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1.
A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. (zucchini) seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[gamma-thio]triphosphate (GTP-gamma-S). Both the binding affinity and the pattern of binding specificity for GTP and guanine nucleoside triphosphate analogs are shared with the more thoroughly characterized animal G-proteins that are known to be involved in signal transduction. The sensitivity of GTP-gamma-S binding to Mg+2 ions and temperature was similar to that reported for rabbit liver G-protein, although the plant complex dissociated more readily. GTP-gamma-S could be recovered unchanged from the binding complex. Proteins (Mr 33 and 50 kDa) present in zucchini membrane preparations were revealed by immunoblotting with antiserum specific for the alpha subunit of platelet GS. These may be homologous to animal G-proteins.  相似文献   

2.
Binding of GTP and its analogue, guanosine 5′-O-[γ-thio]triphosphate (GTP[S]) to G-proteins, and release of GTP[S] from G-proteins are stimulated by muscarinic acetylcholine (mACh) receptors in intact cardiac membranes. Upon solubilization of receptors and G-proteins by membrane extraction with the detergent, 3-[(cholamidopropyl)dimethylammonio]-1-propanesulphonate, followed by sucrose density gradient centrifugation, agonist-liganded mACh receptors stimulated binding of GTP[S] and hydrolysis of GTP by G-proteins with similar requirements as in intact membranes. One soluble agonist-activated mACh receptor induced binding of GTP[S] to several (about seven) soluble G-proteins. In contrast to intact membranes, however, agonist activation of mACh receptors did not induce release of GTP[S] from solubilized G-proteins. The data presented indicate that mACh receptors can interact with and efficiently activate G-proteins even in solution, whereas the possible interaction of receptors with GTP[S]-liganded G-proteins observed in intact membranes is lost upon solubilization of these components.  相似文献   

3.

Background

Guanonine-protein (G-protein) is known as molecular switches inside cells, and is very important in signals transmission from outside to inside cell. Especially in transport protein, most of G-proteins play an important role in membrane trafficking; necessary for transferring proteins and other molecules to a variety of destinations outside and inside of the cell. The function of membrane trafficking is controlled by G-proteins via Guanosine triphosphate (GTP) binding sites. The GTP binding sites active G-proteins initiated to membrane vesicles by interacting with specific effector proteins. Without the interaction from GTP binding sites, G-proteins could not be active in membrane trafficking and consequently cause many diseases, i.e., cancer, Parkinson… Thus it is very important to identify GTP binding sites in membrane trafficking, in particular, and in transport protein, in general.

Results

We developed the proposed model with a cross-validation and examined with an independent dataset. We achieved an accuracy of 95.6% for evaluating with cross-validation and 98.7% for examining the performance with the independent data set. For newly discovered transport protein sequences, our approach performed remarkably better than similar methods such as GTPBinder, NsitePred and TargetSOS. Moreover, a friendly web server was developed for identifying GTP binding sites in transport proteins available for all users.

Conclusions

We approached a computational technique using PSSM profiles and SAAPs for identifying GTP binding residues in transport proteins. When we included SAAPs into PSSM profiles, the predictive performance achieved a significant improvement in all measurement metrics. Furthermore, the proposed method could be a power tool for determining new proteins that belongs into GTP binding sites in transport proteins and can provide useful information for biologists.
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4.
We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.  相似文献   

5.
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.  相似文献   

6.
The presence of G-proteins, interacting with cAMP surface receptors, was investigated in vegetative cells, aggregation-competent cells, and migrating slugs of Dictyostelium discoideum. Our results indicate that G-proteins are present in all stages. In vegetative cells there is a limited number of cAMP receptors but no effect of GTP tau S on cAMP binding could be detected; in addition, no effect of cAMP on GTP tau S binding or GTPase activity was observed. In both aggregation-competent cells and slugs GTP tau S inhibits cAMP binding, while cAMP stimulates GTP tau S binding and high-affinity GTPase. Since the presence of G-proteins coupled to cAMP receptors could be demonstrated in slugs, the involvement of the effector enzymes adenylate cyclase and phospholipase C was investigated. The results show that adenylate cyclase activity is stimulated by GTP tau S in both stages and that in cells from migrating slugs the Ins(1,4,5)P3 production is increased upon stimulation with cAMP. The possible involvement of G-proteins in signal transduction during the slug stage of D. discoideum is discussed.  相似文献   

7.
A model of the nucleotide-binding site in tubulin   总被引:6,自引:0,他引:6  
Tubulin uses GTP to regulate microtubule assembly and is thought to be a member of a class of GDP/GTP-binding proteins (G-proteins) as defined by Hughes [(1983) Febs Lett. 164, 1-8]. How tubulin is structurally related to G-proteins is not known. We use a synthesis of sequence comparisons between tubulin, other G-proteins, and ADP/ATP-binding proteins and topological arguments to identify potential regions involved in nucleotide binding. We propose that the nucleotide-binding domain in the beta-subunit of tubulin is an alpha/beta structure derived from amino acid residues approximately 60-300. Five peptide sequences are identified which we suggest exist as 'loops' that extend from beta-strands and connect alpha-helices in this structure. We argue that GDP binds to four of the five loops in an Mg2+-independent manner while GTP binds in an Mg2+-dependent manner to a different combination of four loops. We propose that this switch between loops upon GTP binding induces a conformational change essential for microtubule assembly.  相似文献   

