scholarly journals GTP gamma S inhibits organelle transport along axonal microtubules.

1993 ◽  
Vol 120 (2) ◽  
pp. 467-476 ◽  
Author(s):  
G S Bloom ◽  
B W Richards ◽  
P L Leopold ◽  
D M Ritchey ◽  
S T Brady
Keyword(s):  
Protein Kinases ◽  
Axonal Transport ◽  
Binding Proteins ◽  
Organelle Transport ◽  
Nucleotide Exchange ◽  
Fast Axonal Transport ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Organelle Motility ◽  
Gamma S

Movements of membrane-bounded organelles through cytoplasm frequently occur along microtubules, as in the neuron-specific case of fast axonal transport. To shed light on how microtubule-based organelle motility is regulated, pharmacological probes for GTP-binding proteins, or protein kinases or phosphatases were perfused into axoplasm extruded from squid (Loligo pealei) giant axons, and effects on fast axonal transport were monitored by quantitative video-enhanced light microscopy. GTP gamma S caused concentration-dependent and time-dependent declines in organelle transport velocities. GDP beta S was a less potent inhibitor. Excess GTP, but not GDP, masked the effects of coperfused GTP gamma S. The effects of GTP gamma S on transport were not mimicked by broad spectrum inhibitors of protein kinases (K-252a) or phosphatases (microcystin LR and okadaic acid), or as shown earlier, by ATP gamma S. Therefore, suppression of organelle motility by GTP gamma S was guanine nucleotide-specific and evidently did not involve irreversible transfer of thiophosphate groups to protein. Instead, the data imply that organelle transport in the axon is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more GTP-binding proteins. Fast axonal transport was not perturbed by AlF4-, indicating that the GTP gamma S-sensitive factors do not include heterotrimeric G-proteins. Potential axoplasmic targets of GTP gamma S include dynamin and multiple small GTP-binding proteins, which were shown to be present in squid axoplasm. These collective findings suggest a novel strategy for regulating microtubule-based organelle transport and a new role for GTP-binding proteins.

scholarly journals Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase.

1993 ◽  
Vol 4 (3) ◽  
pp. 261-269 ◽  
Author(s):  
P G Heyworth ◽  
U G Knaus ◽  
X Xu ◽  
D J Uhlinger ◽  
L Conroy ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Human Neutrophil ◽  
Binding Proteins ◽  
Cysteine Residue ◽  
Posttranslational Processing ◽  
Nucleotide Exchange ◽  
Gtp Binding Proteins ◽  
Phagocyte Nadph Oxidase ◽  
Highly Active ◽  
Gtp Binding

Rac1 and Rac2 are closely related, low molecular weight GTP-binding proteins that have both been implicated in regulation of phagocyte NADPH oxidase. This enzyme system is composed of multiple membrane-bound and cytosolic subunits and when activated catalyzes the one-electron reduction of oxygen to superoxide. Superoxide and its highly reactive derivatives are essential for killing microorganisms. Rac proteins undergo posttranslational processing, primarily the addition of an isoprenyl group to a carboxyl-terminal cysteine residue. We directly compared recombinant Rac1 and Rac2 in a human neutrophil cell-free NADPH oxidase system in which cytosol was replaced by purified recombinant cytosolic components (p47-phox and p67-phox). Processed Rac1 and Rac2 were both highly active in this system and supported comparable rates of superoxide production. Under different cell-free conditions, however, in which suboptimal amounts of cytosol were present in the assay mixture, processed Rac2 worked much better than Rac1 at all but the lowest concentrations. This suggests that a factor in the cytosol may suppress the activity of Rac1 but not of Rac2. Unprocessed Rac proteins were only weakly able to support superoxide generation in either system, but preloading of Rac1 or Rac2 with guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) restored activity. These results indicate that processing is required for nucleotide exchange but not for interaction with oxidase components.

Role of G proteins in the acetylcholine-induced potassium current of canine atrial cells

1989 ◽  
Vol 257 (5) ◽  
pp. H1516-H1522
Author(s):  
S. Sorota ◽  
B. F. Hoffman
Keyword(s):  
Potassium Current ◽  
Binding Proteins ◽  
Membrane Conductance ◽  
Pipette Solution ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Current Voltage ◽  
Gamma S

The acetylcholine-induced opening of potassium channels depends on GTP-binding proteins in the chick, guinea pig, frog, and rat. In contrast, Bubien and Woods (Biochem. Biophys. Res. Commun. 142: 1039-1045, 1987) have recently postulated that the acetylcholine response in cultured canine atrial cells may be independent of GTP-binding proteins. In whole cell patch-clamp experiments using cultured canine atrial cells, we did not detect an effect of GTP (10(-4) M) in the pipette solution on the acetylcholine-induced potassium current. However, 500 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) in the pipette diminished the response to acetylcholine. Pertussis toxin (30 ng/ml for 24 h) blocked the response to acetylcholine. With guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 3 microM) in the patch pipette, acetylcholine irreversibly increased membrane conductance. The current-voltage relationship for the persistently activated current was similar to that induced by acetylcholine. We conclude that the acetylcholine-induced potassium current in canine atrial cells behaves like that seen in other species and depends on GTP-binding proteins.

scholarly journals Retrograde transport from the Golgi region to the endoplasmic reticulum is sensitive to GTP gamma S.

1992 ◽  
Vol 116 (6) ◽  
pp. 1357-1367 ◽  
Author(s):  
A Tan ◽  
J Bolscher ◽  
C Feltkamp ◽  
H Ploegh
Keyword(s):  
Hepg2 Cells ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Brefeldin A ◽  
Retrograde Transport ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Streptolysin O ◽  
Gamma S

The involvement of GTP-binding proteins in the intracellular transport of the secretory glycoprotein alpha 1-antitrypsin was investigated in streptolysin O-permeabilized HepG2 cells. This permeabilization procedure allows ready access to the intracellular milieu of the membrane-impermeant, nonhydrolyzable GTP analog GTP gamma S. In streptolysin O-permeabilized HepG2 cells, the constitutive secretory pathway remains functional and is sensitive to GTP gamma S. Exposure of HepG2 cells to brefeldin A resulted in redistribution of Golgi-resident glycosyltransferases (including both alpha 2----3 and alpha 2----6 sialyltransferases) to the ER. This redistribution was sensitive to GTP gamma S. Our results suggest that GTP-binding proteins are involved in the regulation not only of the anterograde, but also of the retrograde, pathway.

scholarly journals Multiple GTP-binding proteins participate in clathrin-coated vesicle-mediated endocytosis.

1993 ◽  
Vol 120 (1) ◽  
pp. 37-45 ◽  
Author(s):  
L L Carter ◽  
T E Redelmeier ◽  
L A Woollenweber ◽  
S L Schmid
Keyword(s):  
Protein Function ◽  
Binding Proteins ◽  
Coated Vesicle ◽  
Coated Pits ◽  
Vesicle Budding ◽  
Gtp Binding Proteins ◽  
Receptor Mediated Endocytosis ◽  
Gtp Binding ◽  
Gamma S

We have examined the effects of various agonists and antagonists of GTP-binding proteins on receptor-mediated endocytosis in vitro. Stage-specific assays which distinguish coated pit assembly, invagination, and coat vesicle budding have been used to demonstrate requirements for GTP-binding protein(s) in each of these events. Coated pit invagination and coated vesicle budding are both stimulated by addition of GTP and inhibited by GDP beta S. Although coated pit invagination is resistant to GTP gamma S, A1F4-, and mastoparan, late events involved in coated vesicle budding are inhibited by these antagonists of G protein function. Earlier events involved in coated pit assembly are also inhibited by GTP gamma S, A1F4-, and mastoparan. These results demonstrate that multiple GTP-binding proteins, including heterotrimeric G proteins, participate at discrete stages in receptor-mediated endocytosis via clathrin-coated pits.

Fluoroaluminate- and GTP gamma S-induced stress, shortening, and myosin phosphorylation in airway smooth muscle

1993 ◽  
Vol 265 (1) ◽  
pp. L73-L79
Author(s):  
C. M. Hai ◽  
C. B. Ma
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Binding Proteins ◽  
Muscle Length ◽  
Active Stress ◽  
Myosin Phosphorylation ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Muscle Shortening ◽  
Gamma S

GTP-binding proteins in bovine tracheal smooth muscle were activated by fluoroaluminate and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), and the sensitivities of fluoroaluminate- and GTP gamma S-induced active stress and myosin phosphorylation to muscle shortening were compared. Relative to the value of myosin phosphorylation at L0, unloaded shortening induced a 63% decrease in fluoroaluminate-activated steady-state myosin phosphorylation, but had no significant effect on GTP gamma S-activated myosin phosphorylation. These results were consistent with the hypothesis that shortening-sensitive and shortening-insensitive signal-transduction pathways coexist in airway smooth muscle. However, unlike myosin phosphorylation, active stress induced by fluoroaluminate was actually less sensitive to shortening. The amount of shortening necessary to reduce active stress to half of that at Lo was 65% in fluoroaluminate-activated tissues, but was only 34% in GTP gamma S-activated tissues. The observation of different sensitivities of fluoroaluminate-activated myosin phosphorylation and active stress suggests that GTP-binding proteins modulate the dependence of active stress on muscle length in smooth muscle.

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