guanosine-triphosphate and mastoparan

Extracellular signal molecules as diverse as hormones, neurotransmitters and photons use a signal transduction pathway involving a receptor, a G protein and effectors. Compounds that interact directly with G proteins can mimic the receptor-G protein interaction or can block the activation of G proteins by receptors. Several binding sites exist on the G alpha protein that may be exploited for the design of synthetic stimulatory or inhibitory ligands. The effector binding site is regulated by endogenous proteins and appears to be a target for selective exogenous ligands. The GTP binding site presents a large homology within the G protein families and therefore the nucleotide analogs might not be considered as a tool to discriminate between the G protein subclasses. In contrast, different experimental strategies have substantiated the specificity in the interaction between a receptor and a G protein, the receptor binding site of G proteins should be considered as potential drug targets. Drugs interfering with this site such as mastoparan and related peptides, GPAnt-2 and suramin, are lead compounds in the design of selective G protein antagonists. Benzalkonium chloride and methoctramine have agonist or antagonist properties, depending on G protein subtypes. Such compounds would be very useful to delineate the functions of G proteins and G protein-coupled receptors, to understand some side effects of drugs used in therapy and to develop new therapeutic agents.

Topics: Amino Acid Sequence; Animals; Complement C3a; GTP-Binding Protein alpha Subunits, Gs; Guanosine Triphosphate; Hormones; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Neuropeptides; Neurotransmitter Agents; Peptides; Polyamines; Receptors, Drug; Signal Transduction; Wasp Venoms

Recombinant heterotrimeric G-protein α(i1), α(i2) and α(i3) subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β₁γ₂ subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α(i) were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg²⁺, Mn²⁺ inhibited nucleotide binding to all α(i)-heterotrimers studied and accelerated nucleotide release. Mn²⁺ had stabilizing effect on the nucleotide free state of the α(i1) subunit, whereas both Mn²⁺ as well as G-protein activation by mastoparan destabilized the α(i2) subunit.

Topics: Animals; Cell Line; Fluorescence Polarization; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Magnesium; Manganese; Peptides; Protein Multimerization; Protein Stability; Protein Structure, Quaternary; Protein Subunits; Wasp Venoms

Studies demonstrate that cholesterol plays a critical role in the regulation of neurotransmitter release and that secretory vesicle swelling is a requirement for the regulated expulsion of intravesicular contents during cell secretion. In view of this, the involvement of cholesterol in synaptic vesicle swelling was hypothesized and tested in the present study, using isolated synaptic vesicles from rat brain and the determination of their swelling competency in the presence and absence of cholesterol. The involvement of the water channel aquaporin-6 (AQP-6) and proton pump vH(+)-ATPase in GTP-G(alpha o)-mediated synaptic vesicle swelling has been reported previously. Mastoparan, the amphiphilic tetradecapeptide from wasp venom, known to activate the GTPase activity of G(alpha o/i) proteins, stimulates synaptic vesicle swelling in the presence of GTP. In the current study, using nanometer-scale precision measurements of isolated synaptic vesicles, we report for the first time that depletion of cholesterol from synaptic vesicle membrane results in a significant loss of GTP-mastoparan-stimulable synaptic vesicle swelling. In contrast, incorporation of cholesterol into the synaptic vesicle membrane potentiates GTP-mastoparan-stimulable vesicle swelling. Our study further demonstrates that this effect of cholesterol is due, in part, to its involvement in the interactions between AQP-6, vH(+)-ATPase and the GTP-binding G(alpha o) protein at the synaptic vesicle membrane.

Topics: Animals; Aquaporin 6; Brain; Cholesterol; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Microscopy, Atomic Force; Peptides; Proton-Translocating ATPases; Rats; Rats, Sprague-Dawley; Synaptic Vesicles; Wasp Venoms

Muscarinic activation of tracheal smooth muscle (TSM) involves a M(3)AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Galpha(i/o) subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Galpha(q16), whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Galpha(q15/16) stimulated the NPR-GC. Coupling of alpha(q16) to M(3)AChR is supported by MAS decreased [(3)H]QNB binding, being abolished after M(3)AChR-4-DAMP-alkylation. Anti-i(3)M(3)AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i(3)M(3)AChR (M(3)P) was more potent than MAS increasing GTPgamma [(35)S] and decreasing the [(3)H]QNB activities. Coupling between NPR-GC and Galpha(q16) was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Galpha(q16), also showing an immunoreactive heterotrimeric-G-beta-subunit. These data support the existence of a novel transducing cascade, involving Galpha(q16)beta gamma coupling M(3)AChR to NPR-GC.

Topics: Amino Acid Sequence; Animals; Antibodies; Blotting, Western; Cattle; Chromatography, Affinity; Cytoplasm; Enzyme Activation; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine Triphosphate; Guanylate Cyclase; Heterotrimeric GTP-Binding Proteins; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Myocytes, Smooth Muscle; Peptides; Protein Processing, Post-Translational; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Receptors, Atrial Natriuretic Factor; Solubility; Trypsin; Wasp Venoms

Many cells utilize a GTP-dependent pathway to trigger exocytosis in addition to Ca(2+)-triggered exocytosis. However, little is known about the mechanism by which GTP triggers exocytosis independent of Ca(2+). We used dual-color evanescent field microscopy to compare the motion and fusion of large dense core vesicles stimulated by either mastoparan (Mas) in Ca(2+)-free conditions or high K(+) in the presence of Ca(2+). We demonstrate that Mas is hardly effective in triggering the fusion of the predocked vesicles but predominantly mobilizes cytosolic vesicles. In contrast, Ca(2+)-dependent exocytosis is largely due to predocked vesicles. Fusion kinetics analysis and carbon-fiber amperometry reveal that Mas induces a brief 'kiss-and-run' fusion and releases only a small amount of the cargo, whereas Ca(2+) stimulates a more persistent opening of the fusion pore and larger release of the contents. Furthermore, we show that Mas-released vesicles require a much shorter time to reach fusion competence once they approach the plasma membrane. Our data suggest the involvement of different mechanisms not only in triggering and fusion but also in the docking and priming process for Ca(2+)- and GTP-dependent exocytosis.

