Two of the cytosolic NADPH oxidase components, p47-phox and p67-phox, translocate to the plasma membrane in normal neutrophils stimulated with phorbol myristate acetate (PMA). We have now studied the translocation process in neutrophils of patients with chronic granulomatous disease (CGD), an inherited syndrome in which the oxidase system fails to produce superoxide due to lesions affecting any one of its four known components: the gp9l-phox and p22-phox subunits of cytochrome b5m (the membrane-bound terminal electron transporter of the oxidase), p47-phox, and p67-phox. In contrast to normal cells, neither p47-phox nor p67-phox translocated to the membrane in PMA-stimulated CGD neutrophils which lack cytochrome b5!m. In one patient with a rare X-linked form of CGD caused by a Pro --His substitution in gp9l-phox, but whose neutrophils have normal levels of this mutant cytochrome bm, translocation was normal. In two patients with p47-phox deficiency, p67-phox failed to translocate, whereas p47-phox was detected in the particulate fraction of PMAstimulated neutrophils from two patients deficient in p67-phox.Our data suggest that cytochrome b5!4 or a closely linked factor provides an essential membrane docking site for the cytosolic oxidase components and that it is p47-phox that mediates the assembly of these components on the membrane. (J. Clin. Invest. 1991. 87:352-356.)

We investigated the mechanism of H 2 O 2 activation of the Ca 2+ -regulated NADPH oxidase NOX5. H 2 O 2 induced a transient, dose-dependent increase in superoxide production in K562 cells expressing NOX5. Confocal studies demonstrated that the initial calcium influx generated by H 2 O 2 is amplified by a feedback mechanism involving NOX5-dependent superoxide production and H 2 O 2 . H 2 O 2 -NOX5 activation was inhibited by extracellular Ca2+ chelators, a pharmacological inhibitor of cAbl, and over-expression of kinase-dead c-Abl.Transfected kinase-active GFP-c-Abl co-localized with vesicular sites of superoxide production in a Ca 2+ -dependent manner. In contrast to H 2 O 2 , the Ca2+ ionophore ionomycin induced NOX5 activity independently of c-Abl. Immunoprecipitation of cell lysates revealed that active GFP-c-Abl formed oligomers with endogenous c-Abl and that phosphorylation of both proteins was increased by H 2 O 2 treatment. Furthermore, H 2 O 2 -induced NOX5 activity correlated with increased localization of c-Abl to the membrane fraction, and NOX5 proteins could be co-immunoprecipitated with GFP-Abl proteins.Our data demonstrate for the first time that NOX5 is activated by c-Abl through a Ca 2+ -mediated, redox-dependent signaling pathway and suggest a functional association between NOX5 NADPH oxidase and c-Abl.

Chronic granulomatous disease, a syndrome of recurrent infections and failure of oxidative microbicidal activity in phagocytes, results from defects in the gene for one of several components of an oxidase system that can undergo activation. To determine the relative prevalence of certain of the genetic variants of this disorder, we used immunoblotting to detect two specific neutrophil cytosolic proteins of 47 and 67 kd recently shown to be required for oxidase activation. Chronic granulomatous disease is usually an X-linked disorder associated with the absence of membrane cytochrome b558. Of our 94 patients with chronic granulomatous disease, however, 36 had a phenotype characterized by autosomal inheritance, normal membrane oxidase components (including cytochrome b558), and functionally defective cytosolic activity in a cell-free oxidase system. We studied 25 of these 36 patients and found that 22 lacked the 47-kd cytosolic protein, and the remaining 3 were missing the 67-kd component. Patients with chronic granulomatous disease whose functional defect was localized to the neutrophil membrane (classic X-linked cytochrome b-negative type and two other rare variants) had normal amounts of both cytosolic components. We estimate that approximately 33 percent of all patients with chronic granulomatous disease are missing the 47-kd cytosolic oxidase component and about 5 percent of patients are missing the 67-kd component. Chronic granulomatous disease caused by a defect in any cytosolic factors other than the 47-kd and 67-kd proteins, if it exists, is apparently rare.

We have isolated cDNA clones encoding an inositol 1,4,5-trisphosphate receptor type 1 (InsP3R1) from human uteri and a leukaemic cell line, HL-60. Northern-blot analysis showed that approx. 10 kb of InsP3R1 mRNA is expressed in human uteri, oviducts and HL-60 cells. The predicted amino acid sequence of human InsP3R1 (2695 amino acids) has 99% identity with that of the mouse SI-/SII- splicing counterpart. Western-blot analysis with anti-(mouse InsP3R1) antibodies showed that InsP3R1 protein of human uteri and oviducts of approx 220 kDa is immunostained. Northern-blot analysis of HL-60 cell differentiation along the neutrophilic lineage induced by retinoic acid or dimethylsulphoxide showed an accompanying enhanced expression of InsP3R1 mRNA. Immunohistochemical analysis of the cerebella of spinocerebellar degeneration patients showed a variable loss of Purkinje cells with an altered pattern of immunostaining. The InsP3R1 gene (Insp3r1) was localized to the 3P25-26 region of human chromosome 3. The data presented here clearly show that InsP3R1 exists widely in human tissues and may play critical roles in various kinds of cellular functions.

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