Toxin-catalyzed ADP-ribosylation Research Articles

Effects of long-term alcohol intake on ADP ribosylation in rat liver plasma membranes.

Mono-ADP-ribosylation, In which the ADP-ribosyl moiety is transferred from NAD to an acceptor protein, is one of the important posttranslational modifications of cellular proteins. Because mounting evidence suggests significant biological roles of this reaction in transmembrane signal transduction and other cell metabolic reactions, we assessed how long-term alcohol intake alters toxin catalyzed- and endogenous mono-ADP-ribosylation in the liver of a rat model. We first found that thiol-preactivated cholera toxin-catalyzed ADP-ribosylation of the alpha-subunit of the stimulatory GTP-binding protein was enhanced after long-term alcohol intake. Unexpectedly, but interestingly, this enhancement was not accompanied by a concomitant increase of cholera toxin-catalyzed stimulation of the adenylate cyclase activity. We also found that long-term alcohol intake remarkably enhanced endogenous mono-ADP-ribosylation of a 58 kDa protein in plasma membranes. Thus, long-term alcohol intake stimulated endogenous, as well as, toxin-catalyzed mono-ADP-ribosylations. Characterization of the 58 kDa protein may uncover pathophysiological roles of this interesting phenomenon in alcohol-induced liver damage.

Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking

In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertussis toxin (PT). Immunoblot analysis identified the two PT-sensitive target proteins in CHO cells as Gi-2 and Gi-3. In this in vitro ADP-ribosylation assay, the ability of PT and ADP-ribosylate Gi-2 and Gi-3 intact CHO cells was not inhibited by cytochalasin D but was inhibited by chloroquine, monensin, and nocodazole. These data implicated the involvement of a cytochalasin D-independent endocytic mechanism, a pH-sensitive step, and microtubules in the ADP-ribosylation of Gi-2 and Gi-3 by PT in intact CHO cells. Preincubation of CHO cells with cycloheximide, at concentrations that reduced protein synthesis by > 95%, did not inhibit the ability of PT to ADP-ribosylate Gi-2 and Gi-3. Control experiments showed that these agents did not affect either the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, Gt, or the binding of PT to CHO cells, except that monensin slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-mediated endocytosis, probably via a cytochalasin D-independent pathway, which involves intracellular trafficking through late endosomes and the Golgi apparatus. An alternative model predicts the presence of a eukaryotic factor that traffics within cells via this pathway and is required by PT to ADP-ribosylate Gi proteins.

Activation of GTP formation and high-affinity GTP hydrolysis by mastoparan in various cell membranes: G-protein activation via nucleoside diphosphate kinase, a possible general mechanism of mastoparan action

The wasp venom, mastoparan (MP), is a direct activator of reconstituted pertussis toxin-sensitive G-proteins and of purified nucleoside diphosphate kinase (NDPK) [E.C. 2.6.4.6.]. In HL-60 membranes, MP activates high-affinity GTPase [E.C. 3.6.1.-] and NDPK-catalyzed GTP formation, but not photolabeling of G-protein α-subunits with GTP azidoanilide; this suggests that the venom activates G-proteins in this system indirectly via stimulation of NDPK. Moreover, the MP analogue, mastoparan 7 (MP 7), is a much more effective activator of reconstituted G-proteins than MP, whereas with regard to NDPK and GTPase in HL-60 membranes, the two peptides are similarly effective. In our present study, we investigated NDPK- and G-protein activation by MP in membranes of the human neuroblastoma cell line, SH-SY5Y, the human erythroleukemia cell line, HEL, the rat basophilic leukemia cell line, RBL 2H3, and the hamster ductus deferens smooth muscle cell line, DDT 1MF-2. All these membranes exhibited high NDPK activities that were increased by MP. Compared to basal GTP formation rates, basal rates of high-affinity GTP hydrolysis in cell membranes were low. MP activated high-affinity GTP hydrolysis in cell membranes but did not enhance incorporation of GTP azidoanilide into G-protein α-subunits. As with HL-60 membranes, MP and MP 7 were similarly effective activators of NDPK and GTPase in SH-SY5Y membranes. Pertussis toxin inhibited MP-stimulated GTP hydrolyses in SH-SY5Y- and HEL membranes, whereas NDPK activations by MP were pertussis toxin-insensitive. Our data suggest that indirect G-protein activation via NDPK is not restricted to HL-60 membranes but is a more general mechanism of MP action in cell membranes. Pertussis toxin-catalyzed ADP-ribosylation of α-subunits may inhibit the transfer of GTP from NDPK to G-proteins. NDPK may play a much more important role in transmembrane signal transduction than was previously appreciated and, moreover, the GTPase of G-protein α-subunits may serve as GDP-synthase for NDPK.