Topics: Animals; Calcium; Exocytosis; Fluorescent Dyes; Green Fluorescent Proteins; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Membrane Fusion; Microscopy, Fluorescence; PC12 Cells; Peptides; Photochemistry; Rats; Secretory Vesicles; Vesicle-Associated Membrane Protein 2; Wasp Venoms

The peptide toxin mastoparan increased GTP-binding activity of heterotrimeric G proteins in tissues of vertebrate and invertebrate animals, the effect of mastoparan in mussel tissues being less pronounced. The stimulatory effect of mastoparan on GTP binding was not observed after treatment of membranes with pertussis toxin that selectively modulates function of Gi proteins. Activity of mastoparan decreased in the presence of C-terminal peptide 346-355 from the Gi protein alphai2-subunit. Mastoparan dose-dependently decreased the stimulatory effect of hormones on GTP binding in tissues of rats and mussels. The influence of these hormones on the cell is realized via Gi proteins. However, mastoparan did not modulate the effect of Gs protein-activating hormones.

Topics: Amino Acid Sequence; Animals; Bivalvia; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Peptides; Rats; Wasp Venoms

Mastoparan, a hormone receptor-mimetic peptide isolated from wasp venom, stimulates insulin release from pancreatic beta-cells in a Ca(2+)-independent but GTP-dependent manner. In this report, the role of the Rho family GTP-binding protein Cdc42, in the mastoparan stimulus-secretion pathway, was examined. Overexpression of wild-type Cdc42 in beta HC-9 cells, an insulin-secreting mouse-derived cell line, resulted in a 2-fold increase in mastoparan-stimulated insulin release over vector-transfected beta HC-9 cells. This effect was not seen with secretagogues such as glucose that stimulate secretion via Ca(2+)-dependent pathways. GDP/GTP exchange assay data and studies with pertussis (PTX) toxin suggest that mastoparan may work directly to activate Cdc42 and not via PTX-sensitive heterotrimeric GTP-binding proteins. Using bacterial glutathione S-transferase-Cdc42 fusion proteins and co-immunoprecipitation and transient transfection studies, Cdc42 was shown to be an upstream regulator of the exocytotic protein, syntaxin. These results suggest that the GTP-dependent signal underlying the mastoparan effect acts at a "distal site" in stimulus-secretion coupling on one of the SNARE proteins essential for exocytosis. In vitro binding assays, using purified Cdc42 and syntaxin proteins, show that Cdc42 mediates the GTP signal through an indirect association with syntaxin. The H3 domain at the C-terminus of syntaxin, which participates in the formation of the ternary SNARE complex with the core proteins, SNAP-25 and synaptobrevin, is also required for the association with Cdc42. Thus, these studies indicate that Cdc42 could be a putative GTP-binding protein thought to be involved in the mastoparan-stimulated GTP-dependent pathway of insulin release.

Topics: Animals; cdc42 GTP-Binding Protein; Cell Line; Guanosine Diphosphate; Guanosine Triphosphate; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Peptides; Qa-SNARE Proteins; Wasp Venoms

Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein.. Acidification of the endosomal compartment is a prerequisite for intracellular processing of endocytosed complexes. Endosomal acidification is accomplished by an H+-ATPase, in parallel with a Cl- conductance. Previous studies from our laboratory have demonstrated that endosomal acidification is modulated by a pertussis toxin-sensitive mechanism, suggesting that endosomal acidification could be regulated through a self-contained signal transduction pathway. This study was designed to test this hypothesis using the transferrin receptor as a model.. Synthetic peptides corresponding to a region of the cytosolic domain of the transferrin receptor and containing a KPKR sequence were used to stimulate endosomal acidification in a G-protein-dependent manner.. Peptides activated the Gi, as evidenced by stimulation of the rate of GTPgammaS binding. A transferrin receptor peptide that lacked the KPKR sequence did not stimulate endosomal acidification and failed to promote GTPgammaS binding to Gi proteins.. These results demonstrate that regulation of endosomal acidification can be achieved, in part, through a Gi-mediated signal transduction pathway. These findings suggest that regulation of endosomal acidification through such a pathway may facilitate intracellular processing of the transferrin receptor.

Topics: Amino Acid Sequence; Animals; Endosomes; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kidney Cortex; Molecular Sequence Data; Peptide Fragments; Peptides; Proton-Translocating ATPases; Rabbits; Receptors, Transferrin; Sequence Homology, Amino Acid; Signal Transduction; Wasp Venoms

Peptitergent PD1 shows complex effects on GTPase activity of rat brain cortical membranes: inhibition in the presence of lower concentrations of GTP and activation at a higher concentration, above 0.5 microM, of GTP. Its effect is dose dependent and is characterized by an EC50 of 1.8 +/- 0.2 microM and a Hill coefficient of 1.6 +/- 0.3, and it increases both Km and Vmax of the GTP hydrolysis. PD1 that was unable to solubilize G-proteins from the membranes probably acts on them by direct binding near the C-terminal alpha-helical region of the Galpha subunit, similarly to mastoparan.

