ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences |
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| Authors: | Helen E Craig John Coadwell Hervé Guillou Sonja Vermeren |
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| Institution: | 1. Konputazio Zientziak eta A. A. Saila, Informatika Fakultatea, UPV/EHU, E-20018 Donostia-San Sebastián, Spain;2. Departamento de Matemáticas, Universidad Carlos III de Madrid, Leganés (Madrid), Spain;1. INRA, UMR 791 Modélisation Systémique Appliquée aux Ruminants, 16 rue Claude Bernard, F-75005 Paris, France;2. AgroParisTech, UMR 791 Modélisation Systémique Appliquée aux Ruminants, 16 rue Claude Bernard, F-75005 Paris, France;3. UNIP, 11 rue de Monceau, CS 60003, F-75008 Paris, France;1. J. Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapominajek 8, 30-238 Krakow, Poland;2. Department of Theoretical Chemistry, Faculty of Chemistry, M. Curie-Sklodowska University, pl. M. Curie-Sklodowskiej 3, 20-031 Lublin, Poland;1. High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, PR China;2. Heifei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei 230027, PR China;3. Laboratory of Molecular Modeling and Design, State key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Rd., Dalian 116023, PR China |
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| Abstract: | Pleckstrin homology (PH) domains are modules characterised by a conserved three-dimensional protein fold. Several PH domains bind phosphoinositides with high affinity and specificity whilst most others do not. ARAP3 is a dual GTPase activating protein for Arf6 and RhoA which was identified in a screen for phosphatidylinositol-(3,4,5)-trisphophate (PtdIns(3,4,5)P3) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P3, which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to PtdIns(3,4,5)P3 in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P3 in isolation. Instead, the minimal PtdIns(3,4,5)P3 binding fragment comprises ARAP3's N-terminal tandem PH domains, and an N-terminal linker region. For substantial binding, the N-terminal sterile alpha motif (SAM) domain is also required. Site-directed mutagenesis of either of the two N-terminal PH domains within the fragment greatly reduces binding to PtdIns(3,4,5)P3, however, in the context of the full-length protein, point mutations in the second PH domain have a lesser effect on binding, whilst deletion of any one of the five PH domains abolishes PtdIns(3,4,5)P3 binding. We propose a mechanism by which basic residues from the N-terminal tandem PH domains, and from elsewhere in the protein synergise to mediate strong, specific PtdIns(3,4,5)P3 binding. |
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