Protein kinase C-dependent modulation of stimulatory guanine nucleotide binding protein of fetal rat skin keratinocytes.

Although the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) has been known to induce heterologous desensitization of the epidermal adenylate cyclase, the precise mechanism of PMA action remains unknown. Effects of PMA on the receptor-G-protein-adenylate cyclase system of fetal rat skin keratinocytes (FRSK) were investigated. Choleratoxin catalysed the ADP ribosylation of 45 kDa and 52 kDa membrane proteins and islet activating protein (IAP) catalysed the ADP ribosylation of a 40 kDa membrane protein. Incubation of FRSK with PMA decreased the cholera toxin-catalysed ADP ribosylation of the membrane protein, but not the IAP-catalysed ADP ribosylation. The effect of PMA on the cholera toxin-catalysed ADP ribosylation was inhibited by the PKC inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine dihydrochloride). 1-Oleoyl-2-acetylglycerol (OAG), a membrane-permeable diacylglycerol analogue, also decreased the cholera toxin-catalysed ADP ribosylation, but 4-0-methyl PMA, a very weak PKC activator, had no effect. Keratinocytes are known to express the guanine nucleotide binding proteins, Gsalpha, Gi2alpha, and Gi3alpha. Immunoblot analysis of the PMA-treated FRSK showed no detectable difference in the amount of Gsalpha, Gi2alpha, Gi3alpha, or the beta subunit of the G-protein. PMA significantly decreased the beta-adrenergic adenylate cyclase response and cholera toxin-induced cyclic AMP accumulation, while it markedly increased forskolin-induced cyclic AMP accumulation. These results indicate that phorbol esters affect the stimulatory guanine nucleotide binding protein (Gs) of FRSK via a PKC-dependent pathway.

Alterations in inotropy, nitric oxide and cyclic GMP synthesis, protein phosphorylation and ADP-ribosylation in the endotoxin-treated rat myocardium and cardiomyocytes.

To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE 1 receptors and stimulatory G proteins

Chronic exposure of all- trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 μM; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 μM) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE, receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE 1 receptor number ( B max) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPγS (100 μM) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of G sα and S49 cyc − reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE 1 receptors and G s in situ by application of a C-terminal anti-G sα antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE 1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available G sα within the PGE 1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 μM)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE 1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.

P-1017 - Assessment of G protein function in experimental diabetic rat hearts by bacterial toxin-catalyzed ADP-ribosylation and by immunoblot analysis

P-1017 - Assessment of G protein function in experimental diabetic rat hearts by bacterial toxin-catalyzed ADP-ribosylation and by immunoblot analysis

Purification and characterization of the G203T mutant alpha i-2 subunit of GTP-binding protein expressed in baculovirus-infected Sf9 cells.

