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Phosphoinositide-specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTP gamma S. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP-pretreated platelets (A-membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B-membranes). This cAMP-mediated inhibition was abolished in the presence of inhibitors of cAMP-dependent protein kinase (A-kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin-catalyzed ADP-ribosylation among control membranes and the two types of phosphorylated membranes (A- and B-membranes). GTP-binding activities of Gs, Gi and GTP-binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP-binding protein is involved in the GTP gamma S-mediated activation of PLC and that cAMP (plus A-kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240-kDa protein), rather than the GTP-binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP-binding protein from PLC.

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.

Electropermeabilized human neutrophils were used to investigate the possible role of G-proteins in the respiratory burst elicited by concanavalin A (Con A). The Con A-induced activation of the NADPH oxidase was not inhibited by either pertussis toxin or cholera toxin. However, the burst was inhibited by GDP and GDPβS providing evidence for the involvement of a G-protein(s). O 2 consumption in Con A-stimulated cells was dependent on both ATP and Mg 2+ . ATP could be substituted by ATPγS but not by the non-hydrolyzable analog AMP-PNP, suggesting involvement of phosphotransferase reactions. It is concluded that at least two distinct types of G-proteins are capable of inducing the respiratory burst in neutrophils and that accumulation of phosphorylated intermediates may be essential for activation of the respiratory burst by the lectin.

Iodopsin (a red-sensitive cone visual pigment) and rhodopsin (a rod pigment) were isolated from chicken retina. They were separately reconstituted into phosphatidylcholine liposomes and then mixed with rod transducin (T alpha and T beta gamma) purified from bovine retina. Iodopsin enhanced, only when irradiated, the binding of GppNHp to T alpha to a similar extent to irradiated rhodopsin. Furthermore, the binding of GppNHp to T alpha in the presence of a photobleaching intermediate of iodopsin preferably required T beta gamma-2 rather than T beta gamma-1, which is very similar in profile to that in the presence of the intermediate of rhodopsin (J. Biol. Chem., in press). These results indicate that the binding domain for transducin in iodopsin should closely resemble that in rhodopsin.

The 48-kDa protein, a major protein of rod photoreceptor cells, is soluble in the dark but associates with the disk membranes when some (5–10%) of their rhodopsin has absorbed light and if this rhodopsin is additionally phosphorylated by ATP and rhodopsin kinase. If rhodopsin has been phosphorylated and regenerated prior to the protein binding experiment, the binding of 48-kDa protein depends on light but no longer on the presence of ATP. Another photoreceptor protein, GTP-binding protein, associates with both phosphorylated and unphosphorylated rhodopsin upon illumination. Excess GTP-binding protein thereby displaces 48-kDa protein from phosphorylated disks; this indicates competition between these two proteins for binding sites on illuminated phosphorylated rhodopsin molecules.

We describe here the existence of previously undescribed GTP-binding proteins within the human neutrophil. These proteins specifically bind guanine nucleotides under conditions in which the previously characterized G-proteins are unable to bind. We have partially purified these proteins and present both functional and immunologic data which indicate that they are unrelated to Gn, the major neutrophil pertussis toxin substrate. An additional protein, of apparent molecular mass 22 kDa, may be related to the ras G-protein family. Analysis of the structural and functional characteristics of these novel proteins will promote a better understanding of the process of neutrophil activation.

Transducin is a GTP-binding protein which mediates the light activation signal from photolyzed rhodopsin to cGMP phosphodiesterase and is pivotal in the visual excitation process. Biochemical studies suggest that the T alpha subunit of transducin is composed of three functional domains, one for rhodopsin/T beta gamma interaction, another for guanine nucleotide binding, and a third for the activation of phosphodiesterase. The integration of the primary sequence of T alpha along with secondary structure, hydropathy and folding topology predictions, and a comparison with homologous proteins have led to the construction of a three-dimensional model of the T alpha subunit. A molecular mechanism which underlies the coupling action of T alpha is suggested on the basis of this model.

