Interleukin-1 signal transduction. Increased GTP binding and hydrolysis in membranes of a murine thymoma line (EL4)

The post-receptor events which follow the binding of interleukin 1 (IL1) to cells are unclear. The present studies provide evidence for the activation of a guanine nucleotide binding protein (G protein) by IL1 in the membranes of an IL1 receptor-rich strain (NOB-1) of the EL4 murine thymoma line. IL1 alpha and beta increased the binding of the GTP analogue [35S]guanosine 5'-[gamma-thiol]trisphosphate (GTP gamma S) to membranes prepared from these cells. By 1 min after addition of IL1 there was a 2-fold enhancement in binding which was dose dependent in the range 0.1-100 ng/ml. A qualitatively similar result was obtained with IL1 beta although it was 10 times less potent. Specific neutralizing antisera to IL1 alpha and IL1 beta abolished the response. Experiments in which the concentration of [35S]GTP gamma S was varied revealed that IL1 increased the affinity of the binding sites for [35S]GTP gamma S and not their number. IL1 alpha was shown to stimulate GTPase activity in the membranes, the time and concentration dependence of this was similar to that observed for increased [35S]GTP gamma S binding. Half-maximal enhancement of [35S]GTP gamma S binding by IL1 alpha, measured after 4 min, occurred at 5% IL1 receptor occupancy. Maximal stimulation was achieved when 30% of receptors were occupied. Experiments with pertussis and cholera toxins revealed that pretreating membranes with pertussis toxin (100 ng/ml) inhibited by 50% the IL1-induced [35S]GTP gamma S binding and [gamma-32P]GTP hydrolysis. Cholera toxin (100 ng/ml) was without effect. However, both pertussis and cholera toxins at concentrations of 100 ng/ml inhibited IL1-induced IL2 secretion in EL4 NOB-1 cells. These results show that the IL1 receptor of a responsive thymoma line activates, and may be coupled to, a G protein(s). This is a possible mechanism of IL1 signal transduction.     

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)     

Effects of adenyl nucleotides and carbachol on cooperative interactions among G proteins.

Muscarinic agonists and adenyl nucleotides are noncompetitive modulators of sites labeled by [35S]GTP gamma S in washed cardiac membranes from Syrian golden hamsters. Specific binding of the radioligand and its inhibition by either GTP gamma S or GDP reveals three states of affinity for guanyl nucleotides. In the absence of adenyl nucleotide, carbachol promotes an apparent interconversion of sites from higher to lower affinity for GDP; the effect recalls that of guanyl nucleotides on the binding of agonists to muscarinic receptors. In the presence of 0.1 mM ATP gamma S, the binding of [35S]GTP gamma S is increased at concentrations up to about 50 nM and decreased at higher concentrations. At a radioligand concentration of 160 pM, binding exhibits a bell-shaped dependence on the concentration of both ATP gamma S and AMP-PNP; with ADP and ATP, there is a second increase in bound [35S]GTP gamma S at the highest concentrations of adenyl nucleotide. ATP gamma S and AMP-PNP also modulate the effect of GDP, which itself emerges as a cooperative process: that is, binding of the radioligand in the presence of AMP-PNP exhibits a bell-shaped dependence on the concentration of GDP; moreover, the GDP-dependent increase in bound [35S]GTP gamma S is enhanced by carbachol. The interactions among GDP, GTP gamma S, and carbachol can be rationalized quantitatively in terms of a cooperative model involving two sites tentatively identified as G proteins. Both GTP gamma S and GDP exhibit negative homotropic cooperativity; carbachol enhances the homotropic cooperativity of GDP and induces or enhances positive heterotropic cooperativity between GDP and [35S]GTP gamma S. An analogous mechanism may underlie the guanyl nucleotide-dependent binding of agonists to muscarinic receptors. The data suggest that the binding properties of G proteins and their associated receptors reflect cooperative effects within heterooligomeric arrays; agonist-induced changes in cooperativity may facilitate the exchange of GTP for bound GDP and thereby constitute the mechanism of G protein activation in vivo.     

"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.     

