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Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation
T G Gabig1, C D Crean, P L Mantel
1Department of Medicine, Indiana University School of Medicine, Indianapolis 46202-5121.
Blood
|February 1, 1995
Summary
Rac2 and Rap1a are crucial for neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. Rap1a acts as a final switch, while Rac2 mediates component assembly, clarifying their roles in human phagocytes.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation is critical for host defense.
- The roles of low molecular-weight guanosine triphosphatases (GTPases) Rac and Rap1a in this process within intact human phagocytes require further elucidation.
- Previous cell-free studies indicated Rac's requirement and Rap1a's potential participation in NADPH oxidase activation.
Purpose of the Study:
- To investigate the specific functions of Rap1a and Rac2 in the activation of NADPH oxidase in intact human phagocytes.
- To clarify the signaling pathways involving Rap1a and Rac2 as signal transducers from plasma membrane receptors.
- To develop a model for the distinct roles of Rac2 and Rap1a in NADPH oxidase assembly and activation.
Main Methods:
- Oligonucleotide-directed mutagenesis was employed to create mutated human rap1a and rac2 constructs.
- HL60 cells were transfected with wild-type or mutated rap1a/rac2 cDNA and selected for stable expression.
- Differentiated transfected cell lines and control lines were analyzed for NADPH oxidase component expression and superoxide (O2.-) production.
Main Results:
- Stable transfection and differentiation into neutrophil-like cells did not alter NADPH oxidase component levels (p47-phox, p67-phox, gp91-phox).
- Dominant-negative N17 Rap1a and N17 Rac2 inhibited O2.- production, while overexpressing wild-type Rap1a increased it fourfold.
- GTPase-defective V12 Rap1a significantly inhibited O2.- production, suggesting a requirement for Rap1a GTP/GDP cycling.
Conclusions:
- Rac2 is proposed to mediate the assembly of NADPH oxidase components (p47-phox, p67-phox) via cytoskeletal reorganization.
- Rap1a is suggested to function as a downstream activation switch, interacting directly with the flavocytochrome component.
- These findings provide a model for the distinct, sequential roles of Rac2 and Rap1a in neutrophil NADPH oxidase activation.