Photoreceptor outer segments isolated from squid retina are known to contain a light-activated GTP-binding protein. Here it is shown that these photoreceptors contain around 0.01 mol cyclic GMP per mol rhodopsin. Adding GTP in the dark stimulates the production of 0.0003–0.001 mol cyclic GMP/mol rhodopsin per min. GTP and light cause a 2-fold faster increase in cyclic GMP. These results show that either (1) squid rhodopsin activates a guanylate cyclase, or (2) there is a constant guanylate cyclase activity and photoexcited rhodopsin inhibits a cyclic GMP phosphodiesterase.

Transducin, a GTP-binding protein found in the bovine rod outer segment, mediates the signal coupling between rhodopsin and cGMP phosphodiesterase. Recent observations have revealed that transducin consists of two functional subunits: Tα （Mr 39,000） and Tβγ （Mr 35,000 and 8,000）, and that photolyzed rhodopsin catalyzes the exchange of GTP for GDP bound to Tα and, in this case, Tβγ regulates the binding of GTP to Tα. This process, i. e., transducin activation, is an important amplified step to signal the start of transduction during photon absorption by rhodopsin. Therefore, we intended to elucidate the regulatory mechanism of the transducin activation in retina. We found subspecies of Tβγ during the purification of Tβγ from the bovine retinal rod outer segment. Purified Tβγ was separated into three components by chromatography on Mono-Q, an anion exchange column, equipped with FPLC. One of the components caused substantially no enhancement of GppNHp, a nonhydrolyzable analogue of GTP, binding activity to Tα in the presence of photolyzed rhodopsin. These findings indicate that there are functionally two different forms in Tβγ, i. e., active and inactive forms. Each Tβγ subspecies showed the same mobility as Tβ and Tγ in SDS-polyacrylamide gel electrophoresis （PAGE）. The mobility of Tγ was distinctly different, however, between the active and inactive forms in 8M urea-PAGE and 8M urea-SDS-PAGE. Therefore, it is likely that the functional heterogeneity of Tβγ depends on the structural heterogeneity of Tγ. Purified Tγ was further separated into four subspecies （Tγ-1, Tγ-2, Tγ-3, and Tγ-4） by high-performance liquid chromatography on a C18 reverse phase column. ?However, the amino acid sequences of these four Tγ seemed to be identical to each other. These Tγ subspecies consisted of 68 amino acid residues, in which N-terminal amino acid was Pro and C-terminal amino acid was Gly. These results indicate that the structural heterogeneity of Tγ, which was shown by 8M urea electrophoresis, depends on its structural modification which introduces different surface charges on the molecule.

Two GTP-binding proteins (Gi and Go), which were the substrates for islet-activating protein, pertussis toxin, were purified from bovine cerebral cortical membranes. Both Gi and Go completely inhibited calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. The same concentrations of these proteins, however, had no appreciable effect on the basal phosphodiesterase activity. The isolated Gi alpha and beta gamma subunits of GTP-binding proteins were potent inhibitors of the calmodulin-stimulated phosphodiesterase activity, but Go alpha was very weak. Therefore, the beta gamma subunits were likely to be the major active molecules in the brain membranes. GTP-binding proteins were shown to bind directly to calmodulin in a Ca2+-dependent manner by a gel permeation binding experiment.

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.

Exposure of ‘leaky’ adrenal medullary cells to GTP-γ-S inhibits Ca-dependent exocytosis in bovine cells, but stimulates exocytosis in chicken cells. The inhibitory action on bovine cells persists in the presence of TPA suggesting that in this tissue an inhibitory GTP-binding protein may modulate the action of protein kinase C on exocytosis.

The GTP-binding proteins of Swiss 3T3 cell nuclei were analyzed by filter binding assay and UV cross-linking analysis. The results showed the presence of multiple GTP-binding proteins in the nuclei. Scatchard analysis revealed that the K d value for GTP binding to high-affinity components was 69 nM, that to low-affinity components being 2.7 μM. The GTP-binding activities of some nuclear proteins were found to change significantly in response to the growth conditions of the cells. During culture of cells in medium without serum, the GTP-binding activity of a 140 kDa protein clearly decreased, whereas that of a 40 kDa protein increased.

