scholarly journals Opioid peptides promote cholera-toxin-catalysed ADP-ribosylation of the inhibitory guanine-nucleotide-binding protein (Gi) in membranes of neuroblastoma x glioma hybrid cells

1988 ◽  
Vol 252 (2) ◽  
pp. 369-373 ◽  
Author(s):  
G Milligan ◽  
F R McKenzie
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Opioid Peptides ◽  
Hybrid Cells ◽  
Guanine Nucleotide Binding Protein ◽  
Adp Ribosylation ◽  
Guanine Nucleotide Binding

NG108-15 neuroblastoma x glioma hybrid cells express a major 45 kDa substrate for cholera toxin and a 40 kDa substrate(s) for pertussis toxin when ADP-ribosylation is performed in the presence of GTP. In the absence of exogenous GTP, however, cholera toxin was shown to catalyse incorporation of radioactivity into a 40 kDa protein as well as into the 45 kDa polypeptide. In membranes of cells which had been pretreated in vivo with pertussis toxin, the 40 kDa band was no longer a substrate for either pertussis or cholera toxin in vitro, whereas in membranes from cholera-toxin-pretreated cells the 40 kDa band was still a substrate for fresh cholera toxin in vitro and for pertussis toxin. In this cell line, opioid peptides have been shown to inhibit adenylate cyclase exclusively by interacting with Gi (inhibitory G-protein) and with no other pertussis-toxin-sensitive G-protein. Opioid agonists, but not antagonists, promoted the cholera-toxin-catalysed ADP-ribosylation of the 40 kDa polypeptide, hence demonstrating that this cholera-toxin substrate was indeed the alpha-subunit of Gi. These results demonstrate that Gi can be a substrate for either cholera or pertussis toxin under appropriate conditions.

scholarly journals Inhibition by pertussis toxin of the activation of Na+-dependent uridine transport in dimethyl-sulphoxide-induced HL-60 leukaemia cells

1991 ◽  
Vol 280 (2) ◽  
pp. 515-519 ◽  
Author(s):  
J A Sokoloski ◽  
A C Sartorelli ◽  
R E Handschumacher ◽  
C W Lee
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Dimethyl Sulphoxide ◽  
Protein G ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dependent Transport

The effects of pertussis toxin on the Na(+)-dependent transport of uridine were studied in HL-60 leukaemia cells induced to differentiate along the granulocytic or monocytic pathways by dimethyl sulphoxide (DMSO) or phorbol 12-myristate 13-acetate (PMA) respectively. Pertussis toxin at 50 ng/ml completely inhibited the activation of Na(+)-dependent uridine transport and consequently prevented the formation of intracellular pools of free uridine which occurs in HL-60 cells induced to differentiate by DMSO. The inhibition of Na(+)-dependent uridine transport by pertussis toxin in cells exposed to DMSO was associated with a 14-fold decrease in affinity, with no change in Vmax. Pertussis toxin, however, had no effect on Na(+)-dependent uridine transport in PMA-induced HL-60 cells. Furthermore, 500 ng of cholera toxin/ml had no effect on the Na(+)-dependent uptake of uridine in DMSO-treated HL-60 cells. These results suggest that the activation of the Na(+)-dependent transport of uridine in HL-60 cells induced to differentiate along the granulocytic pathway by DMSO is coupled to a pertussis-toxin-sensitive guanine-nucleotide binding protein (G-protein).

Prostaglandin E2 receptors in the heart are coupled to inhibition of adenylyl cyclase via a pertussis toxin sensitive G protein

1992 ◽  
Vol 70 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Richard W. Lerner ◽  
Gary D. Lopaschuk ◽  
Peter M. Olley
Keyword(s):  
Adenylyl Cyclase ◽  
G Protein ◽  
Pertussis Toxin ◽  
Binding Sites ◽  
Cyclase Activity ◽  
Prostaglandin E ◽  
Adenylyl Cyclase Activity ◽  
Adp Ribosylation ◽  
Number Of Binding Sites ◽  
Guanine Nucleotide Binding

