Mechanisms for inhibition of the catalytic activity of adenylate cyclase by the guanine nucleotide-binding proteins serving as the substrate of islet-activating protein, pertussis toxin

Two GTP-binding trimeric proteins (referred to as alpha 41 beta gamma and alpha 39 beta gamma based on the kilodalton molecular weights of their alpha-subunits) were purified from rat brain as the specific substrates of the ADP-ribosylation reaction catalyzed by islet-activating protein, pertussis toxin, and resolved irreversibly into alpha- and beta gamma-subunits by incubation with guanosine 5'-O-(thiotriphosphate) (GTP gamma S). Some of these resolved subunits interacted directly with the adenylate cyclase catalyst partially purified from rat brain in a detergent-containing solution, resulting in inhibition of the cyclase activity as follows. 1) GTP gamma S-bound alpha 41 inhibited the catalyst, but GTP gamma S-bound alpha 39 did not; the inhibition was competitive with GTP gamma S-bound alpha-subunit of Ns, the GTP-binding protein involved in activation of adenylate cyclase. 2) beta gamma from either alpha 41 beta gamma or alpha 39 beta gamma inhibited the catalyst in a manner not competitive with the activator such as forskolin or the alpha-subunit of Ns. 3) The ADP-ribosylation of alpha 41 beta gamma by islet-activating protein did not exert any influence on the subsequent GTP gamma S-induced resolution and the ability of the resolved GTP gamma S-bound alpha 41 to inhibit the catalyst. 4) The beta gamma-induced inhibition of the catalyst was additive to the inhibition caused by GTP gamma S-bound alpha 41. Thus, the direct inhibition of the catalyst by beta gamma or GTP gamma S-bound alpha 41 is a likely mechanism involved in receptor-mediated inhibition of adenylate cyclase, in addition to the previously proposed indirect inhibition due to the reduction of the concentration of the active alpha-subunit of Ns by reassociation with beta gamma.     

A new GTP-binding protein in brain tissues serving as the specific substrate of islet-activating protein, pertussis toxin

A new GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was purified from porcine brain membranes as an alpha beta gamma-heterotrimeric structure. The alpha-subunit of the purified protein (alpha 40 beta gamma) had a molecular mass of 40 kDa and differed from that of Gi (alpha 41 beta gamma) or Go (alpha 39 beta gamma) previously purified from brain tissues. The fragmentation patterns of limited tryptic digestion and immunological cross-reactivities among the three alpha were different from one another. However, the beta gamma-subunit resolved from the three IAP substrates similarly inhibited a membrane-bound adenylate cyclase and their beta-subunits were immunologically indistinguishable from one another. Thus, the alpha 40 beta gamma is a new IAP substrate protein different from Gi or Go, in the alpha-subunit only.     

Subunit interactions of native and ADP-ribosylated alpha 39 and alpha 41, two guanine nucleotide-binding proteins from bovine cerebral cortex

Bovine cerebral cortex contains two major substrates for ADP-ribosylation by pertussis toxin: a 39-kDa protein, alpha 39, and a 41-kDa protein, alpha 41 (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both of these proteins bind guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with a similar affinity (Kd = 30 +/- 10 nM for alpha 39, Kd = 32 +/- 14 nM for alpha 41). Both proteins associate with a beta X gamma subunit made up of a 36-kDa beta component and a 6-kDa gamma component. We have previously shown that the beta X gamma unit is required for pertussis toxin-catalyzed ADP-ribosylation (Neer et al. (1984)). By measuring the amount of beta X gamma required for maximal incorporation of ADP-ribose, we now find that the EC50 for beta X gamma in this reaction is 3 +/- 1 times lower for alpha 41 than for alpha 39. ADP-ribosylation by pertussis toxin does not prevent dissociation of alpha 41 X beta X gamma or alpha 39 X beta X gamma by GTP gamma S. GTP gamma S decreases the sedimentation coefficient of ADP-ribosylated alpha 41 from 4.2 S to 3.0 S and the sedimentation coefficient of ADP-ribosylated alpha 39 from 4.3 S to 2.9 S. The conclusion that GTP gamma S dissociates both ADP-ribosylated heterotrimers was confirmed by the observation that GTP gamma S blocks precipitation of ADP-ribosylated alpha 39 or alpha 41 by anti-beta antibody. Neither alpha 41 X beta X gamma nor alpha 39 X beta X gamma is dissociated by GTP whether or not the proteins are ADP-ribosylated. The observation that alpha 41 more readily associates with beta X gamma than does alpha 39 may explain our earlier observation that alpha 41 is more readily ADP-ribosylated than alpha 39. In most intact membranes, only a 41-kDa ADP-ribosylated protein is seen. However, alpha 39 is also present in most tissues since we can detect it with anti-alpha 39 antibody. The functional consequences of pertussis toxin treatment may depend on whether one or both proteins are ADP-ribosylated. This in turn may depend on the ratio of alpha 41 and alpha 39 to beta X gamma in a given tissue.     

Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from Lubrol extract of sea urchin egg membranes. The partially purified protein possessed two polypeptides of 39 and 37 kDa; the 39 kDa polypeptide was specifically ADP-ribosylated by IAP and the 37 kDa protein cross-reacted with the antibody prepared against purified beta gamma-subunits of alpha beta gamma-heterotrimeric IAP substrates from rat brain. Incubation of this sea urchin IAP substrate with a non-hydrolyzable GTP analogue resulted in a reduction of the apparent molecular mass on a column of gel filtration as had been the case with purified rat brain IAP substrates, suggesting that the sea urchin IAP substrate was also a heterooligomer dissociable into two polypeptides in the presence of GTP analogues. Thus, the 39 and 37 kDa polypeptides of the sea urchin IAP substrate correspond to the alpha- and beta-subunits, respectively, of mammalian IAP substrates which are involved in the coupling between membrane receptor and effector systems.     

Properties of a novel GTP-binding protein which is associated with soluble phosphoinositides-specific phospholipase C

Recently we demonstrated the presence in calf thymocytes of a GTP-binding protein (G-protein) composed of three polypeptides, 54, 41, and 27 kDa, which was physically and functionally associated with a soluble phosphoinositides-specific phospholipase C (PI-phospholipase C). The properties of this G protein were further investigated with the following results. 1) In addition to the ability to bind [35S]guanosine-5'-[gamma-thio]triphosphate (GTP gamma S), the G-protein exhibited GTPase activity, which was enhanced by Mg2+, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, but inhibited by sodium cholate, GTP gamma S and F-.2) The 54-kDa polypeptide was ADP-ribosylated by pertussis toxin and also by endogenous membrane-bound ADP-ribosyltransferase, but none of these three polypeptides was ADP-ribosylated by cholera toxin. 3) The G-protein did not cross-react with either anti-rat brain alpha 1 (alpha-subunit of inhibitory G-protein, G1), alpha 0 (alpha-subunit of other G1-like G-protein, G0) or beta gamma antibodies. 4) Incubation of this G Protein with GTP gamma S caused dissociation of the three polypeptides. 5) The 27 kDa polypeptide showed GTP-binding activity and enhanced the phosphatidylinositol 4,5-bisphosphate hydrolysis by purified PI-phospholipase C. These results suggest that the PI-phospholipase C-associated G-protein in calf thymocytes may be a novel one and that it is involved in the regulation of PI-phospholipase C activity.     

A new GTP-binding protein in differentiated human leukemic (HL-60) cells serving as the specific substrate of islet-activating protein, pertussis toxin