The effect of sulfhydryl modification on the light-induced interaction between rhodopsin and the peripheral GTP-binding protein of the photoreceptor membrane (G-protein) has been investigated by time-resolved near-infrared light-scattering and polyacrylamide gel electrophoresis. It has been found that the modification of rhodopsin with the alkylating agent N-ethylmaleimide (NEM) does not affect its light-induced interaction with the G-protein. Modification of G-protein with NEM or other sulfhydryl agents prevents any light-induced binding to rhodopsin. Dark-associatiion of G to the membrane as well as the light-induced complex with rhodopsin (once formed) is insensitive to NEM.

Regulation of synaptic membrane adenylate cyclase is likely to involve interaction between neurotransmitter receptors, G-proteins and the adenylate cyclase catalytic unit as well as several other membrane proteins and lipids. Despite intensive study of this system, regulation of guanine nucleotide binding by the G-proteins which stimulate [Gs] or inhibit [Gi] adenylate cyclase has been examined only when those proteins have been purified and removed from the influence of the membrane environment. The hydrolysis-resistant photoaffinity GTP-analog, P3-(4-azidoanilido)-P1 5′-GTP (AAGTP) is able to bind specifically to the G-proteins in rat cerebral cortex synaptic membranes and, in this study, we have used this probe to examine the specificity and selectivity of guanine nucleotide binding to each G-protein without removing those proteins from the synaptic membrane. Marked differences were noted between guanine nucleotide binding data obtained with detergent-soluble G-proteins and data from this in situ approach. In these studies it was found that the affinity of the G-proteins binding AAGTP correlated well with the expression of adenylate cyclase activity, the affinity of both forms of Gs, increasing under conditions favoring the stimulation of that enzyme.

The discovery of GTP-binding proteins in mitochondria is a recent event. These regulatory proteins may be participating in membrane fusion and thereby playing important roles in the physiology of the mitochondrion. So far, it has been proposed that GTP-binding protein mediated membrane fusion may be involved in protein import, steroid hormone production and mitochondrial amalgamation during spermatogenesis.

Here we report the isolation of a cDNA encoding the complete human rab18 protein from histamine-stimulated endothelial cells using differential display. The amino acid sequence showed 98% homology with the previously isolated mouse rab18 protein, which is implicated in apical/basolateral endocytosis. Northern blot analysis revealed two transcripts (2.5 kb and 1 kb) ubiquitously expressed in all examined organs, as well as in human umbilical vein endothelial and aortic cells. In blood cells rab18 transcripts were hardly detectable. The histamine-induced time-dependent increase of rab18 mRNA expression in human umbilical vein endothelial cells and peripheral blood mononuclear cells indicates for the first time a link between receptor-mediated signal transduction and the regulation of rab gene expression. This finding might also imply a role for rab proteins in inflammation.

Various adenine nucleotides activated phospholipase C of FRTL-5 cell membranes in the following order of activity, ATP gamma S greater than ATP greater than AppNp greater than AppCp = ADP greater than MeSATP. This order was well consistent with that observed in intact cells. Such activation occurred only in the presence of appropriate concentrations of GTP gamma S and Ca2+, in a way similar to the norepinephrine-induced activation. NaF, a non-specific GTP-binding protein (G-protein) activator, also stimulated the enzyme. These adenine nucleotides, norepinephrine and NaF-induced activations were inhibited by GDP beta S. We conclude that a G-protein is involved in the adenine nucleotides-induced activation of phospholipase C via P2-purinergic receptor in FRTL-5 cells.

