MUTATIONAL ANALYSIS OF NOVEL EFFECTOR DOMAINS IN RAC-1 INVOLVED IN THE ACTIVATION OF NICOTINAMIDE ADENINE-DINUCLEOTIDE PHOSPHATE (REDUCED) OXIDASE

The small molecular weight GTP-binding protein Rac (1 or 2) is an obli gatory participant in the activation of the superoxide-generating NADP H oxidase. Active NADPH oxidase can be reconstituted in a cell-free sy stem, consisting of phagocyte-derived membranes, containing cytochrome b(559). and the recombinant cytosolic proteins p47-phox-, p67-phox, a nd Rac, supplemented with an anionic amphiphile as an activator. The c ell-foe system was used before for the analysis of structural requirem ents of individual components participating in the assembly of NADPH o xidase. In earlier work, we mapped four previously unidentified domain s in Rac1, encompassing residues 73-81 (a), 103-107 (b), 123-133 (c), and 163-169 (d), as important for cell-free NADPH oxidase activation. The domains were defined by assessing the activation inhibitory effect of a series of overlapping peptides, spanning the entire length of Ra c1 [Joseph, C., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082]. We now used the construction of Rac1/H-Ras chimeras, domain deletion, and point mutations, to ascertain the functional relevance of three do mains (b, c, and d) predicted by ''peptide walking'' and to determine the importance of specific residues within these domains. This methodo logy firmly establishes the involvement of domains b and d in the acti vation of NADPH oxidase by Rac1 and identifies H103 and K166, respecti vely, as residues critical for the effector function of these two doma ins. The functional significance of domain c (insert region) could not be confirmed, as shown by the minor effect of deleting this domain on NADPH oxidase activation. Analysis of the three-dimensional structure of Rac1 reveals that residues H103 and K166 are exposed on the surfac e of the molecule. Modeling of the activity-impairing point mutations suggests that the effect on the ability to activate NADPH oxidase depe nds on the side chains of the mutated amino acids and not on changes i n the global structure of the protein, In conclusion, we demonstrate t he existence of two novel effector sites in Rac1, necessary for suppor ting NADPH oxidase activation, supplementing the canonical N-terminal effector region.