Cholera toxin treatment produces down-regulation of the alpha-subunit of the stimulatory guanine-nucleotide-binding protein (Gs) (original) (raw)
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European Journal of Pharmacology: Molecular Pharmacology, 1990
Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [32P]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of G~a. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD + as substrate. However, immunological studies with anfi-peptide antisera which identify all forms of Gsa demonstrated that concentrations of this polypeptide v'ere now substantially reduced in ihe membranes. This effect was specific for Gsa as neither the a-subunits of the pertussis toxin-sensitive G-proteins G~2 and Gi3, nor the ,8 subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP dbosylation did not cause the downregulation of G~a nor of the a-subunits of G~2 or G,3 although it did cause ADP ribosylation of the entire complement of both Gi2 and GI3 in the membranes. Despite the reduction in levels of immunoreactive Gsa from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted. G-proteins; Downregulation; Cholera toxin: Pertussis toxin
Molecular Endocrinology, 1987
ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg 2+ , and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg 2+. GTP analogs (GTP7S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg 2+ , and GTP or GTP analogs such as GTP7S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gj) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively effected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP0S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25-to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.
Molecular Endocrinology, 1987
ADP ribosylation of membranes by pertussis toxin (PT) and cholera toxin (CT) was studied as a function of addition of ATP, various guanine nucleotides, Mg 2+ , and inorganic phosphate (Pi). ADP ribosylation of a 40 kilodalton (kDa) band by PT is markedly enhanced by ATP and GTP and is strongly inhibited by Pi or Mg 2+. GTP analogs (GTP7S and GMP-adenyl-5'-yl imidodiphosphate) were less effective. In contrast, ADP ribosylation of two substrates for CT (of 42 and 50 kDa) is stimulated by Pi, Mg 2+ , and GTP or GTP analogs such as GTP7S, but is unaffected by ATP. These stimulatory conditions correlate well with GTP-mediated activation of stimulated nucleotide-binding regulatory component of adenyl cyclase. Optimal conditions for ADP ribosylation by PT do not correlate simply with conditions thought to lead to stabilization of an inactive form of inhibitory nucleotide-binding regulatory component of adenyl cyclase (Gj) or Gi-like protein; rather, the data suggest the involvement of both a stimulatory nucleotide site on PT (positively effected by either ATP or GTP) and a stabilizing site on the PT substrate (affected by GDP, GDP0S, or GTP). Treatment of membranes with Lubrol PX increased ADP ribosylation by PT by as much as 25-to 30-fold, but inhibited the action of CT. Using defined conditions for ADP ribosylation by PT and CT, distinct labeling patterns were observed in thyroid, brain, corpus luteum, liver, heart, and erythrocytes membranes. All membranes were more intensely labeled by PT rather than CT.
Proceedings of the National Academy of Sciences, 1988
Platelet cytosolic 44-kDa protein is a substrate of cholera toxininduced ADP-ribosylation and is not recognized by antisera against the a subunit of the stimulatory guanine nucleotide-binding regulatory protein (ioprost) ABSTRACT ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADPribosylates a 41-kDa polypeptide. Two antisera against the a subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42-and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with
Biochimica et Biophysica Acta (BBA) - General Subjects, 1984
We have found in water-soluble extracts of rat liver (and RL-PR-C cloned rat hepatocytes), prepared in the absence of detergent, a factor that markedly enhances basal, isoproterenol and cholera toxin activation of adenylate cyclase of rigorously washed hepatocyte membranes, in the absence of added GTP. The factor, which has characteristics of a protein with an Mr of approx. 35000, has been fractionated from crude cytosol by gel filtration, and then further purified over 50-fold by sequential ion-exchange chromatography. The site of action of the protein appears to be at the level of the guanine nucleotide regulatory (G) protein of the plasma membrane adenylate cyclase complex, as the factor, cooperatively with GTP, also permitted cholera toxin to ADP-ribosylate (from 32P-labeled NAD) two integral membrane proteins that migrated on SDS-polyacrylamide gel electrophoresis gels with the mobilities (Mr approx. 46 000 and 48 000) generally observed for the guanine nucleotide regulator protein subunits. In this system, isoproterenol did not stimulate ADP-ribosylation, in either the presence or absence of the liver protein factor.