Topics: Amino Acid Sequence; Animals; Cerebral Cortex; Detergents; GTP Phosphohydrolases; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Intercellular Signaling Peptides and Proteins; Membrane Proteins; Micelles; Models, Chemical; Molecular Sequence Data; Peptides; Protein Structure, Secondary; Rats; Rats, Wistar; Solubility; Wasp Venoms

The chimeric peptide galparan, composed of galanin (1-13) in the N-terminus and mastoparan in the C-terminus, was recently designed and synthesized. The effect of galparan on GTPase activity of rat brain cortical membranes was studied in comparison with the effect of mastoparan and galanin. GTPase was activated by mastoparan but it was noncompetitively inhibited by galparan, while no effect of galanin and galanin (1-13) was found in this tissue. EC50 of 12.1 +/- 2.1 microM and Hill coefficient of 2.1 +/- 0.6 was calculated for galparan from a dose-response curve and Ki of 19.1 +/- 0.3 microM was obtained by fitting the experimental data to the Michaelis-Menten equation valid in the presence of noncompetitive inhibitor. Mastoparan reversed the effect of galparan in a fully competitive manner while benzalkonium chloride did not prevent the inhibition of GTPase activity by galparan. Pertussis-toxin-catalyzed ribosylation of G proteins from rat brain cortical membranes resulted in 15% lower basal GTPase activity of our preparation but did not alter the parameters of the dose-response curve for galparan inhibition. The rate of GTP gamma S binding to G proteins from rat brain cortical membranes was not influenced by galparan. CD spectra revealed predominantly antiparallel beta-structure and unordered secondary structure of galparan in the buffer solution, while in the presence of lipid vesicles it adopted a higher amount of alpha-helix. Critical micelle concentration of galparan in buffer solution of 22 microM was determined. It is suggested that the reversal of GTPase activation by mastoparan to inhibition by galparan is due to different loci of action of these two peptides on G proteins.

Topics: Animals; Cell Membrane; Cerebral Cortex; Galanin; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Peptides; Pertussis Toxin; Protein Binding; Rats; Rats, Wistar; Recombinant Fusion Proteins; Virulence Factors, Bordetella; Wasp Venoms

Mastoparan, a tetradecapeptide component of wasp venom, activates heterotrimeric G-proteins and stimulates exocytosis in several cell types, including the pancreatic beta-cell. In this study, its effects on insulin secretion were assessed in both rat and human pancreatic islets, along with the ability of glucose and alpha-ketoisocaproate (alpha-KIC) to augment mastoparan-stimulated release. In Ca2+-free Krebs-Ringer bicarbonate buffer containing 2.8 mmol/l glucose, 20 micromol/l mastoparan stimulated insulin secretion 12- and 14-fold in rat and human islets, respectively. The inactive analog mastoparan-17 had no effect on release. Under the same Ca2+-free conditions, 11.1 mmol/l glucose had no effect on insulin release alone, but augmented mastoparan-stimulated release by 74% in both rat and human islets. Stimulation of release by mastoparan and augmentation of release by glucose were unaffected by treatment with pertussis toxin. The effect of cellular GTP depletion on the mastoparan stimulation of release and augmentation by alpha-KIC was studied by culturing rat islets in the presence of 25 microg/ml mycophenolic acid for 20 h. In the control islets, alpha-KIC augmented mastoparan-stimulated insulin release by 80%. In the GTP-depleted rat islets, mastoparan-stimulated insulin release was not changed, while the augmentation by alpha-KIC was eliminated. Mannoheptulose completely blocked the augmentation by glucose. In conclusion, mastoparan stimulates insulin release by activation of a signal transduction pathway that can be augmented by nutrients such as glucose and alpha-KIC. Nutrient augmentation of this pathway is heavily dependent on GTP.

Topics: Adenosine Triphosphate; Animals; Calcium; Colforsin; Glucose; Guanosine Triphosphate; Humans; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Keto Acids; Male; Mannoheptulose; Organ Culture Techniques; Peptides; Pertussis Toxin; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetradecanoylphorbol Acetate; Virulence Factors, Bordetella; Wasp Venoms

We investigated whether the specific binding or labeling of 125I-omega-CgTX on crude membranes from chick whole brain was affected when endogenous GTP binding protein (G protein) was activated by GTP analogues, mastoparan (MP) and aluminum fluoride (AIF4-; AICl3 + NaF). Both GTPgammaS and Gpp(NH)p attenuated the inhibitory effect of selective N-type Ca channel inhibitors such as aminoglycoside antibiotics (AGs) or dynorphine (1-13)(Dyn) on specific 125I-omega-CgTX binding in a dose-dependent manner. On the other hand, the inhibitory effects of the divalent metal cations Cd2+, Co2+, Mg2+ and Mn2- on such binding were not attenuated by GTPgammaS. MP and AIF4- also attenuated the inhibitory effect of Neo on this binding similar to GTPgammaS. The attenuating effect of MP was enhanced by the presence of Mg2+ in a dose-dependent manner. However, GTP analogues, MP and AIF4-, did not affect binding or labeling without AGs or Dyn. GTPgammaS, MP and AIF4- also attenuated the specific labeling of a 215-kDa band in crude membranes with 125I-omega-CgTX using the cross-linker DSS (non-reduced condition) in the presence of Neo. These results indicate that there are direct or indirect relationships between N-type Ca channels and G proteins via binding sites for AGs or MP.

Topics: Aluminum Compounds; Animals; Brain; Chickens; Fluorides; GTP-Binding Proteins; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Iodine Radioisotopes; omega-Conotoxin GVIA; Peptides; Wasp Venoms

1. The effects of mastoparan, a wasp venom toxin, on GTP hydrolyzing activity were examined in rat brain membranes. 2. Mastoparan inhibited the low-affinity GTPase activity, defined as the amount of 32Pi released from 0.3 microm [gamma-32P]-GTP in the presence of 100 microM unlabelled GTP, in a concentration-dependent manner. This inhibitory effect of mastoparan on low-affinity GTPase activity was diminished by increasing concentrations of UDP and was completely attenuated at 20 mM, indicating that activation of nucleoside diphosphokinase (NDPK) is inolved in the phenomenon. 3. In the presence of 20 mM UDP, mastoparan stimulated the high-affinity GTPase activity by increasing the Vmax value without affecting the apparent K(M) for GTP. Mastoparan-stimulated high-affinity GTPase activity was apparent at concentrations higher than 1 microM, in a concentration-dependent manner, but without saturation even at 100 microM. 4. Mastoparan-induced high-affinity GTPase activity showed a characteristic sensitivity to MgCl2, quite different from that seen in L-glutamate-stimulated activity, a representative of receptor-mediated G-protein activation. 5. There appeared to be a simple additive interaction between mastoparan- and L-glutamate-stimulated high-affinity GTPase activities, indicting that distinct pools of G-proteins are involved in receptor-independent and receptor-mediated G-protein activation. 6. These results suggest that G-proteins in brain membranes are functionally altered by mastoparan through multiple mechanisms of action and that the mastoparan-induced, direct G-protein activating process lacks a synergistic or antagonistic interaction with an agonist-induced, receptor-mediated activation of G-proteins.

