Characterization of membrane-localized and cytosolic Rac-GTPase-activating proteins in human neutrophil granulocytes: Contribution to the regulation of NADPH oxidase

Miklós Gelszt, Marie Claire Dagher, Gergely Molnár, Andrea Havasi, Julien Faure, Marie Hélène Paclet, Francoise Morel, Erzsébet Ligeti

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Abstract

We have investigated the intracellular localization and molecular identity of Rac-GTPase-activating proteins (Rac-GAPs) in human neutrophils. Immunoblot analysis detected the presence of both pl90RhoGAP and Bcr mainly in the cytosol. An overlay assay performed with [γ-32P]GTP-bound Rac revealed dominant GAP activity related to a 50 kDa protein both in the membrane and cytosol. This activity could be identified by Western blotting and immunoprecipitation with specific antibody directed against the GAP domain of p50RhoGAP. Using a semirecombinant or fully purified cell-free activation assay of the Rac-activated enzyme NADPH oxidase, we demonstrated the regulatory effect of both the membrane-localized and soluble GAPs. We suggest that in neutrophil granulocytes Rac-GAPs have redundant function and represent suitable targets for both the up-regulation and down-regulation of the NADPH oxidase.

Original languageEnglish
Pages (from-to)851-858
Number of pages8
JournalBiochemical Journal
Volume355
Issue number3
Publication statusPublished - May 1 2001

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Keywords

  • Overlay
  • Phagocytes
  • Prenylation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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