A GTP-binding protein serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, was partially purified from human leukemic (HL-60) cells that had been differentiated into neutrophil type. The partially purified protein, referred to as GHL, predominantly consisted of at least two polypeptides with molecular masses of 40,000 daltons (alpha) and 36,000 or 35,000 daltons (beta). The structure was similar to Gi or Go previously purified from rat brain as an alpha beta gamma-heterotrimeric IAP substrate (Katada, T., Oinuma, M., and Ui, M. (1986) J. Biol. Chem. 261, 8182-8191), although the existence of the gamma of GHL was unclear. The 40,000-dalton polypeptide contained the site for IAP-catalyzed ADP-ribosylation and the binding site for guanine nucleotide with a high affinity. The 36,000- and 35,000-dalton polypeptides were cross-reacted with the affinity-purified antibody raised against the beta of brain Gi and Go. Limited proteolysis with trypsin and immunoblot analyses with the use of the affinity-purified antibodies raised against the alpha of brain Gi or Go indicated that the alpha of GHL was different from the alpha of Gi or Go. Kinetics of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding to GHL was also quite different from that to brain Gi or Go. Incubation of GHL with GTP gamma S resulted in a resolution into GTP gamma S-bound alpha and beta(gamma) thus purified had abilities to inhibit a membrane-bound adenylate cyclase activity and to associate with the alpha of brain IAP substrate in a fashion similar to the beta gamma of brain IAP substrates, suggesting that there were no significant differences in the biological activities between the beta(gamma) of GHL and those of Gi or Go. Physiological roles of the new GTP-binding protein, GHL, purified from the neutrophil-like cells in receptor-mediated signal transduction are discussed.     

A truncated recombinant alpha subunit of Gi3 with a reduced affinity for beta gamma dimers and altered guanosine 5'-3-O-(thio)triphosphate binding.

The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Conformations of the alpha 39, alpha 41, and beta.gamma components of brain guanine nucleotide-binding proteins. Analysis by limited proteolysis

We have recently purified two proteins, alpha 39 and alpha 41, from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both proteins bind guanine nucleotides and interact with beta.gamma units. We have used limited proteolysis by trypsin to probe the structure and the conformational states of these proteins. The guanosine 5'-O-(thiotriphosphate) (GTP gamma S)-liganded alpha 41 protein is cleaved into stable 39- and 24/25-kDa products which appear at the same rate. In addition, an 18-kDa peptide is seen. These products are also formed from GDP- or GTP-liganded alpha 41 but are less stable. Cleavage of alpha 39 is different. With GTP gamma S stable 37-kDa product predominates while with GTP or GDP the 37-kDa fragment appears transiently, followed by 24/25-kDa fragments which are stable in the presence of guanine nucleotides but rapidly cleaved without ligand. A 17-kDa peptide is also formed with GTP or GDP. The beta.gamma unit is cleaved by trypsin to stable peptides, a 26/27-kDa doublet and a 14-kDa peptide. Addition of beta.gamma slows tryptic cleavage of alpha 41 but not alpha 39. ADP-ribosylation of alpha 39 and alpha 41 by pertussis toxin affects their conformation in distinct ways which are clearly brought out by the GTP-liganded state. In contrast to unmodified alpha 41, ADP-ribosylated and GTP-liganded alpha 41 is proteolyzed very slowly and without formation of a 39-kDa intermediate. GTP gamma S seems to override the effect of ADP-ribosylation so that cleavage is more rapid and goes via the 39-kDa product. ADP-ribosylation affects alpha 39 more subtly. The GTP-liganded protein is first cleaved to the 37-kDa product and then degraded without forming the 24/25-kDa fragment. These results suggest that ADP-ribosylation might affect the conformation and function of these related proteins differently. The site of [32P]ADP-ribosylation is on the 18-kDa product of alpha 41 and on the 17-kDa product of alpha 39. We have raised polyclonal antibodies against alpha 39 and beta in rabbits and used the antibodies to examine antigenic sites on alpha 39 and beta. The antigenic determinants of alpha 39 are located over most of the native tryptic peptides. Tryptic cleavage of alpha 41 leads to rapid loss of cross-reactivity with anti-alpha 39 antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Characterization of the guinea pig lung membrane leukotriene D4 receptor solubilized in an active form. Association and dissociation with an islet-activating protein-sensitive guanine nucleotide-binding protein

Membrane fractions from the guinea pig lung had high- and low-affinity binding sites for LTD4 with Kd values of 0.016 and 9.1 nM, respectively. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or by prior treatment of the membrane with islet-activating protein (IAP), the high-affinity site shifted to a low-affinity state. Consistently, a 41-kDa protein was ADP-ribosylated by treatment of the lung membranes with IAP, and this event was inhibited by the addition of GTP gamma S. We solubilized the LTD4 receptor from the lung membranes in an active form with 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 10% glycerol. On a gel filtration column, the binding activity was eluted at the volume corresponding to a Mr of 70,000 or over 500,000 in the presence or absence of Mg2+ (5-20 mM), respectively, in solubilizing buffers. The Kd value of [3H]LTD4 binding to the 70-kDa protein was similar to the low-affinity binding constant of the membrane and was insensitive to GTP gamma S. The preparation solubilized in the absence of Mg2+ showed both high- and low-affinity binding sites for LTD4, and the addition of GTP gamma S shifted the high-affinity site to a low-affinity one. Thus, 1) the LTD4 receptor is coupled to an IAP-sensitive GTP-binding protein, 2) this GTP-binding protein is dissociable from the receptor by solubilizing the lung membrane with CHAPS and Mg2+, and 3) the receptor associated to or dissociated from a GTP-binding protein exhibited a high- or low-affinity state, respectively. These data provide an insight into the molecular mechanism of regulation of the LTD4 receptor signaling process by association and dissociation with an IAP-sensitive GTP-binding protein.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

G protein subunit interactions. Studies with biotinylated G protein subunits

Modification of bovine brain G proteins by an N-hydroxysuccinimide ester of biotin has been studied. In the presence of GDP, but in the absence of Mg2+, neither guanine nucleotide binding nor GTPase activity of the protein was altered by modification using less than 1.25 mM biotin derivative with 1 mg/ml G protein. Under these conditions the alpha subunit was modified more extensively than the beta and gamma subunits. However, biotinyl-alpha was less readily bound to streptavidin-agarose than was the less modified beta subunit. Biotinyl-beta gamma was isolated from the modified, intact G protein and further characterized to determine if biotinylation alters its functional properties. Isolated biotinyl-beta gamma and unmodified beta gamma were equivalent based upon: 1) inhibition of the S49 cell membrane adenylyl cyclase, 2) changes in hydrodynamic parameters after being recombined with isolated alpha and treated with guanine nucleotides or complexes of fluoride and aluminum, and 3) competition for isolated alpha binding to biotinyl-beta gamma immobilized previously on streptavidin-agarose. Biotinyl-beta gamma prebound to streptavidin-agarose was 70-100% functional, based upon binding of isolated alpha subunits. Estimates of the affinity of alpha binding to biotinyl-beta gamma indicate that bovine brain alpha 41 has a 10-15-fold higher affinity for beta gamma than does alpha 39. Nonhydrolyzable guanine nucleotides and complexes of fluoride and aluminum decreased binding of either alpha 39 or alpha 41 to biotinyl-beta gamma, and these effects were dependent upon the amount of Mg2+ present. GTP decreased binding of alpha 39, but not alpha 41, to biotinyl-beta gamma. These results indicate that GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact membrane environment or the participation of activating receptors or other membrane-associated proteins. They further indicate that biotinylation of beta gamma does not alter its functional properties and that it can be used for studying G protein subunit interactions.     

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.     

Guanine nucleotides and magnesium dependence of the association states of the subunits of transducin

When GTP gamma S is bound to transducin (T), the two subunits T alpha X GTP gamma S and T beta gamma dissociate, independently of the ionic environment. When GDP is bound, these subunits are associated as a monomeric T alpha X GDP-T beta gamma complex of 75 kDa when the ionic environment is comparable to that of the cytoplasm, but they dissociate in the presence of 10-100 mM Mg2+ or Ca2+. Using this property, the subunits could be separated and purified by a rapid one-step procedure on an ion-exchange column (FPLC), and their molecular masses were verified by neutron small angle scattering. The physiological relevances of the dissociating effect of Mg2+ are discussed.     