Topics: Animals; Brain; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Hydrolysis; Intercellular Signaling Peptides and Proteins; Magnesium; Male; Peptides; Rats; Rats, Sprague-Dawley; Wasp Venoms

The gamma subunits of trimeric G-proteins (gamma1, gamma2, gamma5, and gamma7 isoforms) were found to be methylated at their carboxyl termini in normal rat islets, human islets and pure beta [HIT-T15] cells. Of these, GTPgammaS significantly stimulated the carboxyl methylation selectively of gamma2 and gamma5 isoforms. Exposure of intact HIT cells to either of two receptor-independent agonists--a stimulatory concentration of glucose or a depolarizing concentration of K+--resulted in a rapid (within 30 s) and sustained (at least up to 60 min) stimulation of gamma subunit carboxyl methylation. Mastoparan, which directly activates G-proteins (and insulin secretion from beta cells), also stimulated the carboxyl methylation of gamma subunits in intact HIT cells. Stimulatory effects of glucose or K+ were not demonstrable after removal of extracellular Ca2+ or depletion of intracellular GTP, implying regulatory roles for calcium fluxes and GTP; however, the methyl transferase itself was not directly activated by either. The stimulatory effects of mastoparan were resistant to removal of extracellular Ca2+, implying a mechanism of action that is different from glucose or K+ but also suggesting that dissociation of the alphabetagamma trimer is conducive to gamma subunit carboxyl methylation. Indeed, pertussis toxin also markedly attenuated the stimulatory effects of glucose, K+ or mastoparan without altering the rise in intracellular calcium induced by glucose or K+. Glucose-induced carboxyl methylation of gamma2 and gamma5 isoforms was vitiated by coprovision of any of three structurally different cyclooxygenase inhibitors. Conversely, exogenous PGE2, which activates Gi and Go in HIT cells and which thereby would dissociate alpha from beta(gamma), stimulated the carboxyl methylation of gamma2 and gamma5 isoforms and reversed the inhibition of glucose-stimulated carboxyl methylation of gamma subunits elicited by cyclooxygenase inhibitors. These data indicate that gamma subunits of trimeric G-proteins undergo a glucose- and calcium-regulated methylation-demethylation cycle in insulin-secreting cells, findings that may imply an important role in beta cell function. Furthermore, this is the first example of the regulation of the posttranslational modification of G-protein gamma subunits via nonreceptor-mediated activation mechanisms, which are apparently dependent on calcium influx and the consequent activation of phospholipases releasing arachidonic acid.

Topics: 3-O-Methylglucose; Acetylcysteine; Animals; Calcium; Cells, Cultured; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Glucose; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Male; Methylation; Mycophenolic Acid; Peptides; Pertussis Toxin; Potassium; Protein Methyltransferases; Rats; Rats, Sprague-Dawley; S-Adenosylmethionine; Virulence Factors, Bordetella; Wasp Venoms

Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.

Topics: Amides; Amines; Animals; Bucladesine; Cell Line; Cell Membrane; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; HL-60 Cells; Humans; Indicators and Reagents; Intercellular Signaling Peptides and Proteins; Macromolecular Substances; Molecular Structure; Peptides; Pertussis Toxin; Recombinant Proteins; Spodoptera; Structure-Activity Relationship; Transfection; Virulence Factors, Bordetella; Wasp Venoms

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 alpha-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, DDT1MF-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 alpha-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 alpha-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 alpha-subunits may serve as GDP-synthase for NDPK.

Topics: Animals; Cell Line; Cell Membrane; Cricetinae; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Erythroblastic, Acute; Neuroblastoma; Nucleoside-Diphosphate Kinase; Peptides; Pertussis Toxin; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

The mechanisms underlying mastoparan-induced elevation of the intracellular free calcium concentration ([Ca2+]i) were investigated in the insulin-secreting cell lines RINm5F and HIT. In both cell types, micromolar concentrations of mastoparan induced a prompt increase of [Ca2+]i, measured as an increase in fura-2 fluorescence. This response was dependent on extracellular calcium entry and was suppressed by organic calcium channel blockers; the increase of [Ca2+]i caused by high glucose concentrations or tolbutamide was not enhanced by mastoparan. These data indicate the involvement of voltage-dependent calcium channels and suggest that depolarization, rather than a direct effect on the channels, mediates the response to mastoparan. This proposition was supported by the observation that whole-cell calcium currents measured using the nystatin-permeabilized patch technique were not affected by mastoparan. Mastoparan-induced depolarization was observed using the potentiometric indicator bis-oxonol, and it was shown not to be additive with the depolarization induced by high glucose concentrations or tolbutamide. The mechanism underlying mastoparan-induced depolarization was identified in single-channel patch-clamp experiments, where it was shown that mastoparan caused closure of ATP-sensitive potassium channels [K(ATP) channels] in cell-attached and excised membrane patches. Responsiveness to mastoparan in excised patches demonstrated the membrane-delimited character of K(ATP) channel inhibition. The observation that the response persisted in the absence of exogenous GTP and in the presence of 250 microM GDP or guanosine-5'-O-(2-thio)diphosphate suggested that this effect is not mediated via enhancement of G protein activity. Partial suppression of channel activity by mastoparan did not prevent the action of tolbutamide, which fully suppressed the remaining activity in excised patches. In summary, the increase of [Ca2+]i in the insulin-secreting tumor cell lines RINm5F and HIT in response to mastoparan is mediated via G protein-independent suppression of K(ATP) channel activity, cell depolarization, and activation of voltage-dependent calcium channels.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Guanosine Triphosphate; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Membrane Potentials; Peptides; Potassium Channel Blockers; Rats; Tumor Cells, Cultured; Wasp Venoms

