title: Assays and properties of arfaptin 2 binding to Rac1 and ADP-ribosylation factors (Arfs).
abstract: Arfaptin 1 and 2 were identified as targets for GTP bound ADP-ribosylation factors (Arfs). Arfaptin 1 had no significant effects on guanine nucleotide binding to Arfs, nor enzymatic activities of guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP) acting on Arfs. However, arfaptin 1 inhibited Arf activation of cholera toxin and phospholipase D (PLD) in a dose-dependent manner. Only GTP-bound forms of Arf1, 5, and 6 interacted with arfaptin 1 and 2, but GTP-Arf1 showed the strongest binding to the arfaptins. In contrast to the binding of Arfs to arfaptins, GDP-Rac1 or dominant negative Rac1-N17N bound to arfaptin 2, whereas GTP-Rac1 or dominant active Rac1-Q61L did not bind to arfaptin 2. Neither GTP-Rac1 nor GDP-Rac1 bound to arfaptin 1. Based on our observation, we propose that arfaptin 2 is a target for GDP-Rac1 and for GTP-Arf1, and is involved in interactions between the Rac1 and Arfs signaling pathways. This chapter describes methods for investigating the interactions of arfaptins 1 and 2 with GTP- or GDP-liganded Arfs and Rac1.
authors.affiliation: Center for Basic Neuroscience, University of Texas Southwestern Medical Center, Dallas, USA.
authors.forename: Ok-Ho
authors.initials: OH
authors.lastname: Shin
authors.forename: John H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D005982:Glutathione Transferase / Q000235:genetics; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C105088:ARFIP1 protein, human; C101158:ARFIP2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D011993:Recombinant Fusion Proteins; D013873:Thionucleotides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D005982:Glutathione Transferase; D020727:ADP-Ribosylation Factors; D020830:rac1 GTP-Binding Protein