/JEAP family behaved not as transmembrane but as peripheral membrane proteins. Unexpectedly, the PDZ-binding motives were not necessarily required for their localization to TJs, and dominant negative MUPP1 or Patj did not affect the localization of Amot/JEAP family proteins, suggesting that the interaction with MUPP1/Patj is not necessarily responsible for their proper subcellular distribution.

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