We have put forward the hypothesis that lipopeptides (LPs) activate GTP hydrolysis by Gi-proteins in HL-60 membranes via activation of nucleoside diphosphate kinase (NDPK) as does mastoparan (MP). Therefore, we compared the effects of LPs and MP on NDPK- and GTPase activation in HL-60 membranes. In native membranes, LPs effectively activated GTP hydrolysis and moderately activated GTP formation. In solubilized membranes, the effect of LPs on GTP formation was enhanced whereas the one on GTP hydrolysis was abolished. The NDPK substrate GDP enhanced the relative stimulatory effect of LPs and MP on GTP hydrolysis in HL-60 membranes in the absence of a NTP-regenerating system. A NTP-regenerating system abrogated the potentiating effect of GDP on MP-action, whereas the effect on LP-stimulated GTP-hydrolysis was enhanced. Our data show that LPs activate NDPK in HL-60 membranes and that this activation may account for their G-protein-stimulatory activity. Membrane solubilization may impair the transfer of GTP from NDPK to Gi-protein alpha-subunits and subsequent GTP hydrolysis, whereas GTP formation remains intact, augmenting the effect of LPs on the kinase. Finally, LP- and MP-induced NDPK activation may involve different pools of GDP.

Topics: Amino Acid Sequence; Cell Membrane; Enzyme Activation; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Leukemia, Myeloid; Lipoproteins; Molecular Sequence Data; Nucleoside-Diphosphate Kinase; Peptides; Signal Transduction; Tumor Cells, Cultured; Wasp Venoms

Guanine nucleotide-binding proteins, or G proteins, play an important role in transmitting information from membrane receptors to intracellular effector systems. Activation of G proteins results in the hydrolysis of GTP, and the measurement of GTPase activity represents a means by which the role of G proteins in signal transduction can be investigated. GTPase activity of guinea pig bronchial membranes was measured as the liberation of 32Pi from [gamma-32P]GTP. GTPase activity was divided into two components, one possessing a high affinity and the other a low affinity for GTP. The contribution of high- and low-affinity GTPase to total hydrolysis was dependent on Mg2+. In the presence of submicromolar Mg2+, high-affinity GTPase represented 65-80% of all activity, whereas in the presence of > or = 26 microM Mg2+, all detectable hydrolysis was due to the low-affinity GTPase. High-affinity GTPase was stimulated by Mg2+ in the 0.15-1.1 microM range (2.5-fold maximal stimulation, apparent Km for Mg2+ 0.31 microM). Mastoparan (1-100 microM) caused a concentration-dependent stimulation of high-affinity (but not low-affinity) GTPase (71 +/- 13% maximal stimulation, EC50 0.38 microM), suggesting that high-affinity GTPase may be due to a G protein. Carbachol (10 microM) and fenoterol (10 microM) had no effect on high-affinity GTP hydrolysis, suggesting that under the conditions described, GTPase activity of bronchial membranes is not activated by muscarinic or beta-adrenergic receptors, respectively.

Topics: Animals; Bronchi; Female; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Guinea Pigs; Hydrolysis; Intercellular Signaling Peptides and Proteins; Magnesium; Membranes; Peptides; Signal Transduction; Wasp Venoms

Recent reports of a pertussis-toxin (Ptx)-sensitive inhibition of glucose-induced insulin release by prostaglandin E2 (PGE2) in transformed beta-cells prompted us to look for the presence of prostaglandin-regulatable GTP-binding proteins (G-proteins) on the secretory granules of normal pancreatic islets. PGE2 (but not PGF2 alpha, PGA2, PGB2 or PGD2) stimulated in a concentration-dependent manner a high-affinity GTPase activity in the secretory-granule-enriched fractions of both normal rat and human islets. Similar results were found after sucrose-density-gradient-centrifugation-based isolation of secretory granules to those after a differential-centrifugation procedure. Half-maximal stimulation occurred at 800 nM PGE2, a concentration known to inhibit both phases of glucose-induced insulin secretion from pure beta-cell lines. The GTPase stimulatory effect of PGE2 was blocked virtually totally by Ptx pretreatment; it was not due to an effect on substrate binding since no measurable effect of PGE2 on binding of guanosine 5'-[gamma-[35S]thio]triphosphate was observed in cognate fractions. Other Ptx-sensitive inhibitors of insulin secretion (such as adrenaline or clonidine) also stimulated GTPase activity, suggesting that one (or more) inhibitory exocytotic G-proteins (i.e. a putative GEi) is located on the secretory granules. These studies demonstrate, for the first time in an endocrine gland, the presence of a regulatable G-protein, strategically located on the secretory granules where it might regulate the exocytotic cascade distal to both plasma-membrane events and the generation of soluble mediators of insulin secretion.

