The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation Academic Article uri icon

abstract

  • We have previously established a model of cytosolic phospholipase A 2 (cPLA 2 )-deficient PLB-985 cells and demonstrated that cPLA2-gen- erated arachidonic acid (AA) is essential for re- duced nicotinamide adenine dinucleotide phos- phate (NADPH) oxidase activation and NADPH- dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91 phox (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide- reducing center, to determine if this portion is regulated by AA. The gp91 phox C-terminal reduc- tase domain was expressed in X-CGD PLB-985 cells lacking normal gp91 phox (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabil- ized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA2 protein and activity, as well as AA release after stimulation, supported NADPH-de- pendent diaphorase activity after stimulation, sim- ilar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47 phox and p67 phox to the membrane fractions of both stimulated cell types indicated that the gp91 phox C-terminal region is sufficient to anchor the cytosolic oxidase com- ponents to the membranes. cPLA2 translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane- bound oxidase complex encompassing the flavin domain of gp91 phox provides a docking site for cPLA2 but is not the site of AA-based regulation of oxidase activity. J. Leukoc. Biol. 80: 000-000; 2006.

publication date

  • January 1, 2006