Molecular and Cellular Biochemistry, 1994
ADP-ribosylation factors (ARFs) comprise a family of ~20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.
Journal of Clinical Investigation, 1973
enterotoxin inhibits the antigeninduced, IgE-mediated release of histamine from human leukocytes and the lysis of allogeneic mastocytoma cells by splenic lymphocytes from specifically immunized mice. This effect requires a prolonged preincubation time of the toxin with the lymphocyte/leukocyte preparations: a demonstrable inhibition requires about 30 min of preincubation and the toxin activity is still increasing at 90-180 min. Cholera enterotoxin also stimulates adenyl cyclase and leads to increased levels of cyclic AMP in the lymphocyte/leukocyte preparations. The concentration of toxin required for both cyclic AMP accumulation and inhibition of the biologic responses is about the same (ca. 1 ng/ml), and the time course of cyclic AMP accumulation parallels the development of inhibitory activity. Both activities, inhibition of the in vitro hypersensitivity reactions and cyclic AMP accumulation, are blocked by cholera antitoxin and by a toxoid prepared from the toxin (choleragenoid). These are specific antagonists in that they do not block the inhibiting activity or rise in cyclic AMP levels caused by other adenyl cyclase stimulators. Because cholera enterotoxin has no known activity other than the stimulation of adenyl cyclase and because of its unusual time course and the availability of specific antagonists, this data considerably This is publication 40 from the
Studies on Nucleotide and Receptor Regulation of GiProteins: Effects of Pertussis Toxin
Molecular Endocrinology, 1989
In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of G ; proteins are affected by PT. Experiments with the G s-deficient Gi-regulated adenylyl cyclase of eye" S49 cell membranes indicated that PT blocks G, activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-G; to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of G,. Experiments in which G, was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of G { , but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes. (Molecular Endocrinology 3: 1115-1124, 1989
Cholera toxin affects nuclear ADP-ribosylation in GH1 cells
Biochemical and Biophysical Research Communications, 1988
Incubation of GHl cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceeded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP. 0 1988 Academic Pre*s, Inc. Covalent modification of proteins by ADP-ribosylation modulates a variety of cellular responses (1,2). Poly(ADP-ribosylation) of nuclear proteins has been implicated in cell differentiation and transformation, gene expression, and DNA replication and repair, all of which might involve changes in the structure and function of chromatin (for review see 1,2). Cholera toxin (ChT) enhances adenylate cyclase activity by catalyzing ADP-ribosylation of the stimulatory guanine nucleotide binding protein of the cyclase (3). Although most of the cellular effects of the toxin are directly or indirectly caused by increases in intracellular cyclic AMP (CAMP) levels, recent studies have also shown that some of the effects of ChT appear to be mediated by CAMP-independent mechanisms (4-6). This raises the possibility that internalization of the catalytic subunit of the toxin may modify cellular functions other than the adenylate cyclase system. In this study we examined whether or not ChT alters the poly(ADP-ribosyl)ation of chromatin proteins. Using pituitary GHI cells we report that the toxin can influence poly(ADP-ribosyl)ation of histones and non-histone chromatin proteins and that this effect is, at least partially, due to cyclic nucleotide regulation. ADP-ribosylation assay. GHI cells were grown in monolayers as previously described (4,7). The nuclei were isolated (7) and incubated for 15 min at 37°C in 0.5 ml of 0.1 M Tris-HCI pH 7.9, 2 mM MgCl2, 1 mM dithiothreitol, containing 5-10 pZi adenylate-[32P]NAD