Detection of G Proteins in Purified Bovine Brain Myelin

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal amplification in HL-60 granulocytes. Evidence that the chemotactic peptide receptor catalytically activates guanine-nucleotide-binding regulatory proteins in native plasma membranes

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) [G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal (less than or equal to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (greater than 100-fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive [35S]GTP[S]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.     

Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1.

We describe the reconstitution using purified proteins of the m1 muscarinic cholinergic pathway that activates phosphatidylinositol 4,5-bisphosphate-specific phospholipase C via the G protein Gq/11. Recombinant m1 muscarinic receptor was co-reconstituted in lipid vesicles with either hepatic Gq/11 or with cerebral alpha q/11 and beta gamma subunits. The rate of [35S]GTP gamma S binding to the reconstituted vesicles was stimulated 20-50-fold by agonist. Maximal receptor-catalyzed binding was 7 mol of GTP gamma S bound per mol of receptor. The m2 muscarinic receptor was a poor activator of Gq/11. The binding of [alpha-32P]GTP to [gamma-32P]GTP to m1/Gq/11 vesicles indicated that the receptor could maintain up to 40% of the total coupled Gq/11 in the GTP bound state. The rate of hydrolysis of bound GTP, 0.8 min-1, is consistent with the rate predicted from the GTP binding data but is 3-5-fold lower than rates reported for other trimeric G proteins. Agonist-stimulated photo-affinity labeling with gamma-(4-azidoanilido)-[alpha-32P]GTP indicated that the receptor catalyzed binding to both alpha q and alpha 11 with about equal efficiency. Receptor-catalyzed activation of Gq/11 by GTP gamma S, measured as the ability to activate purified phospholipase C-beta 1, paralleled receptor-catalyzed [35S]GTP gamma S binding. Co-reconstitution of receptor, Gq/11, and phospholipase C-beta 1 restored GTP gamma S-dependent carbachol-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate. The m1 receptor, Gq/11, and phospholipase C-beta 1 are thus sufficient to initiate the hormonal inositol trisphosphate/diacylglycerol signaling pathway without additional proteins.     

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)     

ADP receptor-induced activation of guanine-nucleotide-binding proteins in human platelet membranes.

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[gamma S]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[gamma S] without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1-10 microM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 microM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[gamma S] by maximally about twofold, with half-maximal increase occurring at 0.1 microM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (1S)-adenosine 5'-O-(1-thiotriphosphate) [(Sp)-ATP[alpha S]]. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[gamma S] in human platelet membranes, but without being inhibited by (Sp)-ATP[alpha S]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified.     

Modulation of 5-Hydroxytryptamine1A Receptor Density by Nonhydrolyzable GTP Analogues

Co-incubation of rat cortical membranes with 10(-4) M GTP results in a competitive inhibition of 5-hydroxytryptamine1A (5-HT1A) receptor binding sites labeled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin [( 3H]8-OH-DPAT). Preincubation of cortical membranes with 10(-4) M GTP does not significantly change either KD or Bmax values, indicating that the effect of GTP is reversible. By contrast, GTP gamma S and 5'-guanylylimidodiphosphate (GppNHp) are nonhydrolyzable analogues of GTP which lengthen the time course of guanine nucleotide activation of guanine nucleotide binding proteins (G proteins) and thereby alter G protein-receptor interactions. These nonhydrolyzable GTP analogues were used to characterize the effects of persistent alterations in G proteins on [3H]8-OH-DPAT binding to 5-HT1A receptors. Co-incubation of rat cortical membranes with either 10(-4) M GTP gamma S or GppNHp results in a decrease in both the affinity and apparent density of 5-HT1A binding sites. Co-incubation with the nonhydrolyzable nucleotides reduces the affinity of [3H]8-OH-DPAT binding by 65-70% and lowers the density of the binding site by 53-61%. Similarly, preincubation of membranes with a 10(-4) M concentration of either GTP gamma S or GppNHp significantly increases the KD value and reduces the Bmax value of [3H]8-OH-DPAT binding. These results indicate that GTP gamma S and GppNHp induce persistent changes in 5-HT1A receptor-G protein interactions that are reflected as a decrease in the density of binding sites labeled by [3H]8-OH-DPAT.     