An alpha beta gamma-trimeric GTP-binding protein (Go) serving as the substrate of pertussis toxin-(IAP) catalyzed ADP-ribosylation was purified from rat brain membranes. The constituent alpha-subunit (alpha o) was alkylated with N-ethylmaleimide (NEM), and the functionally important sulfhydryl groups were investigated. There were at least two cysteine residues highly reactive to NEM on the GDP-bound form of alpha o. These alkylations resulted in loss of its ability to be ADP-ribosylated by IAP and to associate with beta gamma, but leaving the GTP-binding site of alpha o intact. The reacted cysteine residues were identified by the sequencing of tryptic fragments of alpha o. One of the alkylation sites was Cys-351, which was four amino acid residues away from the carboxyl-terminus of the molecule. The Cys-351 was proven to be also a site for IAP-catalyzed ADP-ribosylation. Possible roles of cysteine residues on the alpha-subunit of Go are discussed in the functions of the signal transducing protein.

Stimulation by N-formyl-Met-Leu-Phe (fMLP) of rabbit peritoneal neutrophils, in which phosphatidylcholine was preferentially labeled with l-O-[3H]octadecyl lyso platelet-activating factor, activated phospholipase D, resulting in the formation of [3H]PA from [3H]PC. A direct activator of GTP-binding proteins (G-proteins), NaF, also stimulated [3H]PA formation. fMLP-stimulated [3H]PA formation was inhibited by pertussis toxin (IAP) in a time- and dose-dependent manner. IAP also inhibited fMLP-stimulated IP3 formation, but the inhibition of IP3 formation was significantly greater than that of [3H]PA formation. These results indicate that activation of phospholipase D by fMLP in rabbit neutrophils is mediated by an IAP-sensitive G-protein that may be distinct from a phospholipase C-regulating protein.

A synthetic peptide (rab3AL) corresponding to the effector domain of rab3, a small GTP-binding protein, stimulated basal and potentiated Ca(2+)- as well as GTP gamma S-evoked insulin secretion about 2-fold from streptolysin-O permeabilized HIT cells. This effect was specific, since the analogous peptides of ras or rab1 did not affect the exocytotic event. The more than additive effect of rab3AL on Ca2+ or GTP gamma S stimulation indicates a distinct mode of action of the peptide. The partial loss of cytosolic proteins from permeabilized cells was accompanied by a faster run-down of the secretory response to Ca2+ than the one to GTP gamma S. The persistent effect of rab3AL under these conditions points to a membrane localization of its target. These results suggest that rab3 and its effector are involved in the regulation of insulin secretion.

Rab11a is a member of the rab-branch of the ras-like small GTP-binding protein superfamily that is associated with both constitutive and regulated secretory pathways. Using a direct procedure for cDNA cloning of small ras-related GTPases, that is based on the screening of eukaryotic cDNA expression libraries using [α-32P]GTP as a probe, we have isolated two cDNA clones encoding rab11a. Both clones share identical coding sequences, but differ in the length and sequence of their 3′ untranslated regions (3′-UTR). Northern blot hybridisation analysis of various human tissues revealed indeed two mRNA species with lengths of 1.0 and 2.3 kb, respectively. Sequence analysis of the cDNAs identified two different putative polyadenylation signals (AATAAA) at positions 927 and 2302 of the larger transcript. In addition, the 3′-UTR of the larger transcript exhibited several AU-rich elements (ARE) that are believed to control gene expression by regulating the rate of mRNA degradation. Southern blots of human DNA digested with several rare restriction enzymes, and separated by pulse-field gel electrophoresis, yielded the same macro-restriction fragment pattern when hybridised with probes that discriminate between the two transcripts. Taken together, these findings imply that the two mRNA species originate from a single gene, which we have mapped to 15q21.3-q22.31, by the use of diferent polyadenylation sites. As expected, both rab11a-cDNAs yielded the same protein product when transiently expressed in COS-1 cells, and surprisingly, upregulated the proteome expression profile (de novo synthesis or posttranslational modification of preexisting proteins) of a few other, yet unknown GTP-binding proteins.

