MAPPING OF FUNCTIONAL DOMAINS IN P47(PHOX) INVOLVED IN THE ACTIVATIONOF NADPH OXIDASE BY PEPTIDE WALKING

The superoxide generating NADPH oxidase. of phagocytes consists, in re sting cells, of a membrane-associated electron transporting flavocytoc hrome (cytochrome b(559)) and four cytosolic proteins as follows: p47( phox), p67(phox), p40(phox), and the small GTPase, Rac(1 or 2). Activa tion of the oxidase is consequent to the assembly of a membrane-locali zed multimolecular complex consisting of cytochrome b(559) and the cyt osolic components. We used ''peptide walking'' (Joseph, G., and Pick, E. (1995) J. Biol. Chem. 270, 29079-29082) for mapping domains in the amino acid sequence of p47(phox) participating in the molecular events leading to the activation of NADPH oxidase, Ninety-five overlapping p entadecapeptides, with a four-residue offset between neighboring pepti des, spanning the complete p47(phox) sequence, were tested for the abi lity to inhibit NADPH oxidase activation in a cell-free system. This c onsisted of solubilized macrophage membranes, recombinant p47(phox), p 67(phox), and Rad, and lithium dodecyl sulfate, as the activator. Eigh t functional domains were identified and labeled a-h. These were (N- a nd C-terminal residue numbers are given for each domain) as follows: a (21-35); b (105-119): c (149-159); d (193-207); e (253-267); f (305-3 19); g (325-339), and h (373-387). Four of these domains (c, d? e, and g) correspond to or form parts of regions shown before to participate in NADPH oxidase assembly. Thus, domain c corresponds to a region on the N-terminal boundary of the first src homology 3 (SH3) domain, wher eas domains d and e represent more precisely defined sites within the full-length first and second SH3 domains, respectively. Domain g overl aps an extensively investigated arginine-rich region. Domains a and b, in the N-terminal half of p47(phox), and domains f and h, in the C-te rminal half, represent newly identified entities, for which there is n o earlier experimental evidence of involvement in NADPH oxidase activa tion. ''Peptide walking'' was also applied to the identification of do mains in p47(phox) mediating binding to p67(phox). This was done by qu antifying, by enzyme-linked immunosorbent assay, the binding of p67(ph ox), in solution, to a series of 95 overlapping biotinylated p47(phox) peptides, attached to streptavidin-coated 96-well plates. A single pr oline-rich domain (residues 357-371) was found to bind p67(phox) in th e absence and presence of lithium dodecyl sulfate.