Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes
| Autor: | R R, Desrosiers, F, Gauthier, W, Lin, R, Béliveau |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Male
Adenosine Diphosphate Ribose Microvilli Hydrolysis Membrane Proteins Cell Cycle Proteins Cell Biology Kidney Biochemistry Guanine Nucleotides Recombinant Proteins Rats Rats Sprague-Dawley GTP-Binding Proteins Guanosine 5'-O-(3-Thiotriphosphate) Animals Protease Inhibitors cdc42 GTP-Binding Protein rhoA GTP-Binding Protein Molecular Biology Peptide Hydrolases |
| Zdroj: | Biochemistry and Cell Biology. 76:63-72 |
| ISSN: | 1208-6002 0829-8211 |
| DOI: | 10.1139/o98-013 |
| Popis: | Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[β-thio]diphosphate (GDPβS) and guanosine 5'-O-[γ-thio]triphosphate (GTPγS), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTPγS also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTPγS. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTPγS was usually lower for RhoA than for Cdc42. GTPγS also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1β-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTPγS-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.Key words: rho proteins, GTP, proteolysis, kidney. |
| Databáze: | OpenAIRE |
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