Assessment of the Role for Rho Family GTPases in NADPH Oxidase Activation

Rac, a member of the Rho family small GTPases, plays a crucial role in activation of Nox family NADPH oxidases in animals, enzymes dedicated to production of reactive oxygen species such as superoxide. The phagocyte oxidase Nox2, crucial for microbicidal activity during phagocytosis, is activated in a manner completely dependent on Rac. Rac in the GTP-bound form directly binds to the oxidase activator p67 phox , which in turn interacts with Nox2, leading to superoxide production. Rac also participates in activation of the nonphagocytic oxidase Nox1; in this case, GTP-bound Rac functions by interacting with Noxa1, a p67 phox -related protein that is required for Nox1 activation. On the other hand, in the presence of either p67 phox or Noxa1, Rac facilitates superoxide production by Nox3, which is responsible in the inner ear for formation of otoconia, tiny mineralized structures that are required for sensing balance and gravity. All the three mammalian homologs of Rac (Rac1, Rac2, and Rac3), but not Cdc42 or RhoA, are capable of serving as an activator of Nox1–3. Here, we describe methods for the assay of Rac binding to p67 phox and Noxa1 and for the reconstitution of Rac-dependent Nox activity in cell-free and whole-cell systems.