Topics: Adrenergic alpha-Agonists; Animals; Cell Fractionation; Cytoplasmic Granules; Dinoprostone; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Male; Peptides; Pertussis Toxin; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Virulence Factors, Bordetella; Wasp Venoms

We have recently demonstrated a permissive role for GTP in nutrient-induced insulin secretion. One of the possible loci at which GTP might exert its regulatory effects include one (or more) of the GTP-binding proteins which we have identified in subcellular fractions (including secretory granules) of pancreatic islets. Herein, we characterize nucleoside diphosphokinase (NDP kinase) activity, which catalyzes the transphosphorylation of nucleotide diphosphate (e.g., GDP) to nucleotide triphosphates (e.g., GTP) in insulin-secreting cells. The presence of NDP kinase activity in normal rat and human islets, and pure beta (RIN and HIT) cells, was verified by three distinct approaches: first, its catalytic activity (formation of GTP or GTP gamma S from GDP and ATP or ATP gamma S); secondly, by immunologic detection; and third, by quantitating the phosphoenzyme intermediate of NDP kinase, which is involved in a ping-pong phosphotransfer mechanism. Subcellularly, NDP kinase is predominantly cytosolic (with a tetrameric molecular mass of 85-90 kDa) and requires divalent metal ions and thiols for its activity. UDP, which forms an abortive complex with the enzyme, inhibited its activity in a concentration-dependent manner (Ki = 2 mM). The phosphorylated intermediate of NDP kinase was differentially sensitive to heat, acidic pH, and a histidine-selective reagent, diethyl pyrocarbonate, suggesting that (one of) the phosphoamino acid(s) may be histidine. These data demonstrate that in beta cells NDP kinase undergoes transient phosphorylation and suggest that this phosphate, in turn, is transferred to GDP. If the GTP which is formed thereby is bound to, or channelled to, relevant GTP-binding proteins, it would facilitate the formation of active form of these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cytosol; Guanosine Triphosphate; Histidine; Humans; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Macromolecular Substances; Male; Molecular Weight; Nucleoside-Diphosphate Kinase; Peptides; Phosphorylation; Rats; Rats, Sprague-Dawley; Sulfhydryl Reagents; Uridine Diphosphate; Wasp Venoms

Topics: Amino Acid Sequence; Animals; Cations; Detergents; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Lipids; Macromolecular Substances; Mast Cells; Molecular Sequence Data; Peptides; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Tritium; Wasp Venoms

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.

Topics: Adenosine Diphosphate Ribose; Adenosine Triphosphatases; Amino Acid Sequence; Animals; Calcium; Cattle; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Intercellular Signaling Peptides and Proteins; Mitochondria, Liver; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Nucleoside-Diphosphate Kinase; Peptides; Pertussis Toxin; Superoxides; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms

The N-methyl-3'-O-anthranoyl (MANT) guanine nucleotide analogs are useful environmentally sensitive fluorescent probes for studying G protein mechanisms. Both MANT-GTP gamma S (mGTP gamma S) and MANT-GTP (mGTP) displayed a magnesium-dependent increase in fluorescence upon binding to bovine brain G(o). A much greater increase in MANT-guanine nucleotide fluorescence was observed with excitation at 280 nm compared with 350 nm, due to energy transfer from tryptophan in G(o). G(o)-bound mGTP gamma S displays a blue-shift in its emission spectrum indicating a nonpolar environment for the G(o)-bound MANT. For the hydrolyzable analog, mGTP, the increase in fluorescence is followed by a decrease as it is hydrolyzed to mGDP. Unexpectedly, dissociation of mGDP was fast (t1/2 1.7 s) by comparison with GDP itself (t1/2 120 s). Binding of mGTP gamma S to G(o) was slow, but mastoparan increased the rate approximately 4-fold. For mGTP, mastoparan increased both the rate of binding and the peak fluorescence, even at saturating mGTP concentrations. Modeling the mGTP fluorescence kinetics in the presence and absence of mastoparan results in two novel conclusions. First, mGTP does not fully activate the G protein, even when bound. Second, mastoparan appears to increase the rate of the G protein conformational activation step, in addition to its known effect on GDP release.

Topics: Animals; Cattle; Fluorescent Dyes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Models, Chemical; ortho-Aminobenzoates; Peptides; Protein Conformation; Spectrometry, Fluorescence; Wasp Venoms

We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly

Topics: Adenosine Triphosphate; Adrenal Glands; Animals; Arachidonic Acid; Calcium; Catecholamines; Cattle; Cells, Cultured; Chromaffin Granules; Exocytosis; Fatty Acids, Unsaturated; GTP-Binding Proteins; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Neomycin; Peptides; Pertussis Toxin; Phospholipases A; Phospholipases A2; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms

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.

Topics: Aluminum; Aluminum Compounds; Amino Acid Sequence; Coated Pits, Cell-Membrane; Endocytosis; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Peptides; Receptors, Transferrin; Thionucleotides; Tumor Cells, Cultured; Wasp Venoms

Mastoparan is a tetradecapeptide. Mastoparan added to the apical surface of monolayers of Madin-Darby canine kidney (MDCK) epithelial cells, cultured on micropore filters, activated ion transport and increased the permeability of the paracellular pathway across the monolayers. In monolayers of similar MDCK cells in which the basolateral membrane was permeabilized with Staphylococcus aureus alpha toxin (Staph. alpha toxin), the effects of mastoparan on apical membrane ion conductances were dependent on the presence of guanosine triphosphate (GTP). Mastoparan and GTP increased apical membrane chloride conductance more than potassium conductance, with very little change in sodium conductance. In intact monolayers, addition of barium to the apical bath prevented mastoparan activation of ion transport and the increase in paracellular permeability. Increasing bath potassium to 130 mM also reduced ion transport and prevented the increase in paracellular permeability. We hypothesized that these observations could be linked by mastoparan activation of apical chloride and potassium conductances, with consequent decreases in cell volume and resultant increases in paracellular permeability. Addition of 270 mM mannitol to isosmotic media to decrease cell volume decreased MDCK monolayer transepithelial resistance. Addition of mastoparan to monolayers of MDCK cells grown on micropore filters decreased cell volume to the same extent as addition of 270 mM mannitol to isosmotic media. Addition of the potassium channel inhibitor, barium, prevented the decrease in cell volume in response to mastoparan. Mastoparan activates apical membrane chloride and potassium conductances in MDCK cells. The loss of these ions from the cells decreases cell volume, and the decrease in cell volume increases the permeability of the paracellular pathway.

