MAPPING THE DOMAINS OF INTERACTION OF P40(PHOX) WITH BOTH P47(PHOX) AND P67(PHOX) OF THE NEUTROPHIL OXIDASE COMPLEX USING THE 2-HYBRID SYSTEM

The superoxide generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factor s, p67(phox), p47(phox) and p40(phox) as well as a small G protein Rac (for review, see Refs. 1-3). A truncated form of the p40(phox) cDNA w as isolated by a two hybrid screen of a B lymphocyte library using a f ull length clone of p47(phox) as target, This truncated form of p40(ph ox) consisting of the Src Homology 3 (SH3) domain to the 3' stop codon was also shown to interact with p67(phox) in the same system. A Libra ry of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47(phox) or p67(phox). Results show that the SH3 domain of p40(phox) is sufficient for interaction with p47(phox), whereas the C terminus of p40(phox) but not its SH3 domain is involved in the interaction with p67(phox).