Polyphosphoinositide hydrolysis was studied in membranes prepared from a human T cell leukemia line, JURKAT, prelabeled with myo-[2-3H]inositol. The formation of inositol bis- and trisphosphates was stimulated in a buffer with 110 nM free Ca2+ with a nonhydrolyzable GTP analogue, GTP gamma S, and NaF plus AlCl3 in a time- and concentration-dependent manner. GTP gamma S and NaF-AlCl3 had no significant effect on the inositol monophosphate level. AlCl3 enhanced the NaF-stimulated release of inositol polyphosphates. Optimum concentrations of NaF and AlCl3 produced 1.5-fold more inositol polyphosphates than that produced by optimum concentration of GTP gamma S. OKT3 monoclonal antibody, an antibody against the T-cell receptor complex, did not stimulate the inositol polyphosphate formation by JURKAT membranes even in the presence of GTP, although the antibody at the concentrations used markedly stimulated the hydrolysis of polyphosphoinositides in intact JURKAT cells.

Human promyelocytic leukemic (HL-60) cells were induced to differentiate into neutrophil- or macrophage-like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12-myristate 13-acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid-induced differentiation of HL-60 cells was, but the Bt2cAMP- or PMA-induced one was not, inhibited by prior exposure of the cells to islet-activating protein (IAP), pertussis toxin. The IAP-induced inhibition was correlated with the toxin-catalyzed ADP-ribosylation of a membrane GTP-binding protein with a molecular mass of 40 kDa. Thus, the IAP-substrate GTP-binding protein appears to be involved in the retinoic acid-induced differentiation of HL-60 cells.

Melanocytes are pigment producing cells that reside in the basal layer of the epidermis, and form multiple long dendritic processes that transport melanosomes from the melanocyte cell body to the dendritic tips, and then to keratinocytes. Dendrite formation requires actin polymerization in the newly forming dendrite, and dendrite formation in melanocytes is stimulated by hormones and ultraviolet light. The rho-subfamily of monomeric guanosine triphosphate-binding proteins is implicated in remodeling the cellular actin cytoskeleton, resulting in the formation of filopodia, lamellipodia, and stress fibers, as well as in oncogenesis and activation of the Jun/p38 mitogen activated kinase cascade. In this paper we show that rac1 induces the formation of dendrite-like structures when activated mutants are transiently expressed in B16F1 murine melanoma cells and in four human melanoma cell lines. Activated mutants of cdc42 and rhoA induced the formation of filopodia and stress fibers, respectively, in B16F1 cells, but not dendrites. A dominant negative inhibitor of rac1 abrogated the ability of alpha-melanocyte stimulating hormone, a peptide hormone known to stimulate melanocyte dendrite formation, and ultraviolet light, to induce dendrite formation in B16F1 cells, and alpha-melanocyte stimulating hormone and ultraviolet light stimulated the localization of rac1 to dendrite cell membranes. These results suggest that rac1 is an important signaling intermediate in dendrite formation in B16F1 cells, and that rac1 mediates the well-known ability of alpha-melanocyte stimulating hormone and ultraviolet light to induce dendrite formation.

Antibodies generated to a synthetic decapeptide, RMHLRQYELL, representing the carboxyl-terminus of G s -α have been characterized in immunoblots and functional studies. This antibody, designated RM, reacts exclusively with a doublet of proteins of 52 and 45 kDa in immunoblots of bovine brain and wild-type S49 murine lymphoma cell membranes. No such reactivity is seen in membranes from cyc − S49 cells, which lack G s . RM blocks receptor-mediated activation of G s and adenylyl cyclase in membranes from wild-type S49 cells. RM could also immunoprecipitate adenylyl cyclase activity in detergent extracts from GTP[γ]S- or fluoride-preactivated bovine brain membranes; thus binding of α s to effector and carboxyl-terminal antibody was mutually compatible. Such experiments provide an approach for the elucidation of functionally relevant interactions of G-proteins with receptors and electors in the membrane.

Two GTP-binding proteins (G-proteins) of 22 kDa were purified to near homogeneity from a sodium cholate extract of human platelet membranes by successive chromatographies on DEAE-Sephacel, Ultrogel AcA-44, phenyl-Sepharose CL-4B, Mono Q HR5/5 and hydroxyapatite columns. They bound maximally 0.89 mol of [35S]guanosine 5′-(3-O-thio)triphosphate per mol of both purified proteins, and this binding was inhibited by GTP and GDP but not by ATP and AppNHp. Their molecular masses were somewhat lower than that of ras p21 and they were not recognized by an anti-v-Ki-ras p21 antibody. These results indicate that human platelet membranes contain at least two low-molecular-mass G-proteins distinct from ras p21, in addition to the heterotrimeric G-proteins, the α-subunits of which possess molecular mass values of about 40 kDa.

