Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions.
The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.
Bilodeau
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Laboratoire de Médecine Moléculaire, et Centre de Cancérologie Charles Bruneau, Université du Québec a Montréal, Canada.
Lamy
S
S
Desrosiers
R R
RR
Gingras
D
D
Béliveau
R
R
D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D002462:Cell Membrane; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors; D007668:Kidney / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism*; D011189:Potassium Chloride / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D015398:Signal Transduction; D012965:Sodium Chloride / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020743:rhoB GTP-Binding Protein
D018797:Cell Cycle Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D008565:Membrane Proteins; D011994:Recombinant Proteins; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D012965:Sodium Chloride; D011189:Potassium Chloride; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; D020764:cdc42 GTP-Binding Protein; D020743:rhoB GTP-Binding Protein