Mastoparan interacts with the carboxyl terminus of the alpha subunit of Gi

Mastoparan, a peptide toxin from wasp venom, stimulates guanine nucleotide binding and hydrolysis by G proteins. To elucidate the site of mastoparan-G protein interaction, we utilized a polyclonal antibody (R16,17) directed against the carboxyl terminus of the Gi alpha subunit to develop a competitive enzyme-linked immunosorbent assay. We investigated the ability of mastoparan to influence R16,17 antibody binding to G protein alpha subunits in a purified preparation of brain Gi and in neutrophil membrane extracts. Mastoparan antagonized the ability of R16,17 to detect G protein alpha subunits with an IC50 of 15 microM in the purified preparation and with an IC50 of 1 microM for the predominant G protein population in membrane extracts. This reduction was not seen when an unrelated peptide or a peptide of similar charge composition to mastoparan was used in place of mastoparan in the assay. Additionally, antibody R16,17 blocked up to 85% of mastoparan-stimulated GTPase activity. Taken together, these data indicate that the interaction of mastoparan with G protein depends in part on the carboxyl terminus of Gi alpha. Pertussis toxin-catalyzed ADP-ribosylation of Gi alpha markedly inhibited mastoparan-stimulated GTPase activity but only slightly attenuated the ability of mastoparan to recognize G protein. These data suggest that ribosylation inhibits mastoparan-induced G protein activation by a mechanism distinct from the ability of mastoparan to physically interact with G protein. Since mastoparan is thought to mimic hormone-liganded receptors, these findings may be applicable to the mechanism of receptor-Gi protein uncoupling that results from ADP-ribosylation of the G protein.     

Direct activation of GTP-binding regulatory proteins (G-proteins) by substance P and compound 48/80.

The neuropeptide substance P and the polyamine compound 48/80, both known to activate mast cell secretory processes, increased the rate of GTP S binding to G-proteins purified from calf brain (Go/Gi mixture). The GTPase activity of G-proteins was also increased by substance P and compound 48/80 in a dose-dependent and Mg2+-dependent way. These effects were similar to those of the wasp venom peptide mastoparan, another histamine releaser of rat peritoneal and human skin mast cells. This suggests that the secretory property of compound 48/80 and substance P is not due to a receptor-mediated process but, like mastoparan, results from a direct activation of G-proteins.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins)

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.     

Functional Reconstitution of Purified Gi and Go with μ-Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N-Ethylmaleimide

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.     

Amiloride interacts with guanine nucleotide regulatory proteins and attenuates the hormonal inhibition of adenylate cyclase

The effect of amiloride on the hormonal regulation of adenylate cyclase was studied in the rat anterior pituitary. The diuretic did not alter basal adenylate cyclase but augmented the enzyme activity in an irreversible manner in the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S) stimulated adenylate cyclase at lower concentrations and inhibited at higher concentrations. Amiloride treatment enhanced the stimulatory and abolished the inhibitory phase of GTP gamma S action. In addition, amiloride also attenuated the inhibitory effects of atrial natriuretic factor (ANF 99-126) and angiotensin II on cAMP levels and adenylate cyclase activity. On the other hand, amiloride showed an additive effect on the stimulation exerted by corticotropin-releasing factor and vasoactive intestinal peptide on adenylate cyclase in anterior pituitary and on isoproterenol-stimulated cAMP levels in cultured vascular smooth muscle cells. Pertussis toxin, in the presence of [alpha-32 P]NAD, catalyzed the ADP-ribosylation of two protein bands of Mr 41,000 and 39,000, referred to as Gi and Go, respectively, in the anterior pituitary, and 40,000-Da protein in the aorta, referred to as Gi. Amiloride treatment inhibited the labeling of all these bands in a concentration- and time-dependent manner. Similarly, the pertussis toxin-catalyzed ADP-ribosylation of purified Gi from bovine brain was also inhibited by amiloride treatment. However, amiloride had no significant effect on the cholera toxin-catalyzed ADP-ribosylation of Gs. These data suggest that amiloride interacts with the guanine nucleotide regulatory proteins Gi and Go. Modification of Gi results in the attenuation of hormone-induced adenylate cyclase and cAMP inhibition. However, the interaction between amiloride and Go and the consequent Ca2+ mobilization and phosphatidylinositol turnover have to be investigated.     

Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.     

Modification of Brain Guanine Nucleotide-Binding Regulatory Proteins by Tryptamine-4,5-Dione, a Neurotoxic Derivative of Serotonin

We have recently characterized a novel oxidation product of serotonin (5-hydroxytryptamine, 5-HT), tryptamine-4,5-dione, which increases 5-HT efflux from striatum and hippocampus and causes selective neuronal death. Exposure of striatal synaptosomes or the major brain guanine nucleotide-binding regulatory proteins Gi and Go to [3H]tryptamine-4,5-dione resulted in the radiolabeling of a major band with an apparent molecular mass equivalent to that of the alpha subunits of Gi and Go (approximately 40,000). The binding of [35S]guanosine-5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to Gi and Go and pertussis toxin-catalyzed [32P]ADP-ribosylation of the G protein alpha subunits were both inhibited in a dose-dependent manner by tryptamine-4,5-dione. Thus, neurotoxins such as tryptamine-4,5-dione may exert their effects through specific interactions with G proteins.     

Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.     

Modification of the function of pertussis toxin substrate GTP-binding protein by cholera toxin-catalyzed ADP-ribosylation.

The alpha-subunit of Gi-2, in addition to that of Gs (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of Gi-2 were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of Gi-2 were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified Gi-2 displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified Gir2 trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-32P]GTP, the radioactivity was more greatly retained in the modified Gi-2 than in the unmodified protein. 3) The actual catalytic rate (kcat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of Gi-2 for GDP by cholera toxin-induced modification. 5) The modified Gi-2 exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated Gi-2 than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated Gi-2, resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated Gi-2 appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Possible involvement of different GTP-binding proteins in noradrenaline- and thrombin-stimulated release of arachidonic acid in rabbit platelets.

Noradrenaline (NA) stimulated the release of arachidonic acid (AA) from the [3H]AA-labelled rabbit platelets via alpha 2-adrenergic receptors, since the effect of NA was inhibited by yohimbine. The stimulatory effect of NA in digitonin-permeabilized platelets was completely dependent on the simultaneous presence of GTP and Ca2+. The NA- and thrombin-stimulated releases of AA were markedly decreased by the prior ADP-ribosylation of the permeabilized platelets with pertussis toxin. Antiserum directed against the pig brain Go (a GTP-binding protein of unknown function), recognizing both alpha 39 and beta 35,36 subunits, but not alpha 41, of pig brain, reacted with 41 kDa and 40 kDa bands, with not one of 39 kDa, in rabbit platelet membranes. Anti-Go antiserum inhibited guanosine 5'-[gamma-thio]triphosphate-, A1F4(-)-, NA- and thrombin-stimulated AA releases in the membranes. Although the effect of thrombin was inhibited by low concentrations of anti-Go antiserum, high concentrations of the antiserum was needed for inhibition of the NA effect. Antiserum directed against the pig brain G1 (inhibitory G-protein), recognizing both alpha 41 and beta 35,36 subunits, but not alpha 39, of pig brain, reacted with the 41 kDa band in platelets. Anti-G1 antiserum inhibited only the effect of NA. Reconstitution of the platelet membranes ADP-ribosylated by pertussis toxin with Go, not Gi, purified from pig brain restored the thrombin-stimulated release of AA. In contrast, reconstitution of those membranes with Gi, not Go, restored the NA-stimulated release of AA. These results indicate that different GTP-binding proteins, Gi- and Go-like proteins, may be involved in the mechanism of signal transduction from alpha 2-adrenergic receptors and thrombin receptors to phospholipase A2 in rabbit platelets.     