We expressed the Gly203-->Thr (G203T) mutant of Gi2alpha, which was expected to show a dominant-negative phenotype in Gi2-mediated signal transduction, in baculovirus-inefected Sf9 cells and purified the mutant alpha subunit for its characterization. The rate of dissociation of GDP from G203T Gi2alpha was 3- to 4-fold faster than that from wild type Gi2alpha, but their kappacat values for GTP hydrolysis were almost the same. The affinities of the two Gi2alpha proteins for the beta gamma subunits of G proteins to form alpha beta gamma timers, which served as substrates for pertussis toxin-catalyzed ADP-ribosylation, were the same. In marked contrast, G203T Gi2alpha was unable to form a tight complex with a non- hydrolyzable analog (GTP[gammaS) of GTP; bound GTP[gammaS] was readily released from the mutant Gi2alpha even in the presence of a high concentration of Mg2+. Its susceptibility to tryptic digestion also revealed that GTP[gammaS]-bound G203T Gi2alpha formed a conformation apparently different from that of the GTP[gammaS]-bound form of wild-type Gi2alpha. Both the G203T and wild-type Gi2alpha proteins were capable of coupling with membrane-bound alpha2-adrenergic receptors, resulting in the formation of receptor-G protein complexes with high affinity for agonists. However, GTP[gammaS]-dependent uncoupling from high-affinity receptors was markedly attenuated in the case of G203T Gi2alpha. Thus, G203T-mutated Gi2alpha had a unique property in terms of coupling to membrane receptors, in addition to the previously expected defect in the active conformation of the GTP-bound form of Gi2alpha.

Mitogen-activated Protein Kinase Activation Requires Two Signal Inputs from the Human Anaphylatoxin C5a Receptor

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.

Chronic Activation of Inhibitory δ‐Opioid Receptors Cross‐Regulates the Stimulatory Adenylate Cyclase‐Coupled Prostaglandin E1 Receptor System in Neuroblastoma × Glioma (NG108‐15) Hybrid Cells

The present article investigates chronic opioid regulation of the stimulatory adenylate cyclase-coupled prostaglandin E1 (PGE1) receptor system in neuroblastoma x glioma (NG108-15) hybrid cells. Persistent activation of delta-opioid receptors by morphine (10 mumol/L; 3 days) substantially down-regulates the number of PGE1 binding sites by approximately 30%, without affecting their affinity. Radioligand binding studies performed in the presence of GTP gamma S (100 mumol/L) further revealed that the remaining PGE1 binding sites are still capable of interacting functionally with their associated stimulatory G proteins, Gs. On the postreceptor level, neither changes in the abundance nor in the intrinsic activity of the alpha subunit of Gs (Gs alpha) were found during the state of opioid dependence, as has been verified by western blot and S49 cyc- reconstitution experiments, respectively. Evaluation of the functional interaction between PGE1 receptors and Gs by means of receptor-stimulated, cholera toxin-catalyzed ADP-ribosylation of Gs alpha revealed a significant increase in the ability of PGE1 receptors to activate Gs alpha (3.3-fold increase in EC50; p < 0.05) in cells chronically exposed to morphine. This effect was completely blocked by coincubation of the cells together with the opiate antagonist naloxone (100 mumol/L; 3 days), whereas precipitation of morphine withdrawal by naloxone (100 mumol/L) had no further effect on sensitization in PGE1 receptor/Gs coupling. These findings provide evidence that the stimulatory adenylate cyclase-coupled PGE1 receptor system represents a potential target of chronic delta-opioid receptor activation in NG108-15 hybrid cells. They further suggest that sensitization in stimulatory signal transduction plays a critical role in the generation of opioid dependence.

Involvement of N-Myristoylation in Monoclonal Antibody Recognition Sites on Chimeric G Protein α Subunits

Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino-terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.

Trypanosoma brucei: Characterization of a Life Cycle Stage-Specific G-Protein

G-proteins have important and diverse roles in cellular signaling in higher eukaryotes but as yet little is known about the involvement of these GTP-binding proteins in protozoan parasites. Bacterial toxin-catalysed ADP-ribosylation, routinely used in the characterisation of G-proteins in higher eukaryotes, was used to determine whether functional G-protein homologues could be detected in Trypanosama brucei. A 43-kDa polypeptide that was ADP-ribosylated in the presence of pertussis toxin was identified in membrane fractions. This polypeptide was also recognised by an antibody generated against a peptide sequence representing a mammalian GOα G-protein subunit. GTP and GTPγS (a nonhydrolysable analogue of GTP) abolished the ADP-ribosylation reaction, indicating that the 43-kDa polypeptide also binds GTP. Furthermore, this T. brucei G-protein is present in bloodstream but not procyclic life cycle stages. Using an in situ GTP binding assay, a further group of small-molecular-size GTP-binding proteins which are present in both bloodstream and procyclic stages have been identified. This demonstration of G-proteins in T. brucei suggests possible intracellular mechanisms that may be involved in the transduction of host or other parasite signals.

