Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein

• Published in: The Journal of biological chemistry 1991-07, Vol.266 (21), p.13964-13970
• Main Authors: EKLUND, E. A, MARSHALL, M, GIBBS, J. B, CREAN, C. D, GABIG, T. G
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cell physiology; Cell-Free System; Cytosol - metabolism; Enzyme Activation; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - isolation & purification; GTP-Binding Proteins - physiology; Humans; In Vitro Techniques; Molecular and cellular biology; NADH, NADPH Oxidoreductases - metabolism; NADPH Oxidases; Neutrophils - chemistry; Neutrophils - enzymology; Oligonucleotides - chemistry; Polymerase Chain Reaction; rap GTP-Binding Proteins; Recombinant Proteins - metabolism; Signal Transduction; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)92797-3

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.