Pertussis toxin-insensitive effects of mastoparan,a wasp venom peptide,in PC 12 cells

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, modifies the secretion of neurotransmitters and hormones from a variety of cell types. Mastoparan interacts with heterotrimeric guanine nucleotide-binding proteins (G proteins) such as G_i and G_o, which are ADP-ribosylated by pertussis toxin (PTX) and thereby uncoupled from receptors. Previously, some of the effects of mastoparan including secretion were reported to be modified selectively by PTX but not by cholera toxin (CTX). In the present study, we examined the influence of bacterial toxins on the effects of mastoparan in PC12 cells. Mastoparan stimulated [³H]noradrenaline (NA) release from prelabeled PC12 cells in the absence of CaCl₂, although high K⁺ or ATP stimulated the release in a Ca²⁺-dependent manner. Pretreatment with CTX, not PTX, for 24 h inhibited mastoparan-stimulated [³H]NA release. Mastoparan inhibited forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner, although mastoparan had no effect by itself. Pretreatment with PTX completely abolished the inhibitory effect of carbachol via G_i on cyclic AMP accumulation and partially reduced the effect of mastoparan. However, the inhibitory effect of 20 μM mastoparan was not modified by pretreatment with PTX. Thus, we investigated the effect of mastoparan on CTX-catalyzed [³²P]ADP-ribosylation of proteins in PC12 cells. A subunit of CTX (CTX-A) catalyzed [³²P]ADP-ribosylation of many proteins in the cytosolic fraction of PC12 cells. One of these was a 20 kDa protein, named ADP-ribosylating factor (ARF). The addition of mastoparan to assay mixtures inhibited ADP-ribosylation of many proteins including ARF and CTX-A in the presence of the cytosolic fraction. In the absence of the cytosolic fraction, however, mastoparan slightly enhanced ADP-ribosylation of bovine serum albumin and auto-ADP-ribosylation by CTX-A. Mastoparan did not inhibit ADP-ribosylation of the α subunit of G_s in the membrane fraction. These findings suggest that (1) mastoparan interacts with PTX-insensitive and CTX-sensitive factor(s) to stimulate NA release, and (2) mastoparan interacts with ARF inhibiting its activity to enhance the ADP-ribosylation reaction by CTX. ARF may be an exocytosis-linked G protein. © 1996 Wiley-Liss, Inc.     

Regulation of G-proteins in differentiation. Altered ratio of alpha- to beta-subunits in 3T3-L1 cells

The complexion of the adenylate cyclase system and in particular, the regulation of G-proteins was examined in 3T3-L1 cells during differentiation from a fibroblast-like to an adipocyte-like phenotype. Gs alpha (the identified regulatory component of hormone-sensitive adenylate cyclase that mediates stimulation), measured by cholera toxin-catalyzed ADP-ribosylation, increased by approximately 6-fold from day 0 to day 8. Gs alpha, measured by functional reconstitution, increased in specific activity by approximately 3-fold from day 0 to day 8. Both Gi alpha (the G-protein with alpha-subunit Mr 40,000-41,000 whose function is in part the mediation of inhibition of adenylate cyclase) and Go alpha (the highly abundant G-protein first isolated from bovine brain whose effector system remains to be established) measured by pertussis toxin-catalyzed ADP-ribosylation increased by approximately 4-fold over this same period. 3T3-L1 cells possess beta-subunits of G-proteins displaying Mr = 36,000 (beta 36) and Mr = 35,000 (beta 35). The increase in the beta 35 as well as beta 36 subunits was approximately 2-fold. Using quantitative immunoblotting techniques and specific antisera, the total amount of beta-subunits was determined to be 150 as compared to 70 pmol/mg of membrane protein, while the amount of Go alpha was 40 and 10 pmol/mg of membrane protein in adipocytes and fibroblasts, respectively. Since Go alpha is the most abundant G-protein alpha-subunit observed to date in both phenotypes, the overall ratio of beta- to alpha-subunits of G-proteins appears to decrease from approximately 4.7 in fibroblasts to 2.5 in adipocytes. These data suggest that in differentiation not only is the complexion of G-proteins altered but more importantly, the relative amounts of alpha- to beta-subunits are regulated.     

