The leukocyte NADPH oxidase catalyzes the production of O2 from oxygen and NADPH. The yeast 2-hybrid system was used to look for proteins that interact with p67PHOX, one of the cytosolic subunits of the oxidase. This system identified a B lymphocyte cDNA that encoded a 60 kDa protein containing two Ca++- and lipid-binding C2 domains in its C-terminal half. Rim, a Rab3-binding protein involved in synaptic vesicle trafficking, has a homologous N-terminal half and a similar C-terminal C2 domain structure, suggesting that the new protein, formerly named p56C2 but now renamed p62MCSP, might be involved in the trafficking of secretory vesicles in leukocytes. Experiments with recombinant p62MCSP fragments expressed in PC12 cells support this idea. We propose to express p62MCSP and characterize it in terms of its location in neutrophils and EBV- transformed B lymphocytes, its interactions with other leukocyte proteins and with the cortical cytoskeleton, and its association with lipids. The function of p62MCSP will be studied by 1) examining the effects of recombinant p62MCSP and a dominant negative p62MCSP mutant on the cell-free phosphorylation-dependent and SDS-dependent oxidase activating systems; 2) examining oxidase activation in EBV-transformed B lymphocytes expressing a dominant negative mutant of p62MCSP; and 3) examining the effects of a dominant negative mutant on degranulation and oxidase activation in streptolysin O-permeabilized neutrophils. In addition, we will investigate the possibility that p62MCSP interacts with Rab, a family of small guanine nucleotide-binding proteins (GNBP) involved in membrane trafficking, and with Rap1A, a GNBP that copurifies with the membrane-associated subunits of the oxidase. Through these experiments we hope to achieve a deeper understanding of the relationship between membrane vesicle fusion and the activation of the O2-forming oxidase of leukocytes, an enzyme important in host defense and in the harmful side effects of inflammation.
