Guanine nucleotides modulate the effects of brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus

The release of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A (BFA) action, preceding the movement of Golgi membrane into the ER. ATP depletion also causes the reversible redistribution of the 110-kD protein from Golgi membrane into the cytosol, although no Golgi disassembly occurs. To further define the effects of BFA on the association of the 110-kD protein with the Golgi apparatus we have used filter perforation techniques to produce semipermeable cells. All previously observed effects of BFA, including the rapid redistribution of the 110-kD protein and the movement of Golgi membrane into the ER, could be reproduced in the semipermeable cells. The role of guanine nucleotides in this process was investigated using the nonhydrolyzable analogue of GTP, GTP gamma S. Pretreatment of semipermeable cells with GTP gamma S prevented the BFA-induced redistribution of the 110-kD protein from the Golgi apparatus and movement of Golgi membrane into the ER. GTP gamma S could also abrogate the observed release of the 110-kD protein from Golgi membranes which occurred in response to ATP depletion. Additionally, when the 110-kD protein had first been dissociated from Golgi membranes by ATP depletion, GTP gamma S could restore Golgi membrane association of the 110-kD protein, but not if BFA was present. All of these effects observed with GTP gamma S in semipermeable cells could be reproduced in intact cells treated with AlF4-. These results suggest that guanine nucleotides regulate the dynamic association/dissociation of the 110-kD protein with the Golgi apparatus and that BFA perturbs this process by interfering with the association of the 110-kD protein with the Golgi apparatus.     

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

Two distinct populations of ARF bound to Golgi membranes

ADP-ribosylation factor (ARF) is a small molecular weight GTP-binding protein (20 kD) and has been implicated in vesicular protein transport. The guanine nucleotide, bound to ARF protein is believed to modulate the activity of ARF but the mechanism of action remains elusive. We have previously reported that ARF binds to Golgi membranes after Brefeldin A-sensitive nucleotide exchange of ARF-bound GDP for GTP gamma S. Here we report that treatment with phosphatidylcholine liposomes effectively removed 40-60% of ARF bound to Golgi membranes with nonhydrolyzable GTP, presumably by competing for binding of activated ARF to lipid bilayers. This revealed the presence of two different pools of ARF on Golgi membranes. Whereas total ARF binding did not appear to be saturable, the liposome-resistant pool is saturable suggesting that this pool of ARF is stabilized by interaction with a Golgi membrane-component. We propose that activation of ARF by a guanine nucleotide-exchange protein results in association of myristoylated ARF GTP with the lipid bilayer of the Golgi apparatus. Once associated with the membrane, activated ARF can diffuse freely to associate stably with a target protein or possibly can be inactivated by a GTPase activating protein (GAP) activity.     

Thiophosphoryl guanine nucleotide analogues inhibit the (Na+,K+)-ATPase.

The effects of several guanine nucleotide analogues on (Na+,K+)-ATPase activity of membranes isolated from several tissues were analyzed to determine if a G-protein might be involved in the hormonal regulation of the (Na+,K+)-ATPase. Submillimolar concentrations of GTP gamma S, but not GMPPNP, inhibit rat skeletal muscle and axolemma, but not kidney, (Na+,K+)-ATPase activity. Furthermore, GDP beta S does not reverse GTP gamma S inhibition, but rather itself slightly inhibits (Na+,K+)-ATPase activity. Dithiothreitol can block and reverse GTP gamma S inhibition of skeletal muscle (Na+,K+)-ATPase; the results obtained with axolemma membranes are complicated by the inhibition of (Na+,K+)-ATPase activity in these membranes by DTT. Results showing that high membrane concentrations can mute the inhibitory action of GTP gamma S suggest that a minor contaminant in GTP gamma S preparations is responsible for inhibiting (Na+,K+)-ATPase activity. Neither vanadate, a heavy metal, GDP, phosphate, nor thiophosphate, however, is responsible for this inhibition, and the inhibitory activity elutes with GTP gamma S from Sephadex G-10 columns. It is concluded that GTP gamma S or a structural derivative of GTP gamma S inhibits the (Na+,K+)-ATPase, in a tissue-specific manner, not by interaction with a G-protein as a GTP analogue, but through a direct chemical interaction with the (Na+,K+)-ATPase or some regulatory protein. The terminal SH group of the nucleotide analogue is probably required for this interaction.     

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)     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

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 (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical dissection of AP-1 recruitment onto Golgi membranes

Recruitment of the Golgi-specific AP-1 adaptor complex onto Golgi membranes is thought to be a prerequisite for clathrin coat assembly on the TGN. We have used an in vitro assay to examine the translocation of cytosolic AP-1 onto purified Golgi membranes. Association of AP-1 with the membranes required GTP or GTP analogues and was inhibited by the fungal metabolite, brefeldin A. In the presence of GTP gamma S, binding of AP-1 to Golgi membranes was strictly dependent on the concentration of cytosol added to the assay. AP-1 recruitment was also found to be temperature dependent, and relatively rapid at 37 degrees C, following a lag period of 3 to 4 min. Using only an adaptor-enriched fraction from cytosol, purified myristoylated ARF1, and Golgi membranes, the GTP gamma S-dependent recruitment of AP-1 could be reconstituted. Our results show that the association of the AP-1 complex with Golgi membranes, like the coatomer complex, requires ARF, which accounts for the sensitivity of both to brefeldin A. In addition, they provide the basis for a model for the early biochemical events that lead to clathrin-coated vesicle formation on the TGN.     