Chemotactic peptide receptor-supported ADP-ribosylation of a pertussis toxin substrate GTP-binding protein by cholera toxin in neutrophil-type HL-60 cells

A 40-kDa protein, in addition to the alpha-subunits of Gs (a GTP-binding protein involved in adenylate cyclase stimulation), was [32P]ADP-ribosylated by cholera toxin (CT) in the membranes of neutrophil-like HL-60 cells, only if formyl Met-Leu-Phe (fMLP) was added to the ADP-ribosylation mixture. The 40-kDa protein proved to be the alpha-subunit of Gi serving as the substrate of pertussis toxin, islet-activating protein (IAP). No radioactivity was incorporated into this protein in membranes isolated from HL-60 cells that had been exposed to IAP. Gi-alpha purified from bovine brain and reconstituted into IAP-treated cell membranes was ADP-ribosylated by CT plus fMLP. Gi-alpha was ADP-ribosylated by IAP, but not by CT plus fMLP, in membranes from cells that had been pretreated with CT plus fMLP. When membrane Gi-alpha [32P]ADP-ribosylated by CT plus fMLP or IAP was digested with trypsin, the radiolabeled fragments arising from the two proteins were different from each other. These results suggest that CT ADP-ribosylates Gi-alpha in intact cells when coupled fMLP receptors are stimulated and that the sites modified by two toxins are not identical. CT-induced and fMLP-supported ADP-ribosylation of Gi-alpha was favored by Mg2+ and allow concentrations of GTP or its analogues but suppressed by GDP. The ADP-ribosylation did not occur at all, even in the presence of ADP-ribosylation factor that supported CT-induced modification of Gs, in phospholipid vesicles containing crude membrane extract in which Gi was functionally coupled to stimulated fMLP receptors. Thus, Gi activated via coupled receptors is the real substrate of CT-catalyzed ADP-ribosylation. This reaction may depend on additional factor(s) that are too labile to survive the process of membrane extraction.     

The GTP-binding protein of rod outer segments. II. An essential role for Mg2+ in signal amplification

The role of Mg2+ in the GTP hydrolytic cycle was investigated by using purified subunits (G alpha and G beta, gamma) of the GTP-binding protein isolated from Bufo marinus rod outer segments (ROS). Mg2+ markedly stimulated the rate of GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma-s) binding to G alpha. This effect was especially striking in the presence of very small quantities of illuminated ROS disc membranes. GTP hydrolysis could occur in the absence of Mg2+, and Mg2+ increased the rate of GTP hydrolysis only about 50%. These data indicate that Mg2+ plays a fundamental role in amplification of the photon signal by markedly stimulating the rate of formation of GTP X G alpha complexes by very small amounts of illuminated rhodopsin while producing only a modest increase in the rate of GTP hydrolysis. Following hydrolysis of GTP, GDP X G alpha could reassociate with illuminated or unilluminated ROS disc membranes in the presence or absence of Mg2+. In the absence of guanine nucleotides, release of GDP from G alpha bound to illuminated disc membranes was detected in the presence or absence of Mg2+. Moreover, Mg2+ did not affect the rate of GDP release from membrane-bound G alpha. Illumination of B. marinus crude ROS disc membrane preparations markedly reduced pertussis toxin-mediated ADP-ribosylation of a 39,000 Mr (G alpha) protein in the presence but not in the absence, of Mg2+. Moreover, extensive dialysis of illuminated (but not unilluminated) crude ROS disc membranes against a Mg2+-containing buffer caused a marked reduction in the subsequent ADP-ribosylation of G alpha, even when Mg2+ was not present during the ADP-ribosylation step. This reduction was reversed by the addition of GDP or a GDP analogue (but not GMP or hydrolysis-resistant GTP analogues) during the ADP-ribosylation step. Dialysis of crude ROS disc membrane preparations (illuminated or unilluminated) against a Mg2+ -free buffer did not reduce the subsequent ADP-ribosylation of G alpha. These data indicate that Mg2+, in the presence of photolysed rhodopsin, can stimulate the release of GDP from crude preparations of ROS disc membranes. Four lines of evidence suggest that G alpha and G beta, gamma have Mg2+-binding site(s). When stored at 4 degrees C, in the absence of glycerol, G beta, gamma was more stable in the absence than in the presence of Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)