title: Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex.
abstract: The low molecular weight GTP binding protein Rac is essential to the activation of the NADPH oxidase complex, involved in pathogen killing during phagocytosis. In resting cells, Rac exists as a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI). Two types of interactions exist between Rac and Rho-GDI: a protein-lipid interaction, implicating the polyisoprene of the GTPase, as well as protein-protein interactions. Using the two-hybrid system, we show that nonprenylated Rac1 interacts very weakly with Rho-GDI, pointing to the predominant role of protein-isoprene interaction in complex formation. In the absence of this strong interaction, we demonstrate that three sites of protein-protein interaction, Arg66(Rac)-Leu67(Rac), His103(Rac), and the C-terminal polybasic region Arg183(Rac)-Lys188(Rac), are involved and cooperate in complex formation. When Rac1 mutants are prenylated by expression in insect cells, they all interact with Rho-GDI. Rho-GDI is able to exert an inhibitory effect on the GDP/GTP exchange reaction except in the complex in which Rac1 has a deletion of the polybasic region (Arg183(Rac)-Lys188(Rac)). This complex is, most likely, held together through protein-lipid interaction only. Although able to function as GTPases, the mutants of Rac1 that failed to interact with Rho-GDI also failed to activate the NADPH oxidase in a cell-free assay after loading with GTP. Mutant Leu119(Rac)Gln could both interact with Rho-GDI and activate the NADPH oxidase. The Rac1/Rho-GDI and Rac1(Leu119Gln)/Rho-GDI complexes, in which the GTPases were bound to GDP, were found to activate the oxidase efficiently. These data suggest that Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, and presents it to its effector, the p67phox component of the NADPH oxidase.
authors.affiliation: Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (BBSI)-UMR CEA/CNRS/UJF 5092, CEA Grenoble, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France.
authors.forename: N
authors.initials: N
authors.lastname: Di-Poï
authors.forename: J
authors.initials: J
authors.lastname: Fauré
authors.forename: S
authors.initials: S
authors.lastname: Grizot
authors.forename: G
authors.initials: G
authors.lastname: Molnár
authors.forename: E
authors.initials: E
authors.lastname: Pick
authors.forename: M C
authors.initials: MC
authors.lastname: Dagher
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016367:Baculoviridae; D003001:Cloning, Molecular; D004789:Enzyme Activation; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000737:chemistry / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000378:metabolism*; D017368:Protein Prenylation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors
chemical_list: D020730:Guanine Nucleotide Dissociation Inhibitors; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D013481:Superoxides; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins