Search results for "GTP'"

Rac regulation of NtrbohD, the oxidase responsible for the oxidative burst in elicited tobacco cell

2003

Five cDNAs encoding Rac protein homologues to the Rho-related proteins from plants (Rop) were isolated in tobacco, and the function of one of them, Ntrac5, was studied. The Ntrac5 mRNA is repressed when tobacco leaves and cells are treated with the fungal elicitor cryptogein. Tobacco cells were transformed with sense constructs of Ntrac5 or Ntrac5V15, encoding the native GTP/GDP-bound form of this Rac protein homologue or the constitutively active mutant in its GTP-bound form, respectively. Immunological studies indicate that the corresponding protein is continuously located on the plasma membrane (PM). Both types of transformed cells show the same extra-cellular alkalinization as the contr…

FRET biosensor allows spatio-temporal observation of shear stress-induced polar RhoGDIα activation

2018

Rho GDP-dissociation inhibitor α (RhoGDIα) is a known negative regulator of the Rho family that shuts off GDP/GTP cycling and cytoplasm/membrane translocation to regulate cell migration. However, to our knowledge, no reports are available that focus on how the RhoGDIα-Rho GTPases complex is activated by laminar flow through exploring the activation of RhoGDIα itself. Here, we constructed a new biosensor using fluorescence resonance energy transfer (FRET) technology to measure the spatio-temporal activation of RhoGDIα in its binding with Rho GTPases in living HeLa cells. Using this biosensor, we find that the dissociation of the RhoGDIα-Rho GTPases complex is increased by shear stress, and i…

Uncoupling of dynamin polymerization and GTPase activity revealed by the conformation-specific nanobody dynab

2017

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We…

A subset of flavaglines inhibits KRAS nanoclustering and activation.

2020

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these int…

Human oxytocin receptors in cholesterol-rich vs. cholesterol-poor microdomains of the plasma membrane

2000

We analyzed the properties of a G protein-coupled receptor localized in cholesterol-poor vs. cholesterol-rich microdomains of the plasma membrane. For this purpose, the human oxytocin receptor, which is very sensitive against alterations of the membrane cholesterol level, was stably expressed in HEK293 cells. To calculate the total number of receptors independent of ligand binding studies, the oxytocin receptor was tagged with an enhanced green fluorescent protein (EGFP) which did not change the functional properties of the receptor. Only 1% of the oxytocin receptors were present in cholesterol-rich detergent-insoluble domains. In contrast, employing a detergent-free fractionation scheme th…

Dynamics of Ca2+ and guanosine 5'-[gamma-thio]triphosphate action on insulin secretion from alpha-toxin-permeabilized HIT-T15 cells.

1994

The time course of Ca2+ and GTP-analogue effects on insulin secretion was investigated in HIT-T15 cells permeabilized with Staphylococcus alpha-toxin. These cells responded to Ca2+ in the range 0.1-10 microM and could be used in a dynamic perifusion system because of the minimal run-down of the secretory response. High Ca2+ (10 microM) elicited a monophasic ATP-dependent stimulation of insulin secretion that reached a peak within 5 min (approximately 20-fold increase) and rapidly decreased during the subsequent 15 min to a plateau remaining above basal rates (0.1 microM Ca2+). The decrease in Ca(2+)-induced insulin secretion with time could not be attributed to decreased capacity to respond…

6-thio-GTP Derivatives

2006

Binding and/or hydrolysis of purine‐based nucleotides is not required for IM30 ring formation

2021

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms lar…

Regulated expression and phosphorylation of the 23-26-kDa ras protein in the sponge Geodia cydonium.

1990

We have cloned, sequenced and examined the sponge Geodia cydonium cDNA encoding a protein homologous to ras proteins. The sponge ras protein has a more conserved N-terminal region and a less conserved C-terminal region, especially in comparison to Dictyostelium discoideum; the similarity to human c-Ha-ras-1 and to Saccharomyces cerevisiae is less pronounced. The sponge ras cDNA comprises five TAG triplets; at the translational level these UAG termination codons are suppressed by a Gln-tRNA. The sponge ras protein was isolated and partially purified (23-26 kDa) and found to undergo phosphorylation at a threonine moiety, when dissociated cells were incubated in the presence of a homologous ag…

Molecular recognition of nucleotides in water by scorpiand-type receptors based on nucleobase discrimination.

2014

Abstract: The detection of nucleotides is of crucial impor-tance because they are the basic building blocks of nucleicacids. Scorpiand-based polyamine receptors functionalizedwith pyridine or anthracene units are able to form stablecomplexes with nucleotides in water, based on coulombic,p–p stacking, and hydrogen-bonding interactions. This be-havior has been rationalized by means of an explorationwith NMR spectroscopy and DFT calculations. Binding con-stants were determined by potentiometry. Fluorescencespectroscopy studies have revealed the potential of these re-ceptors as sensors to effectively and selectively distinguishguanosine-5’-triphosphate (GTP) from adenosine-5’-triphos-phate (ATP…