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.     

The effects of hydrostatic pressure on pertussis toxin-catalyzed ribosylation of G proteins from deep-living macrourid fishes

To test the effects of hydrostatic pressure on the coupling of receptors to guanyl nucleotide binding reglatory proteins (G proteins) in transmembrane signaling, pertussis toxin (PTX)-catalyzed [32P]ADP-ribosylation was used to probe the guanyl nucleotide-binding proteins Gi and G(o) in brain membranes from four marine teleosts. These macrourids, Coryphaenoides pectoralis, Coryphaenoides cinereus, Coryphaenoides filifer and Coryphaenoides armatus, span depths from 200 to 5400 m. Pertussis toxin specifically labelled proteins of 39-41 kDa. The PTX-catalyzed [32P]ADP-ribosylation reaction was linear for 7 h. Added guanyl nucleotides (guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(3-thiotriphosphate)(GTP[S])) at concentrations up to 1000 microM did not affect ribosylation at atmospheric pressure. Under basal conditions the Gi/G(o) protein population appears to be uncoupled from receptors and bound with GDP. Pressures up to 476 atm were tested in the absence and presence of added guanyl nucleotides, 100 microM GDP and 100 microM GTP[S]. [32P]ADP-ribosylation in brain membranes from the deeper-occurring C. cinereus, C. filifer and C. armatus was not inhibited by increased pressure in the presence of 100 microM GDP. Increasing pressure decreased ribosylation in brain membranes of C. pectoralis. In the presence of 100 microM GTP[S], increased pressure inhibited ribosylation in all species. Pressure appears to enhance the efficacy of GTP[S] in dissociating the heterotrimeric holoprotein.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

Characterization and site-directed mutagenesis of a low M(r) GTP-binding protein, ram p25, expressed in Escherichia coli.

The ram gene encodes a GTP-binding protein with a M(r) of 25,068 (Nagata, K., Satoh, T., Itoh, H., Kozasa, T., Okano, Y., Doi, T., Kaziro, Y., and Nozawa, Y. (1990) FEBS Lett. 275, 29-32). It has a putative effector domain very similar to that of yeast SEC4 protein, and shares 40% identity and 60% homology with it, respectively. In order to analyze the biochemical properties, ram cDNA was engineered and inserted into a bacterial expression vector; this allowed the production at a high level of soluble recombinant ram p25 in Escherichia coli. The purified ram p25 contained an equimolar amount of GDP. The purified protein bound approximately 1 mol of [35S]guanosine 5'-O-(thiotriphosphate) GTP gamma S)/mol of protein, with a Kd value of 120 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by ATP and ADP. In the presence of 10 mM Mg2+, the dissociation of [8,5'-3H]GDP and [35S]GTP gamma S from ram p25 occurred with rates of 0.015 min-1 and 0.004 min-1, respectively, showing that the ram p25 has a higher affinity for GTP than GDP. The rate of release of Pi from [gamma-32P]GTP-bound ram p25 was calculated to be 0.011 min-1. The contribution of guanine nucleotide-binding and GTP-hydrolysis domains of the protein to its biochemical activities was investigated by site-directed mutagenesis. Substitution of Val for Gly at position 19 resulted in disappearance of [35S]GTP gamma S- and [3H]GDP-binding activity in spite of good expression of the protein. Mutations of Thr41 to Ser, Ala76 to Thr, and Asn133 to His slightly increased the rates of [35S] GTP gamma S binding and [3H]GDP dissociation, but had almost no effects on the manner of [gamma-32P]GTP hydrolysis. Replacement of Gln78 with Leu significantly increased the [3H]GDP dissociation rate (7-fold) and decreased GTP hydrolytic activity considerably.     

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.     