Differential activation of G i and G S proteins by E- and I-type prostaglandins in membranes from the human erythroleukaemia cell line, HEL

The group of prostaglandin (PG) E 2- and prostacyclin receptors consists of different subtypes, which exibit different affinities for prostaglandins and synthetic analogues. PGE 2 activates the E-type PG receptor subtypes EP 1, EP 2 and EP 3, whereas the PGE 2 analogue, sulprostone, binds only to the EP 1 and EP 3 receptor subtypes. The stable PGI 2 analogues, iloprost and cicaprost, both activate the PGI 2 receptor (IP) and iloprost, additionally, bind to the EP 1 subtype. Using these subtype-selective PG receptor agonists, we studied the interaction of PG receptor subtypes with G s and G i-type heterotrimeric guanine nucleotide-binding proteins (G proteins) in membranes from the human erythroleukaemia cell line, HEL. Sulprostone stimulated high-affinity GTPase in HEL membranes in a pertussis toxin (PTX)-sensitive manner. In contrast, the stimulations induced by PGE 2, iloprost and cicaprost were only partially inhibited by PTX. PGE 2, sulprostone, iloprost and cicaprost stimulated cholera toxin-catalysed ADP-ribosylation as well as labelling with GTP azidoanilide of membrane proteins comigrating with immunologically identified G i protein α subunits. Furthermore, PGE 2, iloprost and cicaprost enhanced GTP azidoanilide-labelling of G s protein α subunits, whereas sulprostone did not. We suggest that in HEL cells (1) EP 1, and EP 3 receptor subtypes activate G i proteins, that (2) the EP 2 receptor subtype activates G s proteins and that (3) the IP receptor activates both G i and G s proteins.

The inhibition of adenylyl cyclase activity in isolated lung membranes by muscarinic and α-adrenoreptor agonists: Role of G-protein α and βγ sub-units

Forskolin (10 μM) failed to abolish the GppNHp- (0.1 nM) dependent inhibition of adenylyl cyclase activity in isolated membranes. Whilst clonidine (1 μM), an α 2-adrenoceptor agonist, inhibited adenylyl cyclase activity it did not protentiate the GppNHp-dependent inhibition. This indicates that low concentrations of the guanine-nucleotide activate sufficient G i to inhibit adenylyl cyclase activity maximally and that clonidine inhibits this enzyme via a similar route. These data support a role of α i sub-unit inhibition of adenylyl cyclase activity. In contrast, forskolin (10 μM) abolished the GppNHp (0.001–0.1 nM) dependent inhibition of adenylyl cyclase activity in membranes where adenylyl cyclase activity is limited, i.e. where activity has been depleted by approximately 80%. In this case, inhibition of adenylyl cyclase by βγ sub-units is implicated and only becomes evident under these conditions. Adenylyl cyclase is also inhibited by muscarinic receptor agonist, methacholine and by the α 2-adrenoreptor agonists, clonidine and nor-adrenaline. Both classes of agonist also elicit an increase in the cholera toxin-catalysed ADP-ribosylation of the splice variant forms of G s α and of a polypeptide of 41,000 M r. The ADP-ribosylation of the 41,000 M r polypeptide is inhibited by GTP (100 μM) and therefore displays characteristics similar to G i α. Muscarinic receptor and α 2-adrenoceptor agonists appear to inhibit adenylyl cyclase activity in lung membranes predominantly via G i α. Lung expresses both type II and IV adenylyl cyclase which are stimulated by direct interaction with βγ sub-units and this is conditional upon the co-incident activation of G s α. Inhibition of adenylyl cyclase by G i α provides a means by which muscarinic receptor and α 2-adrenoceptor agonists can inhibit these isoforms independently of the βγ sub-unit effects.

