Neutrophils play a key role in many types of inflammatory diseases, including chronic lung disease, atherosclerosis, and rheumatoid and acute inflammatory arthritis. One of the primary agents of damage in these diseases appears to be the neutrophil-generated superoxide anion. Activation of the superoxide generating system in human neutrophils is thought to involve the interaction or assembly of cytochrome b-559, the terminal component of this system, with other cytosolic and membrane proteins. Previously, we reported the association of a ras-related protein, Rap1A, with neutrophil cytochrome b-559 (Quinn, M.T., et al. (1989) Nature 342: 198-200). The association of this quanosine triphosphate (GTP)-binding protein with cytochrome b-559 suggested a possible role for this protein in the structure and/or function of the cytochrome and, hence, in the regulation of neutrophil superoxide production. To address this possibility, the proposed studies will focus on investigating the fundamental hypothesis that the rap1 protein plays a role in the function of cytochrome b-559, possibly as a molecular switch that regulates the cytochrome b-559 interaction with other components of the NADPH oxidase system. Specifically, this proposal describes strategies for: 1) Characterization of the cytochrome b:Rap1A association. The stability of these complexes will be examined with respect to the effects of salts, Ph, quanine nucleotides and detergents. 2) Analysis of the effect of neutrophil activation state on the stability, quantity, and stoichiometry of the complexes. The phosphorylation state of Rap1A during stages of cell activation will also be determined. 3) Determination of the structural basis of the Rap1A:cytochrome b association, including a determination of which cytochrome b subunit associates with Rap1A and the sequences of the regions of this interaction. 4) Determination of the functional role of system. 5) Structural analysis of the Rap1A-cytochrome b association to evaluate conformational changes induced in Rap1A and cytochrome b y their association and by the binding of GTP. Analyses will include affinity chromatography, fluorescence spectroscopy, Fourier-transform infrared spectroscopy, and fluorescence energy transfer to analyze conformational changes. The accomplishment of these studies will provide a basis for the understanding of the role f Rap1A in the structure and function of the cytochrome and the role of this complex in the neutrophil superoxide generating system. This understanding may eventually lead to potential therapeutic strategies for reducing superoxide production in inflammatory disease. In addition, these studies will provide a basis for the understanding of the role of ras-related proteins in cells and their possible interaction with functional effector systems.
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