The NADPH oxidase of human phagocytes is responsible for the formation of superoxide anion and other oxidants used by these cells for bacterial killing, and is also an important component of the inflammatory response. This is a competing renewal application based on progress gained to date on the following original specific aims: to identify the GTP-binding protein (Gox) associated with stimulation of NADPH oxidase, to investigate the role of Gox in regulating the NADPH oxidase, and to characterize the interaction of Gox with regulatory proteins. The Applicant showed that Gox was most likely Rac2 using cell-free activation of the NADPH oxidase using purified protein components and using Rac2-specific antibodies. Posttranslational isoprenylation of Rac is necessary for Rac2 to be activated by a membrane-associated guanine nucleotide exchange factor, but is not required for interaction with the oxidase itself. The ability of Rac to stimulate superoxide production is dependent upon its conversion from the inactive GDP-bound form to the active GTP-bound form. Translocation to the membrane requires nucleotide exchange by a membrane- bound exchange factor (GEF), and is accompanied by release from a cytosolic complex with Guanine dissociation inhibitors (GDI(s)) that normally maintains Rac in the cytosol in unstimulated cells. Interaction with GDI can also inhibit intrinsic and GTPase activating protein (GAP)-stimulated GTP hydrolysis by Rac. Oxidant production in vitro is inhibited by Rac GAPs and they have recently shown that the Bcr protein, which is a Rac GAP is a physiological regulator of oxidant production in vivo. Activation of Rac by chemoattractant receptors requires the action of protein tyrosine kinases, inhibitors of which can dissociate Rac translocation from movement of the cytosolic p47phox and p67phox components of the oxidase to the membrane. Thus, instead of hypothesizing that Rac acts to mediate oxidase assembly from the cytosol, the Applicant now hypothesizes that Rac must be regulating the system at the level of the plasma membrane. In CGD patients lacking the cytochrome b component, but not in those lacking p47 or p67, they found that Rac translocation was decrease by >75%. Lastly, the Applicant has used mutational analysis of Rac2 to determine regions important for activation of the NADPH oxidase, GAPs, and actin assembly, leading to the hypothesis that Rac may utilize a second effector binding site distinct from the classical site found in Ras-related GTPases, and may therefore provide a link among phagocyte oxidant production, phagocytosis, and adherence/motility. The first Specific Aim will be to focus on Rac2 (and Rap1A) regulation of NADPH oxidase at the membrane/membrane-associated cytoskeleton level. The hypothesis to be tested is that Rac facilitates the transfer of e- to O2 in neutrophil membranes using published assays to measure this activity. Once Rac has been found to directly modulate a step of e- transfer, membranes containing specific cytochrome mutations will be used to determine the relative roles of Rac, p47 and p67 in the process. The Applicant will also evaluate oxidase regulation in vivo using a viral system for protein expression in myeloid cells. Dominant active and inhibitory Rac mutations will be constructed and transfected into these cells to determine effects on oxidase activity, translocation of p47 and p67, and GTP hydrolysis. In addition, constructs of p47 and p67 containing isoprenylation-directing motifs will be transfected into cells to provide stimulation-independent membrane association of these proteins. The question to be addressed is whether these membranes are active in the absence of added Rac2- GTPgammaS to evaluate if an active oxidase system will assemble without Rac2. The applicant has shown that Pak kinase is able to phosphorylate p47. The question as to its requirement is oxidase activation will be addressed using this dominant mutant strategy using a Pak-1 mutant which is catalytically inactive and is already in hand. The cells to be used in these studies are mature human neutrophils (if they work) and HL 60 and/or THP1 macrophage cell lines. The second Specific Aim is to study the interaction of low molecular- weight G proteins with GDI(s) on a molecular level, since GDI(s) appear to be critical regulators of the membrane-to-cytosol trafficking cycle that GTP-binding proteins of the Rho family undergo in all cells. Recombinant proteins will be used in a biosensor assay to study the efficiency and binding kinetics of different protein-protein interactions, in the absence and presence of lipid modifiers. The third Specific Aim is to use molecular biological approaches to identify, clone and characterize phagocyte-specific guanine nucleotide exchange factors which are active on Rac2 and which may be regulated during the course of oxidase activation in phagocytes. This will be done by either a PCR cloning approach using primers based on the murine Tiam1 gene (the only GEP found to be catalytically active on Rac1 or Rac2), or potentially by using the yeast two-hybrid system employing Rac 2 mutants in the """"""""bait"""""""" construct to probe a differentiated HL 60 cDNA library fused with a gene which can activate gene transcription.
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