title: A PH domain in the Arf GTPase-activating protein (GAP) ARAP1 binds phosphatidylinositol 3,4,5-trisphosphate and regulates Arf GAP activity independently of recruitment to the plasma membranes.
abstract: ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3))-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P(3) binding. Here, we tested the hypothesis that PtdIns(3,4,5)P(3)-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P(3), but with relatively low affinity (approximately 1.6 microm), and the PH domains did not mediate PtdIns(3,4,5)P(3)-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P(3) binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P(3), the subsequent production of which triggers enzymatic activity.
authors: Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors: Fanny
authors: F
authors: Campa
authors: Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors: Hye-Young
authors: HY
authors: Yoon
authors: Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
authors: Vi Luan
authors: VL
authors: Ha
authors: Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
authors: Zsofia
authors: Z
authors: Szentpetery
authors: Program for Developmental Neuroscience, NICHD, National Institutes of Health, Bethesda, Maryland 20892.
authors: Tamas
authors: T
authors: Balla
authors: Laboratory of Cellular and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892. Electronic address: randazzo@helix.nih.gov.
authors: Paul A
authors: PA
authors: Randazzo
mesh_terms: D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: C446513:ARAP1 protein, human; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D018129:Phosphatidylinositol Phosphates; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D020727:ADP-Ribosylation Factors