ARAP3 binding to phosphatidylinositol-(3,4,5)-trisphosphate depends on N-terminal tandem PH domains and adjacent sequences.
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)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 PtdIns(3,4,5)P(3) in an unusual, PH domain-dependent manner. None of the five PH domains are sufficient to bind PtdIns(3,4,5)P(3) in isolation. Instead, the minimal PtdIns(3,4,5)P(3) 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)P(3), 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 PtdIns(3,4,5)P(3) binding.
Craig
Helen E
HE
The Babraham Institute, Cambridge, UK.
Coadwell
John
J
Guillou
Hervé
H
Vermeren
Sonja
S
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002448:Cell Adhesion; D002522:Chlorocebus aethiops; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
C446513:ARAP1 protein, human; C446514:ARAP2 protein, human; C446512:ARAP3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D018129:Phosphatidylinositol Phosphates; C060974:phosphatidylinositol 3,4,5-triphosphate