In previous studies we have identified and isolated a prostaglandin E2 (PGE2) receptor from cardiac sarcolemmal (SL) membranes. Binding of PGE2 to this receptor in permeabilized SL vesicles inhibits adenylyl cyclase activity. The purpose of this study was to determine if the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive guanine nucleotide binding inhibitory (Gi) protein. Incubation of permeabilized SL vesicles in the presence of 100 μM 5′-guanylamidiophosphate, Gpp(NH)p, a nonhydrolyzable analogue of GTP, resulted in a shift in [3H]PGE2 binding from two sites, one of high affinity (KD = 0.018 ± 0.003 nM) comprising 7.7% of the total available binding sites and one of lower affinity (KD = 1.9 ± 0.7 nM) to one site of intermediate affinity (KD = 0.52 ± 0.01 nM) without a significant change in the total number of PGE2 binding sites. A shift from two binding sites to one binding site in the presence of Gpp(NH)p was also observed for [3H]dihydroalprenolol binding to permeabilized cardiac SL. When permeabilized SL vesicles were pretreated with activated pertussis toxin, ADP-ribosylation of a 40- to 41-kDa protein corresponding to Gi was observed. ADP-ribosylation of SL resulted in a shift in [3H]PGE2 binding to one site of intermediate affinity without significantly changing the number of binding sites. In alamethicin permeabilized SL vesicles, 1 nM PGE2 significantly decreased (30%) adenylyl cyclase activity. Pretreatment with activated pertussis toxin overcame the inhibitory effects of PGE2. These results demonstrate that the cardiac PGE2 receptor is coupled to adenylyl cyclase via a pertussis toxin sensitive Gi protein. They also demonstrate that the interaction of this Gi protein with the PGE2 receptor is important in the regulation of PGE2 binding to its receptor.Key words: prostaglandin E2, sarcolemma, heart, adenylyl cyclase, G protein.

Tonic inhibition of renal response to vasopressin by a pertussis toxin substrate

1988 ◽  
Vol 255 (6) ◽  
pp. F1107-F1115
Author(s):  
W. B. Jeffries ◽  
R. Fallet ◽  
G. D. Gong ◽  
P. Van Dreal ◽  
W. A. Pettinger
Keyword(s):  
Camp Accumulation ◽  
Guanine Nucleotide Binding Protein ◽  
Renal Response ◽  
Collecting Tubule ◽  
Adenylate Cyclase Stimulation ◽  
V2 Receptor ◽  
Guanine Nucleotide Binding

The putative role of the inhibitory guanine nucleotide binding protein (Gi) in modulating the renal response to vasopressin was investigated using islet activating protein (IAP). IAP treatment in rats in vivo abolished the capacity of alpha 2-adrenoceptors to reverse vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in microdissected cortical collecting tubule (CCT) segments. IAP pretreatment also caused a marked upward shift in the dose-response curve of vasopressin (10(-10) to 10(-4) M)-induced cAMP accumulation. Augmentation of the response to vasopressin in rat CCT was dependent on the in vivo dose of IAP and paralleled the loss in alpha 2-adrenoceptor responsiveness. In the isolated perfused kidney the antinatriuretic and antidiuretic effects of the V2-receptor agonist desamino-8-D-arginine vasopressin (DDAVP) (1 pM) were enhanced following IAP pretreatment. alpha 2-Adrenoceptor stimulation (30 nM epinephrine) inhibited the renal effects of DDAVP (1 pM) in kidneys from control but not IAP-pretreated rats. Interestingly, IAP pretreatment alone caused increased urine flow rate and enhanced excretion of sodium and chloride without affecting potassium excretion or renal hemodynamics in vitro. Our results suggest that an IAP substrate, probably Gi, 1) is required for signal transduction by renal alpha 2-adrenoceptors, 2) may tonically modulate the response to vasopressin in the CCT but not of parathyroid hormone in the proximal convoluted tubule, and 3) participates in renal water and electrolyte reabsorption independent of exogenous adenylate cyclase stimulation.

Pertussis toxin inhibition of T-cell hybridoma invasion is reversed by manganese-induced activation of LFA-1

1994 ◽  
Vol 107 (3) ◽  
pp. 551-559
Author(s):  
G. La Riviere ◽  
J.W. Klein Gebbinck ◽  
M.H. Driessens ◽  
E. Roos
Keyword(s):  
T Cell ◽  
G Protein ◽  
Pertussis Toxin ◽  
Hybridoma Cells ◽  
Autocrine Motility Factor ◽  
Motility Factor ◽  
Serum Component ◽  
Extracellular Factor

