The Rac Target NADPH Oxidase p67 Interacts Preferentially with Rac2 Rather Than Rac1

NADPH oxidase is a plasma membrane enzyme of phagocytes generating superoxide anions which serve as bactericidal agents. Activation of this multimolecular enzyme minimally requires assembly at the membrane with flavocytochrome b of cytosolic components p47 , p67 , and Rac proteins. Rac1 and Rac2 are 92% homologous cytosolic small GTPase proteins. Both Rac1 and Rac2 have been implicated with NADPH oxidase activation in vitro ; however, Rac2 is largely predominant in human phagocytes. Here, using the yeast two-hybrid system, we provide data demonstrating in vivo interactions between human p47 , p67 , and Rac proteins. Rac proteins interact with p67 in a GTP-dependent manner, but do not interact with p47 . Moreover, Rac effector site mutants, which are known to be inactive in NADPH oxidase, lose their interaction with p67 ; Rac2L61 mutant, which has an increased NADPH oxidase affinity, shows an increased affinity for p67 . Finally, we observe that p67 interacts 6-fold better with Rac2 than with Rac1. We also show a strong intracellular interaction between p47 and p67 . These results indicate that activated Rac can regulate NADPH oxidase by interacting with p67 and that Rac2 is the main p67 -interacting GTPase in human cells.