| Abstrakt: |
Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rapla may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1-166 truncated form of Rapla was functional in the cell-free system, and thus, clarification of the function of Rapla and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligo-nucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1aor rac2in the mammalian expression vector pSRαneo. HL60 cells transfected with wild-type or mutated rac2or rap1acDNA constructs and control HL60 cells transfected with the pSRaneo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47-phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2-in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O-2. production by differentiated cell lines expressing mutated N17 Rapla or N17 Rac2 dominant-negative proteins was inhibited, whereas 0 production by the subline overexpressing wild-type Rapla was increased by fourfold. O-2production by the differentiated cell line expressing GTPase-defective V12 Rapla was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rapla for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rapla functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase. |