Bovine rod outer segment (ROS) membranes contain in addition to the heterotrimeric G protein transducin, several small GTP-binding proteins (23–27 kDa). Furthermore, these membranes contain two substrate proteins (about 22 and 24 kDa) for botulinum C3 ADP-ribosyltransferase known to ADP-ribosylate small G proteins in any mammalian cell type studied so far. Most interestingly, [32P]ADP-ribosylation of ROS membrane small G proteins by C3 is regulated by light and guanine nucleotides in a manner similar to pertussis toxin-catalyzed [32P]ADP-ribosylation of the α-subunit of transducin. These findings suggest that not only the heterotrimeric G protein transducin but also the C3 substrate small G proteins present in ROS membranes interact with photoexcited rhodopsin and thus contribute to its signalling action.

Botulinum ADP-ribosyltransferase C3 modified 21-24 kDa proteins in a guanine nucleotide-dependent manner similar to that described for botulinum neurotoxin C1 and D. Whereas GTP and GTP gamma S stimulated C3-catalyzed ADP-ribosylation in the absence of Mg2+, in the presence of added Mg2+ ADP-ribosylation was impaired by GTP gamma S. C3 was about 1000-fold more potent than botulinum C1 neurotoxin in ADP-ribosylation of the 21-24 kDa protein(s) in human platelet membranes. Antibodies raised against C3 blocked ADP-ribosylation of the 21-24 kDa protein by C3 and neurotoxin C1 but neither cross reacted with neurotoxin C1 immunoblots nor neutralized the toxicity of neurotoxin C1 in mice. The data indicate that the ADP-ribosylation of low molecular mass GTP-binding proteins in various eukaryotic cells is not caused by botulinum neurotoxins but is due to the action of botulinum ADP-ribosyltransferase C3. The weak enzymatic activities described for botulinum neurotoxins appear to be due to the contamination of C1 and D preparations with ADP-ribosyltransferase C3.

To assign the GTP-binding protein (G-protein) subtype involved in the signal transduction from exogenous receptors to phospholipase C in the Xenopus oocyte translation system, antisense DNA complementary to rat G-protein alpha-subunit mRNA was designed and injected together with rat brain poly(A)+ RNA. Current response of mRNA-injected oocytes to acetylcholine (ACh) was suppressed dose-dependently by a co-injection of Gil alpha-antisense DNA, but response of the same oocytes to serotonin (5-HT) was not inhibited. In the oocytes co-injected with Go alpha-antisense DNA, the 5-HT response was more effectively suppressed than the ACh response. These results suggest that Go alpha but not Gil alpha intermediates brain 5-HT1C receptor function, and in contrast, muscarinic receptors derived from rat brain utilize Gil alpha rather than Go alpha to activate phospholipase C.

CaCl2, EGTA, GTP gamma S and anti-alpha-fodrin antibodies were injected into fibroblast-like IMR-33 cells and Madin-Darby bovine kidney (MDBK) epithelial cells cultured both in the presence and absence of cycloheximide and fetal calf serum. EGTA, GTP gamma S antifodrin antibody induced fusion of MDBK cells within one hour after injection. The cells formed polykaryons with up to 15 nuclei, reaching an average fusion index of 20%. IMR-33 cells fused at a slower kinetics and only upon injection of GTP gamma S or antifodrin antibodies. No fusions were seen in serum-deprived, quiescent cells. On the other hand, cycloheximide treatment did not prevent the fusions. The results show that cells can be induced to fuse by using agents that interfere with the regulation of the G-proteins, intracellular calcium level or membrane skeleton. We suggest that the putative fusogens are resident proteins of the plasma membrane which become exposed upon destabilization of the membrane skeleton.