Nucleotide regulatory protein-mediated activation of phospholipase C in human polymorphonuclear leukocytes is disrupted by phorbol esters

Polymorphonuclear leukocytes (PMNs) activate phospholipase C via a guanine nucleotide regulatory (G) protein. Pretreatment of the PMNs with pertussis toxin (PT) or 4-beta-phorbol 12-myristate 13-acetate (PMA) inhibited chemoattractant-induced inositol trisphosphate generation. To determine the loci of inhibition by PT and PMA, G protein-mediated reactions in PMN plasma membranes were examined. Plasma membranes prepared from untreated and PMA-treated PMNs demonstrated equivalent ability of a GTP analogue to suppress high affinity binding of the chemoattractant-N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) to its receptor. The rate, but not the extent, of high affinity binding of GTP gamma[35S] to untreated PMN membranes was stimulated up to 2-fold by preincubation with 1 microM fMet-Leu-Phe. The ability of fMet-Leu-Phe to stimulate the rate of GTP gamma S binding was absent in membranes prepared from PT-treated PMNs, but remained intact in membranes from PMA-treated cells. Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) via phospholipase C could be activated in untreated PMN membranes by either fMet-Leu-Phe plus GTP or GTP gamma S alone at low concentrations of Ca2+ (0.1-1 microM). Membranes prepared from PT-treated PMNs degraded PIP2 upon exposure to GTP gamma S, but not fMet-Leu-Phe plus GTP. In contrast, membranes prepared from phorbol ester-treated PMNs did not hydrolyze PIP2 when incubated with GTP gamma S. Treatment with PT or PMA did not affect the ability of 1 mM Ca2+ to activate PIP2 hydrolysis in PMN membranes, indicating that neither treatment directly inactivated phospholipase C. Therefore, PT appears to block coupling of the chemoattractant receptors to G protein activation, while phorbol esters disrupt coupling of the activated G protein to phospholipase C. The phorbol ester-mediated effect may mimic a negative feedback signal induced by protein kinase C activation by diacylglycerol generated upon activation of phospholipase C.     

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.     

Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.     

Synergistic inhibition of human platelet adenylate cyclase by stable GTP analogs and epinephrine

Adenylate cyclase inhibition by stable GTP analogs and their interaction with epinephrine were studied in human platelet membranes. Whereas basal enzyme activity was increased by these nucleotides, the stable GTP analogs decreased the adenylate cyclase activity stimulated by fluoride or forskolin by maximally 60 to 70%, with the potency order, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanyl-5'-ylimidodiphosphate greater than guanyl-5'-ylmethylenediphosphate. The inhibition of the forskolin-stimulated enzyme by GTP gamma S was half-maximal at about 4 nM, occurred after a time lag period, which was inversely related to the GTP gamma S concentration, and was resistant to washing of the membranes. Prostaglandin E1-stimulated activity exhibited a biphasic response towards GTP gamma S, with activation occurring at low (1 nM) and inhibition at higher GTP gamma S concentrations. The inhibitory effect of GTP gamma S was competitively antagonized by GTP. This antagonism was prevented by epinephrine, which inhibited the stimulated platelet adenylate cyclase in the presence of GTP to the same degree as observed with GTP gamma S alone. In the absence of GTP, epinephrine largely diminished the time lag required for the inhibitory action of GTP gamma S. Furthermore, the decrease in final activity induced by GTP gamma S was amplified by epinephrine. Whereas the acceleration of the inhibitory action of GTP gamma S was observed at low and high GTP gamma S concentrations, the amplification by epinephrine was observed only at submaximally effective concentrations of GTP gamma S.     

Calcium and GTP: essential components in vesicular trafficking between the endoplasmic reticulum and Golgi apparatus

Ca2+ and GTP hydrolysis are shown to be required for the transport of protein between the ER and the cis-Golgi compartment in semiintact cells, an in vitro system that reconstitutes transport between intact organelles. Transport was inhibited rapidly and irreversibly in the presence of micromolar concentrations of the nonhydrolyzable GTP analogue, GTP gamma S. The transport block in the presence of GTP gamma S was found to be distal to a post-ER, pre-Golgi compartment where proteins accumulate during incubation at 15 degrees C. In addition, transport was completely inhibited in the absence of free Ca2+. A sharp peak defining optimal transport between the ER and the cis-Golgi was found to occur in the presence of 0.1 microM free Ca2+. Inhibition of transport in the absence of free Ca2+ was found to be fully reversible allowing the step inhibited by GTP gamma S to be assigned to a position intermediate between the ER and the Ca2+ requiring step. The results suggest that GTP hydrolysis may trigger a switch to insure vectorial transport of protein along the ER/Golgi pathway, and that a free Ca2+ level similar to the physiological levels found in interphase cells is essential for a terminal step in vesicle delivery to the cis-Golgi compartment.     

