Mapping the Domains of Interaction of p40 with Both p47 and p67 of the Neutrophil Oxidase Complex Using the Two-hybrid System

The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67phox, p47phox and p40phox as well as a small G protein Rac (for review, see (1Morel F. Doussière J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (523) Google Scholar, 2Babior B. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 49-95PubMed Google Scholar, 3Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar)). A truncated form of the p40phox cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47phox as target. This truncated form of p40phox consisting of the Src Homology 3 (SH3) domain to the 3′ stop codon was also shown to interact with p67phox in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47phox or p67phox. Results show that the SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C terminus of p40phox but not its SH3 domain is involved in the interaction with p67phox. The superoxide-generating NADPH oxidase complex in phagocytic cells is constituted of a heterodimeric flavocytochrome b and cytosolic factors, p67phox, p47phox and p40phox as well as a small G protein Rac (for review, see (1Morel F. Doussière J. Vignais P.V. Eur. J. Biochem. 1991; 201: 523-546Crossref PubMed Scopus (523) Google Scholar, 2Babior B. Adv. Enzymol. Relat. Areas Mol. Biol. 1992; 65: 49-95PubMed Google Scholar, 3Thrasher A.J. Keep N.H. Wientjes F. Segal A.W. Biochim. Biophys. Acta. 1994; 1227: 1-24Crossref PubMed Scopus (212) Google Scholar)). A truncated form of the p40phox cDNA was isolated by a two hybrid screen of a B lymphocyte library using a full length clone of p47phox as target. This truncated form of p40phox consisting of the Src Homology 3 (SH3) domain to the 3′ stop codon was also shown to interact with p67phox in the same system. A library of smaller fragments of the truncated p40 cDNA was constructed and screened against either p47phox or p67phox. Results show that the SH3 domain of p40phox is sufficient for interaction with p47phox, whereas the C terminus of p40phox but not its SH3 domain is involved in the interaction with p67phox.