Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex.
Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [alpha32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Département de Biologie Moléculaire et Structurale, CEA Grenoble, France.
J
J
Fauré
P V
PV
Vignais
M C
MC
Dagher
D000244:Adenosine Diphosphate / Q000378:metabolism; D002070:Butadienes / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020730:Guanine Nucleotide Dissociation Inhibitors; D006160:Guanosine Triphosphate / Q000378:metabolism; D045782:Hemiterpenes; D006801:Humans; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D010420:Pentanes; D010716:Phosphatidylinositols / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D017368:Protein Prenylation; D012263:Ribonucleosides / Q000378:metabolism; D020741:rho GTP-Binding Proteins; D062950:rho Guanine Nucleotide Dissociation Inhibitor gamma; D020742:rhoA GTP-Binding Protein
C569643:ARHGDIG protein, human; D002070:Butadienes; D020730:Guanine Nucleotide Dissociation Inhibitors; D045782:Hemiterpenes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010420:Pentanes; D010716:Phosphatidylinositols; D010761:Phosphorus Radioisotopes; D012263:Ribonucleosides; D062950:rho Guanine Nucleotide Dissociation Inhibitor gamma; C005059:isoprene; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein