Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions

1999 ◽  
Vol 77 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Diane Bilodeau ◽  
Sylvie Lamy ◽  
Richard R Desrosiers ◽  
Denis Gingras ◽  
Richard Béliveau
Keyword(s):  
Ionic Strength ◽  
Brush Border ◽  
Rat Kidney ◽  
Regulatory Protein ◽  
Rho Proteins ◽  
Glutathione S Transferase ◽  
Free System ◽  
Brush Border Membranes ◽  
A Cell ◽  
Membrane Bound

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.Key words: rhoGDI, rho proteins, ionic strength, kidney.

Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by βγ subunits of heterotrimeric G-proteins

2000 ◽  
Vol 352 (2) ◽  
pp. 491-499 ◽  
Author(s):  
Hervé LE STUNFF ◽  
Lien DOKHAC ◽  
Sylvain BOURGOIN ◽  
Marie-France BADER ◽  
Simone HARBON
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Ammonium Sulphate ◽  
Phospholipase D ◽  
Tyrosine Kinases ◽  
Glutathione S Transferase ◽  
Free System ◽  
Adp Ribosylation ◽  
A Cell ◽  
Membrane Bound

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629–637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5ƀ-[γ-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6(2–13)] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1(2–17) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF-4 and this inhibition was counteracted by the fusion protein glutathione S-transferase-β-adrenergic receptor kinase 1 (495–689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein βγ-subunit scavengers. It is concluded that G-protein subunits βγ are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.

Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes

1998 ◽  
Vol 76 (1) ◽  
pp. 63-72
Author(s):  
Richard R Desrosiers ◽  
France Gauthier ◽  
Wei Lin ◽  
Richard Béliveau
Keyword(s):  
Proteolytic Activity ◽  
Brush Border ◽  
Rat Kidney ◽  
Proteolytic Degradation ◽  
Rat Tissues ◽  
Rho Proteins ◽  
Brush Border Membranes ◽  
Membrane Treatment ◽  
Kidney Liver

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.

Binding of lysozyme to brush border membranes of rat kidney

1983 ◽  
Vol 732 (2) ◽  
pp. 372-376 ◽  
Author(s):  
Gabriele Beyer ◽  
Folkert Bode ◽  
Karl Baumann
Keyword(s):  
Brush Border ◽  
Rat Kidney ◽  
Brush Border Membranes

The binding and degradation of insulin by brush-border membranes from rat kidney

1982 ◽  
Vol 10 (4) ◽  
pp. 220-220
Author(s):  
J. HYWEL THOMAS ◽  
PHILIP G. DAVEY ◽  
CHRISTOPHER D. G. JENKINS ◽  
DESPINA K. PAPACHRISTODOULOU
Keyword(s):  
Brush Border ◽  
Rat Kidney ◽  
Brush Border Membranes

scholarly journals Characterization of neutral endopeptidase 24.11 in dog glomeruli

1993 ◽  
Vol 291 (3) ◽  
pp. 773-779 ◽  
Author(s):  
C Landry ◽  
P Santagata ◽  
W Bawab ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
...  
Keyword(s):  
Cell Surface ◽  
Brush Border ◽  
Neutral Endopeptidase ◽  
Glycosylation Pattern ◽  
Electrophoretic Mobilities ◽  
Mammalian Tissues ◽  
Dog Kidney ◽  
A Cell ◽  
Membrane Bound ◽  
Triton X

Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.

The properties of free and membrane bound polysomes studied in a cell free system of Bombix mori silkglands

FEBS Letters ◽  
1971 ◽  
Vol 13 (6) ◽  
pp. 321-324 ◽  
Author(s):  
J. Daillie ◽  
L. Grasset ◽  
J.-C. Beck ◽  
J.-P. Prudhomme ◽  
J.-P. Ebel
Keyword(s):  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Membrane Bound
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