Regulation of brain phosphatidylinositol-4-phosphate kinase by GTP analogues. A potential role for guanine nucleotide regulatory proteins

Incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4,5-bisphosphate (PIP2) in membranes isolated from rat brain was enhanced in a concentration-dependent manner by the GTP analogue guanosine 5'-O-(thio)triphosphate (GTP gamma S). In contrast, neither the labeling of phosphatidylinositol 4-phosphate in the same membranes nor PIP kinase activity in the soluble fraction were stimulated by GTP gamma S. Synthesis of [32P]PIP2 was not stimulated by GTP, GDP, GMP, or ATP; however, the stimulatory effects of GTP gamma S were antagonized by GTP, GDP, and guanosine 5'-O-thiodiphosphate (GDP beta S). The nucleotide-stimulated labeling of PIP2 was not due to protection of [gamma-32P] ATP from hydrolysis, activation of PIP2 hydrolysis by phospholipase C, or inhibition of PIP2 hydrolysis by its phosphomonoesterase. Therefore, phosphatidylinositol 4-phosphate kinase activity in brain membranes may be regulated by a guanine nucleotide regulatory protein. This system may enhance the resynthesis of PIP2 following receptor-mediated activation of phospholipase C.     

Stimulation and inhibition of human platelet adenylylcyclase by thiophosphorylated transducin beta gamma-subunits.

The effect of beta gamma-dimers isolated from the retinal guanine nucleotide-binding protein (G protein) transducin eluted from illuminated bovine rod outer segment membranes with GTP, guanosine 5'-O-(beta, gamma-imino)triphosphate (Gpp(NH)p), or guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) on basal and forskolin-stimulated adenylylcyclase activities in membranes of human platelets was studied. beta gamma-Subunits isolated from transducin eluted with GTP gamma S (TD beta gamma GTP gamma S) had a concentration-dependent stimulatory effect on basal adenylylcyclase activity. The stimulatory agonist prostaglandin E1 increased the potency and the maximum extent of stimulation due to TD beta gamma GTP gamma S). With a similar concentration dependence, TD beta gamma GTP gamma S exerted an inhibitory influence on forskolin-stimulated adenylylcyclase activity. At the same concentrations, beta gamma-dimers isolated with either GTP or Gpp(NH)p did not alter enzyme activities. The observed effects of TD beta gamma GTP gamma S were similar to those of directly added GTP gamma S with regard to maximum levels, time dependence, and persistence; however, TD beta gamma GTP gamma S was approximately 10-fold more potent than GTP gamma S. Treatment of TD beta gamma GTP gamma S, but not of free GTP gamma S, with hydroxylamine caused a loss of adenylylcyclase regulation by TD beta gamma GTP gamma S. The data presented indicated that TD beta gamma GTP gamma S potently and efficiently activates the stimulatory and inhibitory G proteins of adenylylcyclase in human platelet membranes. Furthermore, evidence is provided suggesting that the observed effects of TD beta gamma GTP gamma S, which can be thiophosphorylated by GTP gamma S at the beta-subunit (Wieland, T., Ulibarri, I., Gierschik, P., and Jakobs, K. H. (1991) Eur. J. Biochem. 196, 707-716), are due to formation of GTP gamma S at the G proteins.     

A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.     

2'(3')-O-(N-methylanthraniloyl)-substituted GTP analogs: a novel class of potent competitive adenylyl cyclase inhibitors

2'(3')-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTP gamma S) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on G alpha(s)- and G alpha(i)-protein-mediated signaling. MANT-GTP gamma S/MANT-GppNHp had lower affinities for G alpha(s) and G alpha(i) than GTP gamma S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G alpha fusion proteins. MANT-GTP gamma S was much less effective than GTP gamma S at disrupting the ternary complex between the formyl peptide receptor and G alpha(i2). MANT-GTP gamma S/MANT-GppNHp non-competitively inhibited GTP gamma S/GppNHp-, AlF(4)(-)-, beta(2)-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in G alpha(s)-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP gamma S/MANT-GppNHp was not due to G alpha(s) inhibition because it was also observed in G alpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked AC inhibition by GTP gamma S/GppNHp in S49 cyc(-) membranes but enhanced the potency of MANT-GTP gamma S/MANT-GppNHp at inhibiting AC by approximately 4-8-fold. MANT-GTP gamma S and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 and 160 nm, respectively. The K(i) value for MANT-GppNHp at insect cell AC was 155 nm. Collectively, MANT-GTP gamma S/MANT-GppNHp bind to G alpha(s)- and G alpha(i)-proteins with low affinity and are ineffective at activating G alpha. Instead, MANT-GTP gamma S/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.     

Activation of signal-transducing guanine-nucleotide-binding regulatory proteins by guanosine 5'-[gamma-thio]triphosphate. Information transfer by intermediately thiophosphorylated beta gamma subunits

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific epidermal growth factor receptors on porcine thyroid cell membranes

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.