Pertussis toxin treatment in vivo is associated with a decline in G-protein beta-subunits

The effects of pertussis toxin on the steady-state levels of G-protein alpha- and beta-subunits were investigated both in vitro and in vivo. The steady-state level Go alpha, a major substrate for pertussis toxin-catalyzed ADP-ribosylation, was unaltered by pertussis toxin treatment for periods up to 100 h for 3T3-L1 cells in culture or up to 3 days in vivo. In 3T3-L1 cells pertussis toxin treatment did not alter levels of Gs alpha-subunits; in S49 cells the level of Gs alpha-subunits declined moderately following by pertussis toxin treatment. The steady-state levels of G beta-subunits, in contrast, were found to decline to less than 50% of the normal cellular complement following pertussis toxin treatment in vitro and in vivo. Inhibitory control of adenylate cyclase, pertussis toxin-catalyzed ADP-ribosylation of Gi alpha and Go alpha, and the GTP-dependent shift in agonist-specific binding to beta-adrenergic receptors were attenuated or abolished within 5 h of pertussis toxin treatment, representing "early" effects of the toxin. Stimulatory regulation of adenylate cyclase, in contrast, displayed a progressive enhancement that was first observed 4 h after pertussis toxin treatment, increasing thereafter up until 100 h, the last time point measured. This progressive enhancement of the stimulatory pathway of adenylate cyclase was not manifest at the level of stimulatory receptors, since the Kd and Bmax for one such receptor, the beta-adrenergic receptor, were shown to be unaltered in toxin-treated cells. Furthermore, the potentiation of stimulation of adenylate cyclase was observed in cells stimulated by the beta-adrenergic agonist isoproterenol and PGE1 alike. The progressive enhancement of the stimulatory pathway correlated best with the decline in G beta-subunit levels that occurs following pertussis intoxication. The changes in both of these parameters occur "late" (12-48 h), as compared to the early events that occur within 5 h. Pertussis toxin action appears to be composed of two, temporally distinct, groups of effects. Pertussis toxin-catalyzed ADP-ribosylation of G alpha-subunits, attenuation of the inhibitory regulation of adenylate cyclase, and attenuation of the ability of GTP to induce an agonist-specific shift in receptor affinity are members of the early group of effects. The second group of late effects includes the decline in G beta-subunit levels and the progressive enhancement of the stimulatory pathway of adenylate cyclase. This enhanced stimulatory control at these later times cannot be explained by the attenuation of the inhibitory pathway occurring early, but rather appears as G beta-subunit levels decline.     

Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Characterization of four G0-type proteins purified from bovine brain membranes

Recently we reported there were at least four types of G0 or G0-like proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an alpha-monomeric form, and the others as alpha beta gamma-trimers. The four proteins, of which alpha-subunits were confirmed to be a family of G0-type by an immunoblot analysis, were thus referred to as alpha (0)1, G(0)2, G(0)3 and G(0)4, respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four alpha-subunits, together with their fragmentation patterns containing radiolabeled ADP-ribose that had been incorporated by pertussis toxin-catalyzed ADP-ribosylation, suggested that the four G0-alpha were classified into either of two groups such as alpha (0)1 and G(0)2-alpha, or G(0)3-alpha and G(0)4-alpha. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between alpha (0)1 and G(0)2-alpha or G(0)3-alpha and G(0)4-alpha. Thus, the four G0-type proteins appeared to be different entities from one another.