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   Activation of NADPH-oxidase dependent superoxide generation requires the participation of several cytosolic factors including p47phox, p67phox, p40phox and Rac (1 or 2). In the resting cell, p47phox, p67phox, and p40phox form a large molecular weight complex which can be recovered from the cytosolic fraction. Activation of the NADPH oxidase is initiated by the assembly of p47phox, p67phox, and Rac with cytochrome b558 in a 1:1:1:1 complex in the plasma membrane. p47phox, p67phox, and Rac exhibit cooperative binding, since increasing the concentration of any one component lowers the EC50 of the other components. A great deal of current research involves understanding the protein protein interactions, and how these change with the activation state.

Molecular Interactions. This component contains a tandem repeat of two SH3 domains near the center of the molecule. The first of these binds to a proline-rich sequence within the C-terminus of p22phox in the assembled oxidase complex. In addition, this SH3 domain can interact with the C-terminal proline-rich sequence in the same molecule (possibly in the non-activated p47phox.). The second of these appears to bind to a proline-rich sequence in p67phox. near the center of this molecule. SH3 interactions with proline-rich regions, while undoubtedly important, do not appear to be the sole determinants of protein-protein interactions among oxidase components.

Phosphorylation. In vivo, p47phox becomes phosphorylated at multiple serines within its C-terminus in response to cellular activation. Phosphorylation appears to be part of the activation signal for the oxidase, although phosphorylation of other components may also be involved. A cell-free system showing activation by phosphorylation of p47phox as well as a membrane component has recently been described (Park, et al. (1997) JBC 272, 11035-11043).

Function. p47phox is not essential for the in vitro function of the NADPH oxidase, provided very high concentrations of p67phox, and Rac are present. Functionally, the effect of p47phox is to increase the binding for p67phox by approximately 100-fold and the binding of Rac by about 50-fold, both based on EC50 values. Thus, p47phox appears to be a regulated adaptor protein, and does not seem to participate directly in regulating the activity of the NADPH oxidase. Thus, either Rac or p67phox is the direct regulator of oxidase activity.

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Molecular Interactions. p67phox contains a central proline-rich region followed by an SH3 domain, with a second SH3 domain at the C-terminus. The function of the first SH3 domain is unknown. p67phox binds to p47phox via a "tail-to-tail" interaction, utilizing the SH3 domain in the C-terminus of the former to bind to the proline-rich sequence in the C-terminus of the latter. In addition, the second SH3 domain of p47phox binds to the proline-rich region of p67phox. The N-terminal half of p67phox contains a binding region for Rac, since p67phox (1-198) binds to Rac with the same affinity as full-length p67phox. The exact location of the Rac binding site is unknown, and the sequence in this region does not correspond to other known Rac binding sites on other proteins.

Function. Other than binding to p47phox and Rac, the function of p67phox is currently unknown. In the absence of p47phox, p67phox fails to assemble with the NADPH oxidase, supporting an adaptor function for p47phox in the binding of p67phox. Corresponding effects of p67phox on the binding of p47phox have also been demonstrated, indicating mutually facilitated binding of these cytosolic components.

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Molecular Interactions. p40phox was initially characterized as a component of a complex with p67phox and p47phox in cytosol from non-activated neutrophils. It can interact with the C-terminal proline-rich domain of p47phox, although it is not clear that it does so in vivo. This component has been reported to translocate to the plasma membrane upon cellular activation, along with p47phox and p67phox..

Function. In a cell-free system p40phox is not essential for activity. It has recently been reported to inhibit activity of the oxidase system. It may therefore play some down-regulatory role, or may help maintain the cytosolic factors in an "off" state.

Burnham, D.N., Uhlinger, D.J., and Lambeth, J.D. (1990) "Diradylglycerol Synergizes with an Anionic Amphiphile to Activate Superoxide Generation and Phosphorylation of p47phox in a Cell-free System from Human Neutrophils" J. Biol. Chem. 265, 17550-17559.

Tyagi, S.R., Neckelmann, N., Uhlinger, D.J., Burnham, D.N. and Lambeth, J.D. (1992) "Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: Effects of guanine 5'O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile" . Biochemistry 31, 2765-2774.

Uhlinger, D.J., Inge, K.L., Kreck, M.L., Tyagi, S.R., Neckelmann, N. and Lambeth, J.D. (1992) "Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane" Biochem. Biophys. Res. Commun. 186, 509-516.

Uhlinger, D.J., Tyagi, S.R., Inge, K.L. & Lambeth, J.D. (1992) "Guanine nucleotides regulate the assembly of the Human Neutrophil Respiratory Burst Oxidase: Evidence for G Protein Regulation of the Binding of p67-phox and p47-phox" J. Biol. Chem. 268, 8624-8631.

Uhlinger, D.J., Taylor, K.L. and Lambeth, J.D. (1994) "p67-phox enhances the binding of p47-phox to the human neutrophil respiratory burst oxidase complex" J. Biol. Chem. 269, 22095-22098.

Freeman, J.L. and Lambeth, J.D. (1996) "NADPH oxidase activity is independent of p47-phox in vitro" J. Biol. Chem. 271, 22578-22585.