Muscarinic acetylcholine receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes

Receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]), to guanine-nucleotide-binding proteins (G-proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholine (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1-1 microM. In contrast, the GDP analog, guanosine 5'-O-(2-thiodiphosphate), could not replace GDP in promoting carbachol-induced increase in [35S]GTP[S] binding. In addition to GDP, agonist-induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required the presence of Mg2+, being half-maximally and maximally effective at about 30 microM and 300 microM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist-stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5-fold, with half-maximal and maximal increase being observed at 5-10 microM and 100 microM, respectively. The agonist-induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two--three-fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of the membranes with pertussis toxin under conditions leading to 95% ADP-ribosylation of the toxin-sensitive G-protein alpha-subunits markedly reduced agonist-stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist-stimulated binding of [35S]GTP[S] to G-proteins can be a sensitive assay for measuring receptor-regulated G-protein activation in native membranes and, furthermore, suggest that one agonist-activated mACh receptor can activate two or three cardiac G-proteins, being mainly members of the pertussis-toxin-sensitive G-proteins.     

Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils.

Arachidonic acid (20:4) and other cis-unsaturated fatty acids exert direct effects on a variety of cells, effects that do not depend on the metabolism of fatty acids via cyclooxygenase or lipoxygenase pathways. In these studies arachidonic acid and other cis-unsaturated fatty acids (but not trans-unsaturated or saturated fatty acids) increased the specific binding of the nonhydrolyzable analog of GTP, [35S]GTP gamma S, to purified neutrophil membrane preparations and elicited superoxide anion generation from intact neutrophils. There was a positive correlation (r = 0.70) between the capacity of fatty acids to increase nucleotide binding and to elicit the respiratory burst. Scatchard plot analysis of binding at equilibrium demonstrated an increase in the number of available GTP binding sites in the presence of 50 microM arachidonic acid. Nonsteroidal antiinflammatory agents interfered with the arachidonic acid effect on [35S]GTP gamma S binding. ADP-ribosylation of the pertussis toxin substrate Gi alpha within the plasmalemma-reduced specific [35S]GTP gamma S binding and blocked arachidonate-dependent enhancement of binding. Moreover, pertussis toxin treatment of intact neutrophils inhibited arachidonic acid-induced superoxide anion generation. The data indicate that arachidonic acid directly activates a GTP binding protein in the neutrophil plasma membrane and may thereby act as a second messenger in signal transduction.     

Affinity labeling of eukaryotic initiation factor 2 and elongation factor 1 alpha beta gamma with GTP analogs

As part of an attempt to understand the specific function and role of each subunit in multisubunit protein synthesis factors, we have attempted to identify the nucleotide binding peptides of eukaryotic initiation factor 2 (eIF-2). To ensure that the interactions were of a specific nature, two general controls were used: first, other protein factors with characterized GTP binding activity were tested; second, all affinity labeling was checked for nucleotide specificity by protection with the authentic nucleotide at a 10-fold molar excess over the affinity reagent. Results with a number of GTP modifying reagents ([alpha-32P]GTP, [alpha-32P]GDP, oxidized [alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GTP, 3'-p-azidobenzoyl-[alpha-32P]GDP, and 5'-p-[8-3H]fluorosulfonylbenzoyl guanosine) indicate that appropriate conditions for both nucleotide and subunit specific labeling have been achieved. Under these conditions all reagents modified the beta subunit of eIF-2. Complementary studies with subunit-deficient forms of eIF-2 also suggest that the beta subunit of eIF-2 is involved with GTP binding. Coupled with other data suggesting that the gamma subunit of eIF-2 might be involved in GTP binding and amino acid sequence data of eIF-2 gamma from which a part of a GTP binding consensus sequence can be localized, support is provided for the concept of alternate GTP binding domains or a GTP binding domain shared between different subunits of eIF-2.     

Cannabinoid receptor stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding in rat brain membranes

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [³⁵S]GTPγS binding in rat brain membranes. Assays were conducted with 0.05 nM [³⁵S]GTPγS, incubated with rat cerebellar membranes, 1–30 μM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [³⁵S]GTPγS binding increased with increasing concentrations of GDP, with 10–30 μM GDP providing approximately 150–200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [³⁵S]GTPγS binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [³⁵S]GTPγS binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [³⁵S]GTPγS binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [³⁵S]GTPγS autoradiography. These results demonstrate that [³⁵S]GTPγS binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.