Microinjection of the constitutively active recombinant Val-14 p21rho A into Xenopus oocytes induced dramatic morphological changes with re-distribution of pigments from the animal pole resulting in spotted oocytes. The effects induced by Val-14 p21rho A were regulated by progesterone in a dose-dependent manner whereas prior ADP-ribosylation of the rho protein blocked its activity. About 30 min after microinjection, p21 rho was associated with the plasma membrane. The membrane association of p21rho and its biological activity were inhibited by lovastatin, an inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase. The findings suggest that membrane attachment and biological activity of p21rho depend on isoprenylation of the GTP-binding protein.

The 27 kDa platelet membrane protein (Gn27) that binds [α-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617–620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22–23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431–439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

A novel enzymatic activity, the hydrolysis of linkages between mono(ADP-ribose) and cysteine residues in Gi prepared by eukaryotic ADP-ribosyl-transferase C [(1988) J. Biol. Chem. 263, 5485-5489] was found in the cytosol of human erythrocytes. The mono(ADP-ribosyl) Gi hydrolase, tentatively named ADP-ribosyI protein hydrolase C was partially purified by sequential chromatographies on DEAE-cellulose and Blue Sepharose. This enzyme catalyzes the release of ADP-ribose from mono(ADP-ribosyl) Gi Its activity was enhanced by Ca2+ and inhibited by ADP-ribose. The presence of this enzyme in eukaryotic cells suggests that endogenous mono(ADP-ribosyl)ation of Gi is a reversible post-translational modification.

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.

The molecular mechanism of light signal transduction in plants mediated by the photosensor phytochrome is not well understood. The possibility that phytochrome initiates the signal transduction chain by modulating a G-protein-like receptor is examined in the present work. Etiolated Avena seedlings contain G-proteins as examined in terms of the binding of GTP as well as by cross-reaction with mammalian G-protein antibodies. The binding of GTP was regulated in vivo by red/far-red light. The possible involvement of G-proteins in the phytochrome-mediated signal transduction in etiolated Avena seedlings has been implicated from the study of the light regulated expression of the Cab and phy genes.

Mono-glucosylation of (H/K/N)Ras by Clostridium sordellii lethal toxin (TcsL) blocks critical survival signaling pathways, resulting in apoptotic cell death. One yet unsolved problem in studies on TcsL is the lack of a method allowing the specific detection of (H/K/N)Ras glucosylation. In this study, we identify the Ras(Mab 27H5) antibody as a glucosylation-sensitive antibody capable for the immunoblot detection of (H/K/N)Ras glucosylation in TcsL-treated cells. Alternative Ras antibodies including the K-Ras(Mab F234) antibody or the v-H-Ras(Mab Y13-159) antibody recognize Ras proteins regardless of glucosylation. (H/K)Ras are further shown to be more efficaciously glucosylated by TcsL than Rac1 in rat basophilic leukemia cells as well as in a cell-free system.Structured summaryMINT-7261742: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) H-RAS (uniprotkb:P01112) by enzymatic studies (MI:0415)MINT-7261729: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) Rac1 (uniprotkb:P63000) by enzymatic studies (MI:0415)MINT-7261772: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) K-RAS (uniprotkb:P01116) by enzymatic studies (MI:0415)MINT-7261784: TcsL (uniprotkb:Q46342) enzymaticly reacts (MI:0414) N-RAS (uniprotkb:P01111) by enzymatic studies (MI:0415)

Pertussis toxin, islet-activating protein (IAP), and cholera toxin ADP-ribosylated 40 kDa and 45 kDa proteins in membrane preparations from Caenorhabditis elegans. Proteins with the same molecular weights were recognized in the same membranes by an antibody that had been raised against a peptide common to alpha-subunits of mammalian alpha beta gamma-heterotrimeric G proteins. The antibody produced immunoprecipitation with the 40 kDa protein 32P-labeled by IAP. A 35 kDa protein immunochemically indistinguishable from the beta-component of mammalian G proteins was also found in C. elegans membranes. The membranes displayed adenylate cyclase activity which was highly sensitive to forskolin and GTP analogues, whose action was antagonized by GDP beta S. Receptor-coupled regulation of adenylate cyclase thus appears to be mediated by mammalian-type G proteins in C. elegans as well.