Pertussis toxin (PT) inhibits invasiveness of T-cell hybridomas in vitro and metastasis formation in vivo. We present evidence for the hypothesis that PT interferes with functional activation of LFA-1. Invasion by TAM2D2 T-cell hybridoma cells of fibroblast monolayers was completely blocked by PT pretreatment, but the cells regained invasiveness in the presence of Mn2+, which activates LFA-1. This invasion was blocked by anti-LFA-1 mAb, and Mn2+ did not stimulate invasiveness of LFA-1-deficient TAM2D2 mutants. TAM2D2 cells did not adhere to surfaces coated with the LFA-1 counterstructure ICAM-1, but Mn2+ induced adhesion. Hence, LFA-1 on TAM2D2 cells requires activation before it can participate in the invasion process. The hypothesis is further supported by the slightly different results obtained with the TAM8C4 T-cell hybridoma. PT inhibited invasion strongly but not completely. This reduced invasion was increased by Mn2+. TAM8C4 cells did adhere to ICAM-1, but Mn2+ enhanced adhesion. Thus, part of LFA-1 on TAM8C4 cells is constitutively active, allowing for some PT-insensitive invasion, but further activation is required for optimal adhesion and invasion. PT blocks G-protein-mediated signals, suggesting that an extracellular factor is involved. This is not a serum component or an autocrine motility factor, since the PT effect was serum-independent, and PT did not inhibit motility. Therefore, it is probably produced by the fibroblasts, and either secreted or associated with the cell surface. These results are in line with the hypothesis that a fibroblast constituent activates LFA-1 via a PT-sensitive G-protein and thus stimulates invasion of T-cell hybridomas into the fibroblast monolayer.

scholarly journals Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms

1989 ◽  
Vol 260 (2) ◽  
pp. 427-434 ◽  
Author(s):  
K R McLeish ◽  
P Gierschik ◽  
T Schepers ◽  
D Sidiropoulos ◽  
K H Jakobs
Keyword(s):  
Cholera Toxin ◽  
G Protein ◽  
Leukotriene B4 ◽  
Alpha Subunit ◽  
Gtpase Activity ◽  
Protein Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Gtp Hydrolysis ◽  
Fmlp Receptor ◽  
Guanine Nucleotide Binding

Differentiated HL-60 cells were found to respond to the chemoattractants leukotriene B4 (LTB4) and N-formylmethionyl-leucyl-phenylalanine (FMLP), in a manner similar to neutrophils. Membranes of myeloid differentiated HL-60 cells were used (a) to examine the ability of LTB4 receptors to interact with a guanine-nucleotide-binding protein (G-protein), and (b) to compare this G-protein with that which is coupled to the FMLP receptor. LTB4 stimulated a dose-dependent increase in GTP hydrolysis and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) binding, demonstrating that LTB4 receptors on HL-60 cells are coupled to a G-protein. Both pertussis toxin and cholera toxin inhibited stimulation of GTPase activity and GTP[S] binding by either LTB4 or FMLP, indicating that both receptors are coupled to a G-protein containing a 40 kDa alpha-subunit. That the two receptors share a common G-protein was shown by FMLP enhancement of cholera-toxin-induced inhibition of GTPase activity stimulated by either FMLP or LTB4. However, LTB4 did not enhance cholera-toxin-induced inhibition of GTPase activity, suggesting that the receptors interacted differently with this G-protein. This difference was confirmed by showing that FMLP, but not LTB4, stimulated receptor-specific [32P]ADP-ribosylation of the 40 kDa alpha-subunit. Concentrations of LTB4 and FMLP which produced maximal responses produced enhanced stimulation in both assays. This additive effect was not abolished by inactivation of up to 80% of G-protein activity by N-ethylmaleimide or cholera toxin. We conclude that LTB4 and FMLP receptors in HL-60 cells are coupled to a common G-protein. The receptor-G-protein interaction is different for the two receptors, and G-proteins not coupled to both receptors may account for the additive response.

scholarly journals The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol

2006 ◽  
Vol 74 (4) ◽  
pp. 2259-2267 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Danielle Sentz ◽  
Randall K. Holmes
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Toxic Activity ◽  
Hydrophobic Domain ◽  
Vesicular Traffic ◽  
Adp Ribosylation ◽  
Quality Control Mechanism ◽  
Adp Ribosylation Factor

ABSTRACT Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsα, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A13 subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA13 subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A13 subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A13 subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A13 subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.

scholarly journals Gαi1and Gαi3regulate macrophage polarization by forming a complex containing CD14 and Gab1

