GTPγS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor

In a cell-free system from neutrophil cytosol GTPγS can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTPγS stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPγS·G(iα2) or G(β1γ2) subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPγS. When recombinant, constitutively activated and GTPγS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations > 100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPγS. The action of GTPγS was blocked by mM [Mg²⁺], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPγS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPγS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P₂-binding peptide did not interfere with the response to GTPγS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.