Topics: Animals; Cell Count; Cell Membrane Permeability; Cells, Cultured; Chlorides; Cholera Toxin; Cyclic AMP; Dogs; Epithelium; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kidney; Membrane Potentials; Peptides; Potassium; Second Messenger Systems; Signal Transduction; Wasp Venoms

Guanosine triphosphate (GTP)-binding proteins are required for intracellular vesicular transport. Mastoparan is a peptide component of wasp venom that increases nucleotide exchange in some classes of G alpha subunits of regulatory heterotrimeric GTP-binding proteins (G proteins). Mastoparan and other compounds that increase nucleotide exchange by G proteins inhibited endosome fusion in vitro and reversed the effects of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S), a nonhydrolyzable GTP analog. Addition of beta gamma subunits of G proteins to the fusion assay antagonized the stimulatory effect of GTP-gamma-S, confirming the participation of G proteins. These results indicate that GTP-binding proteins are required for endosome fusion and in particular that a G protein is involved. Given the function of G proteins in signal transduction, these findings may provide insight into the mechanism by which endosomal vesicles become competent for fusion after their formation at the cell surface.

Topics: Biological Transport; Endocytosis; Endosomes; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Intracellular Membranes; Macromolecular Substances; Membrane Fusion; Organelles; Peptides; Wasp Venoms

We have recently reported that glucagon activated the L-type Ca2+ channel current in frog ventricular myocytes and showed that this was linked to the inhibition of a membrane-bound low-Km cAMP phosphodiesterase (PDE) (Méry, P. F., Brechler, V., Pavoine, C., Pecker, F., and Fischmeister, R. (1990) Nature 345, 158-161). We show here that the inhibition of membrane-bound PDE activity by glucagon depends on guanine nucleotides, a reproducible inhibition of 40% being obtained with 0.1 microM glucagon in the presence of 10 microM GTP, with GTP greater than GTP gamma S, while GDP and ATP gamma S were without effect. Glucagon had no effect on the cytosolic low-Km cAMP PDE, assayed with or without 10 microM GTP. Glucagon inhibition of membrane-bound PDE activity was not affected by pretreatment of the ventricle particulate fraction with cholera toxin. However, it was abolished after pertussis toxin pretreatment. Mastoparan, a wasp venom peptide known to activate G(i)/G(o) proteins directly, mimicked the effect of glucagon. PDE inhibition by glucagon was additive with the inhibition induced by Ro 20-1724, but was prevented by milrinone. This was correlated with an increase by glucagon of cAMP levels in frog ventricular cells which was not additive with the increase in cAMP due to milrinone. We conclude that glucagon specifically inhibits the cGMP-inhibited, milrinone-sensitive PDE (CGI-PDE). Insensitivity of adenylylcyclase to glucagon and inhibition by the peptide of a low-Km cAMP PDE were not restricted to frog heart, but also occurred in mouse and guinea pig heart. These results confirm that two mechanisms mediate the action of glucagon in heart: one is the activation of adenylylcyclase through Gs, and the other relies on the inhibition of the membrane-bound low-Km CGI-PDE, via a pertussis toxin-sensitive G-protein.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenosine Triphosphate; Adenylyl Cyclases; Animals; Cyclic AMP; Cyclic GMP; Cytosol; Glucagon; GTP-Binding Proteins; Guanine Nucleotides; Guanosine Triphosphate; Guanylate Cyclase; Heart Ventricles; Intercellular Signaling Peptides and Proteins; Kinetics; Myocardium; Peptides; Pertussis Toxin; Rana esculenta; Virulence Factors, Bordetella; Wasp Venoms

Topics: Animals; Cell Membrane; Cell Membrane Permeability; Cells, Cultured; Chlorides; Dogs; Electrophysiology; Epithelial Cells; Epithelium; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Ion Channels; Kidney; Peptides; Potassium; Wasp Venoms

The amphiphilic agents melittin, compound 48/80 and mastoparan inhibit ADP-ribosylation of porcine brain rho protein by Clostridium botulinum exoenzyme C3. However, ADP-ribosylation of recombinant rhoA expressed in E.coli was not inhibited by these agents. Accordingly, steady state GTP hydrolysis by recombinant rhoA was not stimulated by mastoparan, whereas GTP hydrolysis by porcine brain rho was stimulated 2.5-fold in the presence of this wasp venom. After microinjection of recombinant rhoA into Xenopus laevis oocytes the inhibitory effect of mastoparan on C3 ADP-ribosylation was restored. The data suggest that the amphiphilic agents tested are only active at the posttranslationally processed form of rho and that they exert their effects via the C-terminal end.

Topics: Adenosine Diphosphate Ribose; Animals; Autoradiography; Brain; Carbon Radioisotopes; Cloning, Molecular; Cytosol; Escherichia coli; GTP-Binding Proteins; Guanosine Triphosphate; Hemiterpenes; Intercellular Signaling Peptides and Proteins; Melitten; NAD; Oocytes; Organophosphorus Compounds; p-Methoxy-N-methylphenethylamine; Peptides; Protein Processing, Post-Translational; Recombinant Proteins; rhoA GTP-Binding Protein; Swine; Wasp Venoms; Xenopus laevis

The effects of mastoparan and compound 48/80 on the activities of alpha beta gamma-trimeric GTP-binding proteins (G proteins) were studied with purified Go and Gi-1 which had been reconstituted into phospholipid vesicles. Pertussis toxin-catalyzed ADP-ribosylation of Go or Gi-1 was inhibited by mastoparan or compound 48/80, suggesting that the G proteins were dissociated into their constituent alpha- and beta gamma-subunits in the presence of these compounds. The steady-state rate of GTP hydrolysis catalyzed by Go or Gi-1 was stimulated by the two compounds. Both the stimulations were due to increases in the rate of the GDP-GTP exchange reaction occurring on the G proteins. However, the modes stimulation of the GTPase activity depended on the type of G protein used, and the stimulations caused by the two compounds were differently affected by pertussis toxin-catalyzed ADP-ribosylation of G proteins. Moreover, the mastoparan-induced stimulation of the GTPase activity was partially inhibited by compound 48/80. Thus, the two histamine secretagogues mastoparan and compound 48/80 appear to activate G proteins differently, though they interact with the signal-transducing proteins, at least partly, at a common binding site.

