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Mechanism of NADPH oxidase activation by the Rac/Rho-GDI complex
1Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (BBSI)-UMR CEA/CNRS/UJF 5092, CEA Grenoble, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France.
Biochemistry
|August 22, 2001
Summary
Rho GDP dissociation inhibitor (Rho-GDI) binding to Rac GTPase is crucial for NADPH oxidase activation. Protein-lipid interactions dominate this binding, with specific protein-protein interactions playing a supporting role.
Area of Science:
- Cellular Biology
- Molecular Biology
- Immunology
Background:
- The low molecular weight GTP binding protein Rac is essential for activating the NADPH oxidase complex, a key component in pathogen killing during phagocytosis.
- In resting cells, Rac forms a heterodimeric complex with Rho GDP dissociation inhibitor (Rho-GDI), involving both protein-lipid and protein-protein interactions.
Purpose of the Study:
- To elucidate the specific roles of protein-lipid and protein-protein interactions in the Rac-Rho-GDI complex formation.
- To determine how these interactions influence Rac's ability to activate the NADPH oxidase complex.
Main Methods:
- Utilized the two-hybrid system to analyze Rac-Rho-GDI interactions.
- Employed site-directed mutagenesis to create nonprenylated and prenylated Rac1 mutants.
- Assessed NADPH oxidase activation in cell-free assays.
Main Results:
- Nonprenylated Rac1 shows weak interaction with Rho-GDI, highlighting the importance of protein-lipid interactions (prenylation).
- Specific protein-protein interaction sites (Arg66-Leu67, His103, and the C-terminal polybasic region) cooperate in complex formation when prenylation is absent.
- Rac1 mutants unable to interact with Rho-GDI failed to activate NADPH oxidase, even when prenylated.
- Rho-GDI binding, particularly to the polybasic region, is critical for inhibiting the GDP/GTP exchange reaction and stabilizing Rac in an active conformation.
Conclusions:
- Protein-lipid interaction, mediated by Rac prenylation, is the predominant force in Rac-Rho-GDI complex formation.
- Rho-GDI stabilizes Rac in an active conformation, even in the GDP-bound state, facilitating NADPH oxidase activation.
- The Rac1-Rho-GDI complex, irrespective of GDP or GTP binding, efficiently activates the NADPH oxidase, suggesting a crucial role for Rho-GDI in effector presentation.