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  • Dissociation of Rac1(GDP).RhoGDI complexes by the cooperative action of anionic liposomes containing phosphatidylinositol 3,4,5-trisphosphate, Rac guanine nucleotide exchange factor, and GTP.

Dissociation of Rac1(GDP).RhoGDI complexes by the cooperative action of anionic liposomes containing phosphatidylinositol 3,4,5-trisphosphate, Rac guanine nucleotide exchange factor, and GTP.

The Journal of biological chemistry (2008-05-29)
Yelena Ugolev, Yevgeny Berdichevsky, Carolyn Weinbaum, Edgar Pick
ABSTRACT

Rac plays a pivotal role in the assembly of the superoxide-generating NADPH oxidase of phagocytes. In resting cells, Rac is found in the cytosol in complex with Rho GDP dissociation inhibitor (RhoGDI). NADPH oxidase assembly involves dissociation of the Rac.RhoGDI complex and translocation of Rac to the membrane. We reported that liposomes containing high concentrations of monovalent anionic phospholipids cause Rac.RhoGDI complex dissociation ( Ugolev, Y., Molshanski-Mor, S., Weinbaum, C., and Pick, E. (2006) J. Biol. Chem. 281, 19204-19219 ). We now designed an in vitro model mimicking membrane phospholipid remodeling during phagocyte stimulation in vivo. We showed that liposomes of "resting cell membrane" composition (less than 20 mol % monovalent anionic phospholipids), supplemented with 1 mol % of polyvalent anionic phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) in conjunction with constitutively active forms of the guanine nucleotide exchange factors (GEFs) for Rac, Trio, or Tiam1 and a non-hydrolyzable GTP analogue, cause dissociation of Rac1(GDP).RhoGDI complexes, GDP to GTP exchange on Rac1, and binding of Rac1(GTP) to the liposomes. Complexes were not dissociated in the absence of GEF and GTP, and optimal dissociation required the presence of PtdIns(3,4,5)P(3) in the liposomes. Dissociation of Rac1(GDP).RhoGDI complexes was correlated with the affinity of particular GEF constructs, via the N-terminal pleckstrin homology domain, for PtdIns(3,4,5)P(3) and involved GEF-mediated GDP to GTP exchange on Rac1. Phagocyte membranes enriched in PtdIns(3,4,5)P(3) responded by NADPH oxidase activation upon exposure in vitro to Rac1(GDP).RhoGDI complexes, p67(phox), GTP, and Rac GEF constructs with affinity for PtdIns(3,4,5)P(3) at a level superior to that of native membranes.

MATERIALS
Product Number
Brand
Product Description

Avanti
18:1 PI(3,4)P2, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′-bisphosphate) (ammonium salt), powder
Avanti
18:1 PI(3,5)P2, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,5′-bisphosphate) (ammonium salt), powder
Avanti
18:1 (Δ9-Cis) PC (DOPC), Avanti Research - A Croda Brand
Avanti
18:1 PI(3,4,5)P3, 1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′,5′-trisphosphate) (ammonium salt), powder
Avanti
18:1 (Δ9-Cis) PC (DOPC), Avanti Research - A Croda Brand
Avanti
18:1 PI(4,5)P2, Avanti Research - A Croda Brand