Neutrophils and other phagocytic blood cells generate high levels of reactive oxygen species in response to a variety of infectious or inflammatory stimuli in a process known as the respiratory burst. This activity is attributed to an enzyme complex called NADPH oxidase, which uses molecular oxygen and NADPH to produce superoxide anion, a precursor of microbicidal oxidants. These oxidants play a key role in host defense against microbial infections and serve as mediators of inflammatory signals. Patients with chronic granulomatous disease have NADPH oxidase deficiencies which result in enhanced susceptibility to microbial infections and dysregulated inflammatory responses. This project is exploring the structural basis for NADPH oxidase function and the cellular mechanisms underlying regulation of the respiratory burst. Oxidase activation is a stepwise process that begins with stimulation of G-protein-coupled receptors and activation of various kinases and phospholipases. Active oxidase assembly involves phosphorylation of several protein components that transmigrate to specific membrane domains. We have shown that interacting sites (SH3 domains) within p47phox and p67phox play a central role in regulating oxidase assembly and that these domains can both inhibit and promote formation of the active oxidase complex. Inhibitory mechanisms include: 1) intramolecular SH3 contacts within p47 and p67phox, which maintain these proteins in inactive conformations, 2) competitive binding of p40phox, another SH3 domain-containing cytosolic factor, and 3) inhibition by a rac-activated kinase, PAK2, which down-regulates the oxidase by binding p47phox in a phosphorylation-dependent process. The formation of the membrane-bound oxidase complex is necessary for oxidase activation, but not sufficient, since cells deficient in cytosolic phospholipase A2 (p85) will assemble the oxidase complex but not produce superoxide unless arachidonic acid is provided. In related work we are examining the role of phospholipase D and its products in phagocyte activation. Information on the structure and function of NADPH oxidase may provide a basis for therapeutic strategies designed to either inhibit or enhance respiratory burst activity.