Topics: Adenosine Diphosphate Ribose; Animals; Binding Sites; Cattle; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Hydrolysis; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kinetics; Liposomes; p-Methoxy-N-methylphenethylamine; Peptides; Wasp Venoms

Histamine release induced by the introduction of a nonhydrolyzable analogue of GTP, GTP-gamma-S, into ATP-permeabilized mast cells, is associated with phosphoinositide breakdown, as evidenced by the production of phosphatidic acid (PA) in a neomycin-sensitive process. The dependency of both PA formation and histamine secretion on GTP-gamma-S concentrations is bell shaped. Whereas concentrations of up to 0.1 mM GTP-gamma-S stimulate both processes, at higher concentrations the cells' responsiveness is inhibited. At a concentration of 1 mM, GTP-gamma-S self-inhibits both PA formation and histamine secretion. Inhibition of secretion can, however, be overcome by the basic secretagogues compound 48/80 and mastoparan that in suboptimal doses synergize with 1 mM GTP-gamma-S to potentiate secretion. Secretion under these conditions is not accompanied by PA formation and is resistant both to depletion of Ca2+ from internal stores and to pertussis toxin (PtX) treatment. In addition, 48/80, like mastoparan, is capable of directly stimulating the GTPase activity of G-proteins in a cell-free system. Together, our results are consistent with a model in which the continuous activation of a phosphoinositide-hydrolyzing phospholipase C (PLC) by a stimulatory G-protein suffices to trigger histamine secretion. Basic secretagogues of mast cells, such as compound 48/80 and mastoparan, are capable of inducing secretion in a mechanism that bypasses PLC by directly activating a G-protein that is presumably located downstream from PLC (GE). Thereby, these secretagogues induce histamine secretion in a receptor-independent manner.

Topics: Adenosine Triphosphate; Animals; Brain; Cell Membrane Permeability; Exocytosis; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Histamine Release; Intercellular Signaling Peptides and Proteins; Male; Mast Cells; Membranes; p-Methoxy-N-methylphenethylamine; Peptides; Pertussis Toxin; Phosphatidic Acids; Rats; Rats, Inbred Strains; Thionucleotides; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms

Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimulates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein alpha subunits and alpha beta gamma trimers. Relative stimulation was greater with alpha beta gamma trimers and beta gamma subunits could increase net MP-stimulated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 2-4. The membrane-bound alpha-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of Go to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on Go. 4) MP stimulated steady state GTPase activity at less than 1 microM Mg2+ and stimulated the dissociation of both GDP and guanosine 5'-O-(3-thiotriphosphate) at less than 1 nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the alpha subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC50 more than 10-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chloride competitively antagonized the stimulation of Gi by MP but potently stimulated nucleotide exchange on Go. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.

Topics: Amines; Amino Acid Sequence; Animals; Bee Venoms; Brain; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Liver; Macromolecular Substances; Magnesium; Molecular Sequence Data; Oligopeptides; Peptides; Rabbits; Receptors, Muscarinic; Structure-Activity Relationship; Swine; Thionucleotides; Wasp Venoms

Mastoparan inhibited [3H]inositol phosphate accumulation induced by carbachol as well as cyclic AMP accumulation induced by isoproterenol in 1321N1 human astrocytoma cells. Mastoparan inhibited GTP gamma S-induced, but not Ca2(+)-induced, [3H]inositol phosphate accumulation in membrane preparations with an IC50 of approximately 10 microM. The inhibitory effect of mastoparan on carbachol-induced [3H]inositol phosphate accumulation was resistant to pertussis toxin (IAP) treatment in intact cells. These results suggest that mastoparan inhibits phospholipase C in human astrocytoma cells via a GTP binding protein, which is not a substrate for IAP.

Topics: Astrocytoma; Bee Venoms; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Peptides; Pertussis Toxin; Phosphatidylinositols; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella; Wasp Venoms

The effects of mastoparan on phospholipase C-catalysed phosphoinositide hydrolysis were examined in [3H]inositol-labelled human neuroblastoma SH-SY5Y cells. [3H]Inositol phosphate formation in intact cells was not altered by 20 microM mastoparan. In contrast, [3H]inositol phosphate formation in electrically permeabilized cells stimulated with guanosine 5'-[gamma-thio]triphosphate and/or carbachol was inhibited by mastoparan with half-maximal effects at approx. 3 microM. The peptide was much less effective in inhibiting stimulatory effects of Ca2+. Similar but less potent inhibitory effects were observed with the cations, neomycin and spermine, indicating that direct interaction of mastoparan with polyphosphoinositides might account for its inhibitory effects on inositol phosphate formation.

Topics: Bee Venoms; Calcium; Carbachol; Cell Membrane Permeability; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Hydrolysis; Inositol Phosphates; Intercellular Signaling Peptides and Proteins; Neuroblastoma; Peptides; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Receptors, Muscarinic; Thionucleotides; Tumor Cells, Cultured; Type C Phospholipases; Wasp Venoms

Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.

Topics: Adenosine Diphosphate Ribose; Animals; Bee Venoms; Brain; Cattle; Cell Membrane; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Liver; Peptides; Pertussis Toxin; Rabbits; Receptors, Drug; Thionucleotides; Virulence Factors, Bordetella; Wasp Venoms