2015 ◽  
Vol 112 (15) ◽  
pp. 4731-4736 ◽  
Author(s):  
Xianjing Li ◽  
Duowei Wang ◽  
Zhen Chen ◽  
Ermei Lu ◽  
Zhuo Wang ◽  
...  
Keyword(s):  
Binding Protein ◽  
Macrophage Polarization ◽  
Growth Factor Receptor ◽  
Guanine Nucleotide Binding Protein ◽  
Akt Activation ◽  
Proteasome Pathway ◽  
Guanine Nucleotide Binding ◽  
Underlying Mechanisms

Heterotrimeric G proteins have been implicated in Toll-like receptor 4 (TLR4) signaling in macrophages and endothelial cells. However, whether guanine nucleotide-binding protein G(i) subunit alpha-1 and alpha-3 (Gαi1/3) are required for LPS responses remains unclear, and if so, the underlying mechanisms need to be studied. In this study, we demonstrated that, in response to LPS, Gαi1/3form complexes containing the pattern recognition receptor (PRR) CD14 and growth factor receptor binding 2 (Grb2)-associated binding protein (Gab1), which are required for activation of PI3K-Akt signaling. Gαi1/3deficiency decreased LPS-induced TLR4 endocytosis, which was associated with decreased phosphorylation of IFN regulatory factor 3 (IRF3). Gαi1/3knockdown in bone marrow-derived macrophage cells (Gαi1/3KD BMDMs) exhibited an M2-like phenotype with significantly suppressed production of TNF-α, IL-6, IL-12, and NO in response to LPS. The altered polarization coincided with decreased Akt activation. Further, Gαi1/3deficiency caused LPS tolerance in mice. In vitro studies revealed that, in LPS-tolerant macrophages, Gαi1/3were down-regulated partially by the proteasome pathway. Collectively, the present findings demonstrated that Gαi1/3can interact with CD14/Gab1, which modulates macrophage polarization in vitro and in vivo.

scholarly journals Identification of Binding Proteins for TSC22D1 Family Proteins Using Mass Spectrometry

2021 ◽  
Vol 22 (20) ◽  
pp. 10913
Author(s):  
Ryouta Kamimura ◽  
Daisuke Uchida ◽  
Shin-ichiro Kanno ◽  
Ryo Shiraishi ◽  
Toshiki Hyodo ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Binding Proteins ◽  
Guanine Nucleotide ◽  
Binding Assays ◽  
Guanine Nucleotide Binding Protein ◽  
Cell Systems ◽  
Guanine Nucleotide Binding

TSC-22 (TGF-β stimulated clone-22) has been reported to induce differentiation, growth inhibition, and apoptosis in various cells. TSC-22 is a member of a family in which many proteins are produced from four different family genes. TSC-22 (corresponding to TSC22D1-2) is composed of 144 amino acids translated from a short variant mRNA of the TSC22D1 gene. In this study, we attempted to determine the intracellular localizations of the TSC22D1 family proteins (TSC22D1-1, TSC-22 (TSC22D1-2), and TSC22(86) (TSC22D1-3)) and identify the binding proteins for TSC22D1 family proteins by mass spectrometry. We determined that TSC22D1-1 was mostly localized in the nucleus, TSC-22 (TSC22D1-2) was localized in the cytoplasm, mainly in the mitochondria and translocated from the cytoplasm to the nucleus after DNA damage, and TSC22(86) (TSC22D1-3) was localized in both the cytoplasm and nucleus. We identified multiple candidates of binding proteins for TSC22D1 family proteins in in vitro pull-down assays and in vivo binding assays. Histone H1 bound to TSC-22 (TSC22D1-2) or TSC22(86) (TSC22D1-3) in the nucleus. Guanine nucleotide-binding protein-like 3 (GNL3), which is also known as nucleostemin, bound to TSC-22 (TSC22D1-2) in the nucleus. Further investigation of the interaction of the candidate binding proteins with TSC22D1 family proteins would clarify the biological roles of TSC22D1 family proteins in several cell systems.

scholarly journals Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3710-3718 ◽  
Author(s):  
Ikuo Uchida ◽  
Ryoko Ishihara ◽  
Kiyoshi Tanaka ◽  
Eiji Hata ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Salmonella Enterica ◽  
Phage Type ◽  
Chinese Hamster ◽  
Adp Ribosylation ◽  
Dependent Modification

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB – encoded by a prophage in S. Typhimurium DT104 – are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [32P]NAD and ArtA, and the label was released by HgCl2, which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA6Arg-Ala and ArtA115Glu-Ala, in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

Sign in / Sign up

Export Citation Format

Share Document