Two different methods were used to study directly α-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo -[su3H]inositol prior to membrane isolation; in the other we used exogenous [ 3 H]PIP 2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca 2+ -dependent PIP 2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-γ-S. Of the two mitogens, α-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only α-thrombin is a potent activator of PIP 2 breakdown in intact cells. Consistent with this observation, α-thrombin but not EGF potentiated GTP-γ-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples α-thrombin receptor to PIP 2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP 2 -phosphodiesterase activity.

GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus , yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [α- 32 P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2′,3′-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [α- 32 P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H- ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. themophilus and initiation factor 2 (1F2) from T. thermophilus . The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras -proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.

Exogenous sphingosine 1-phosphate (S1P) induced Ca2+ mobilization, in association with an increase in inositol polyphosphate production reflecting activation of phospholipase C in HL60 leukemia cells. The increase in intracellular Ca2+ concentration ([Ca2+]i) induced by S1P was inhibited by an appropriate treatment of the cells with pertussis toxin (PTX), U73122 (a phospholipase C inhibitor) or phorbol 12-myristate 13-acetate (PMA). In parallel with the Ca2+ response, these agents also inhibited inositol polyphosphate production. The S1P-induced Ca2+ response was also attenuated in the dibutyryl cAMP-induced differentiated cells, where GTP-binding protein-induced Ca2+ response is suggested to be enhanced. Lysophosphatidic acid (LPA) also increased [Ca2+]i in the cells, but the maximal response was about half of that of S1P, and furthermore PTX and dibutyryl cAMP treatment hardly affected the LPA-induced Ca2+ mobilization. We conclude that exogenous S1P mobilizes Ca2+ through phospholipase C activation. The S1P-induced enzyme activation is at least partly mediated by PTX-sensitive GTP-binding protein-coupled receptors which may be different from LPA receptors.

The complete amino acid sequence of the gamma-subunit of the GTP-binding protein from cattle retina has been established. The polypeptide chain of the gamma-subunit consists of 69 amino acid residues and contains the unusual sequence Cys35-Cys36. The Mr of the gamma-subunit is 8008.7.

Department of Biochemistry, Gijii University School of Medicine, Gifu 500, *Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Yokohama 227, +Institute of Medical Science, University of Tokyo, Tokyo 108 and “Department of Physiological Chemistry, Faculty of Pharmaceutical Science, University of Tokyo, Tokyo 113, Japan Received 15 July 1988 Two GTP-binding proteins serving as the specific substrate of islet-activating protein (IAP), pertussis toxin, were purified from human platelet membranes as heterotrimers with an c&-subunit structure. The a of the major IAP substrate had a molecular mass of 40 kDa and differed from that of Gil or G. previously purified from brain membranes. The partial amino acid sequences of the 40 kDa a completely matched with the sequences which were deduced from the nucleotide sequences of the human Gi2 a gene. On the other hand, the a of the minor IAP substrate purified from human platelets was about 41 kDa and cross-reacted with an antibody raised against a of brain Gi i (Gi i a). These results indicate that the major IAP substrate present in human platelet membranes is a product of the G1r a gene. GTP-binding protein; Islet-activating protein; Pertussis toxin; (Human platelet)

Type C1 and D toxins produced by Clostridium botulinum caused ADP-ribosylation of a protein of 24 kDa in membrane preparations of rat clonal pheochromocytoma cells (PC12) and of proteins of 25 and 26 kDa in neuron-rich culture of fetal rat brain cells. The ADP-ribosylation reaction was dependent on the presence of MgCl2, GTP and GTPγS. The results obtained suggested that the ADP-ribosylation reaction is responsible for the development of the biological activity of the botulinum neurotoxins and that the target of this reaction may be novel GTP-binding proteins localized on cell membranes.