ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences

摘要

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 (Ptdlns(3,4,5)P-3) binding proteins. It is a regulator of cell shape and adhesion, and is itself regulated by PtdIns(3,4,5)P-3, which acts to recruit ARAP3 to the plasma membrane and to catalytically activate it. We show here that ARAP3 binds to Ptdlns(3,4,5)P-3 in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind Ptdlns(3,4,5)P-3 in isolation. Instead, the minimal PtdIns(3,4,5)P3 binding fragment comprises ARAPTs 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)P-3 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 Ptdlns(3,4,5)P-3 binding.