Analysis of Pertussis Toxin-Sensitive Receptor: G-Protein Interactions in Native Porcine Endothelial Cells

Endothelium-dependent relaxations evoked by activation of α2-adrenergic and serotonergic receptors are reduced by pertussis toxin, which irreversibly inhibits the activity of certain G-proteins. The present experiments were conducted in order to further characterize the inhibitory effect of the toxin and to directly analyze receptor: G-protein interactions in native porcine endothelial cells. Cell membranes, prepared from freshly harvested endothelial cells, were incubated with pertussis toxin in the presence of 32P-NAD and labelled proteins were separated on SDS-PAGE. Pertussis toxin catalyzed the transfer of 32P-ADP-ribose from NAD to a 40 kD protein. The toxin-catalyzed ADP-ribosylation was not significantly affected by bradykinin but was reduced by serotonin, mastoparan (a direct G-protein activator) or UK 14, 304, an α2-adrenergic agonist. Western blot analysis demonstrated that porcine endothelial cells express Giα-2 and Giα-3 protein. Immunoprecipitation of solubilized, 32P-ADP ribosylated protein ...

Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with nucleoside diphosphate kinase.

The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.

Differential activation of dibutyryl cAMP-differentiated HL-60 human leukemia cells by chemoattractants

Dibutyryl cAMP-differentiated HL-60 human leukemia cells possess receptors for the chemoattractants N- formyl- L-methionyl- L-phenylalanine (fMLP), C5a and leukotriene B 4 (LTB 4). We compared the effects of these chemoattractants in HL-60 membranes and in intact HL-60 cells. fMLP, C5a and LTB 4 stimulated GTP hydrolysis and guanosine 5′- O-[3-thio]triphosphate (GTP[γS]) binding in HL-60 membranes with similar effectiveness and in a pertussis toxin (PTX)-sensitive manner. They also stimulated photolabeling of the α-subunits of the guanine nucleotide-binding proteins (G-proteins), G i2 and G i3 with similar effectiveness. Chloride salts of monovalent cations differentially enhanced and inhibited chemoattractant-induced GTP hydrolyses. C5a was less effective than fMLP in enhancing cholera toxin-catalysed ADP-ribosylation of G iα2 and G iα3, and LTB 4 was ineffective. fMLP was more effective than C5a and LTB 4 in stimulating Ca 2+ influx in HL-60 cells. C5a- and LTB 4-induced rises in cytosolic Ca 2+ concentration ([Ca 2+] i) were PTX-sensitive, whereas the effect of fMLP was partially PTX-insensitive. LTB 4-induced rises in [Ca 2+] i were more sensitive towards homologous desensitization than those induced by C5a, and the effect of fMLP was resistant in this regard. C5a was considerably less effective than fMLP in activating superoxide anion formation and azurophilic granule release, and LTB 4 was ineffective. Our data suggest that fMLP, C5a and LTB 4 effectively activate the G-proteins, G i2 and G i3, in HL-60 cells and that fMLP may additionally activate PTX-insensitive G-proteins. fMLP, C5a and LTB 4 are full, partial and incomplete secretagogues, respectively, and these differences may be due to differences in homologous receptor desensitization and qualitative G i-protein activation.

G-proteins and adenylyl cyclase in ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus)

The involvement of guanine nucleotide-binding regulatory proteins (G-proteins) and adenylyl cyclase in the gonadotropin stimulation of cAMP was investigated using crude membrane fractions from granulosa cells of amago salmon (Oncorhynchus rhodurus) postvitellogenic ovarian follicles. Although cholera toxin-catalyzed ADP ribosylation occurred in 45- and 50-kDa proteins, only the former was recognized by an antibody against the α-subunit of Gs. With pertussis toxin, only the 41-kDa protein was ADP- ribosylated. This 41-kDa protein was recognized by an antibody against the α-subunit of Gi. Partially purified chum salmon gonadotropin (SGA) stimulated adenylyl cyclase activity in crude membrane preparations of granulosa cells only in the presence of pertussis toxin in the incubation medium. Adenosine inhibited adenylyl cyclase in the presence of GTP and pertussis toxin reversed it. Unlike SGA, forskolin, which acts upon adenylyl cyclase without G-protein interaction, markedly stimulated adenylyl cyclase activity in the absence of pertussis toxin. These results provide evidence that both stimulatory (Gs) and inhibitory (Gi) regulation by adenylyl cyclase operates in the granulosa cells of amago salmon postvitellogenic ovarian follicles. It is possible that, although a stimulatory receptor interacts with Gs, its activity is influenced by the functional state of Gi.

Age-related changes in beta-adrenergic neuroeffector systems in the human heart.

Aging decreases cardiac beta-adrenergic responsiveness in model systems and in humans in vivo. The purpose of this study was to comprehensively evaluate the age-related changes in the beta-receptor-G protein-adenylyl cyclase complex in nonfailing human hearts. Twenty-six nonfailing explanted human hearts aged 1 to 71 years were obtained from organ donors and subjected to pharmacological investigation of beta-adrenergic neuroeffector systems. When the population was subdivided into the 13 youngest and 13 oldest subjects, total beta-receptor density assessed by maximum [125I]ICYP binding (beta max) was reduced in older hearts by 37% in left ventricles and 31% in right ventricles (both P < .05), and the downregulation was confined to the beta 1 subtype (r = .78 left ventricle beta 1 density versus donor age). Older donor hearts exhibited a 3- to 4-fold rightward shift of ICYP-isoproterenol (ISO) competition curves and demonstrated 43% fewer receptors in a high-affinity agonist binding state (P < .05). Older hearts exhibited decreased adenylyl cyclase stimulation by ISO, by zinterol (beta 2-agonist), and by the G protein-sensitive probes forskolin, Gpp(NH)p, and NaF. In contrast, there was no change in response to manganese, a specific activator of the adenylyl cyclase catalytic subunit. Toxin-catalyzed ADP ribosylation in membranes prepared from older versus younger hearts revealed a 29% to 30% reduction (P < .05) with cholera toxin (Gs) but no difference with pertussis toxin (Gi). The systolic contractile response of isolated right ventricular trabeculae to ISO was decreased by 46%, with a 10-fold increase in ISO EC50 in older relative to younger donor hearts. There is a profound decrease in cardiac beta-adrenergic responsiveness with aging. This occurs by multiple mechanisms including downregulation and decreased agonist binding of beta 1-receptors, uncoupling of beta 2-receptors, and abnormal G protein-mediated signal transduction.

Protein kinase C-dependent modulation of stimulatory guanine nucleotide binding protein of fetal rat skin keratinocytes

Although the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA) has been known to induce heterologous desensitization of the epidermal adenylate cyclase, the precise mechanism of PMA action remains unknown. Effects of PMA on the receptor-G-protein-adenylate cyclase system of fetal rat skin keratinocytes (FRSK) were investigated. Choleratoxin catalysed the ADP ribosylation of 45 kDa and 52 kDa membrane proteins and islet activating protein (IAP) catalysed the ADP ribosylation of a 40 kDa membrane protein. Incubation of FRSK with PMA decreased the cholera toxin-catalysed ADP ribosylation of the membrane protein, but not the IAP-catalysed ADP ribosylation. The effect of PMA on the cholera toxincatalysed ADP ribosylation was inhibited by the PKC inhibitor, H-7 (1-(5-isoquinolinesulfonyl)-2-methyl piperazine dihydrochloride). 1-Oleoyl-2-acetylglycerol (OAG), a membrane-permeable diacylglycerol analogue, also decreased the cholera toxin-catalysed ADP ribosylation, but 4-O-methyl PMA, a very weak PKC activator, had no effect. Keratinocytes are known to express the guanine nucleotide binding proteins, Gsα, Gi2α and Gi3α. Immunoblot analysis of the PMA-treated FRSK showed no detectable difference in the amount of Gsα, Gi2α, Gi3α or the β subunit of the G-protein. PMA significantly decreased the β-adrenergic adenylate cyclase response and cholera toxin-induced cyclic AMP accumulation, while it markedly increased forskolin-induced cyclic AMP accumulation. These results indicate that phorbol esters affect the stimulatory guanine nucleotide binding protein (Gs) of FRSK via a PKC-dependent pathway.