8.
Heterotrimeric GTP-binding proteins (G-proteins) are involved in a diverse array of signalling pathways. They are generally thought to be membrane-bound proteins, which disassociate on receptor activation and binding of GTP. A model to explain this has been proposed, which is often described as 'the G-protein cycle'. The 'G-protein cycle' is discussed in the present paper in relation to evidence that now exists regarding the non- membranous localization of G-proteins. Specifically, the experimental evidence demonstrating association of G-proteins with the cytoskeleton and the nucleus, and the mechanisms by which G-proteins translocate to these sites are reviewed. Furthermore, the possible effector pathways and the physiological function of G-proteins at these sites are discussed.  相似文献   

9.
We describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [3H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [3H]fMet-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, we also demonstrated fMet-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils.  相似文献   

10.
The molecular mechanism of light perception through phytochrome is not well understood. This red-light photosensor has been implicated in various physiological processes, including the photoinduction of flowering. A few recent studies have shown that phytochrome initiates signal transduction chains via guanosine triphosphate (GTP)-binding proteins (G-proteins). We show here by different approaches that G-proteins exist in spinach (Spinacia oleracea L. cv. Nobel). Binding of GTP on the plasmalemma has been partially characterized and its possible regulation by red light examined by in-vitro assays. These experiments indicate a clear regulation of GTP binding by red light and also by Mastoparan. At least three G-proteins or protein subunits were found to be associated with the plasmalemma of leaf cells. The use of an antibody raised against an animal Gβ subunit confirmed the presence of heterotrimeric G-proteins. Separation of a crude membrane extract by free-flow electrophoresis also showed that some G-proteins could exist on the tonoplast.  相似文献   

11.
G-protein oncogenes in acromegaly.   总被引:1,自引:0,他引:1  
A Spada  L Vallar 《Hormone research》1992,38(1-2):90-93
G-proteins belong to a family of proteins which share the common properties of GTP binding and hydrolysis. Heterotrimeric G-proteins are composed of alpha-, beta- and gamma-subunits. The alpha-subunit which differs from one G-protein to another contains the GDP/GTP binding site and has intrinsic GTPase activity. The receptor occupancy causes displacement of bound GDP by GTP, dissociation of free beta gamma-dimer and alpha-GTP complex, interaction of the activated alpha-GTP complex with intracellular effectors, such as enzymes and ion channels. The turn off of the reaction is due to the GTPase activity which causes the hydrolysis of GTP to GDP. G-proteins are essential for transferring hormonal signals from cell surface receptors to intracellular effectors. Since G-proteins generate intracellular effectors involved in cell growth, G-protein genes have the propensity to be converted into oncogenes. In fact, mutations in the alpha-subunit of Gs (the G-protein involved in the activation of adenylyl cyclase) have been demonstrated in 40% of human GH secreting pituitary adenomas. Single amino acid substitutions replacing Arg 201 with either Cys or His or Gln 227 with either Arg or Leu cause constitutive activation of adenylyl cyclase by inhibiting GTPase (gsp oncogene). The same mutations were identified in about 10% of thyroid adenomas and in the McCune-Albright syndrome.  相似文献   

12.
The exsitence of GTP-binding regulatory proteins (for short term, often refered as G-proteins) in higher plant cells is certain. G-proteins are classified into two groups based on their molecular structures, which are the heterotrimeric G-proteins (big G-proteins) that contain three different subunits and the small G-proteins that have only one subunit (monomeric G-proteins). All G-proteins are characterized by their properties to bind with and hydrolyze GTP, by which G-proteins function as transmembrane and intracellular signalling molecules. As a distinguished participant in signal transduction, G-proteins directly and/or indirectly regulate a number of physiological processes, such as regulation of phytochrome-related physiological processes and gene expression, involvement in blue-light response, K+-channel regulation, stomatal movement, hormone regulation, protein phosphrylation dephosphorylation, etc. Although G-proteins in plant cells have not been purified, the genes for a subunit of heterotrimeric G-proteins have been cloned. More evidences for the importance of G-proteins in plant signalling processes are rapidly accumulating.  相似文献   

13.
EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.  相似文献   

14.
GTP-binding regulatory proteins (G-proteins) were identified in chemosensory membranes from the channel catfish, Ictalurus punctatus. The common G-protein beta-subunit was identified by immunoblotting in both isolated olfactory cilia and purified taste plasma membranes. A cholera toxin substrate (Mr 45,000), corresponding to the G-protein that stimulates adenylate cyclase, was identified in both membranes. Both membranes also contained a single pertussis toxin substrate. In taste membranes, this component co-migrated with the alpha-subunit of the G-protein that inhibits adenylate cyclase. In olfactory cilia, the Mr 40,000 pertussis toxin substrate cross-reacted with antiserum to the common amino acid sequence of G-protein alpha-subunits, but did not cross-react with antiserum to the alpha-subunit of the G-protein from brain of unknown function. The interaction of G-proteins with chemosensory receptors was determined by monitoring receptor binding affinity in the presence of exogenous guanine nucleotides. L-Alanine and L-arginine bind with similar affinity to separate receptors in both olfactory and gustatory membranes from the catfish. GTP and a nonhydrolyzable analogue decreased the affinity of olfactory L-alanine and L-arginine receptors by about 1 order of magnitude. In contrast, the binding affinities of the corresponding taste receptors were unaffected. These results suggest that olfactory receptors are functionally coupled to G-proteins in a manner similar to some hormone and neurotransmitter receptors.  相似文献   

15.
A novel GTP-binding protein, GIR, along with insulin receptor (IR), has been partially purified from human placenta. A non-hydrolyzable substrate, GTP gamma S which is known to bind to GTP-binding proteins with high affinity, reduces insulin binding to IR-GIR fraction by approximately 29% and 100 nM insulin stimulates GTP gamma S binding to IR-GIR fraction by approximately five-fold. The molecular weight of the protein (may be subunit) that binds to 8-azido-GTP, a photoaffinity label for G-proteins, is approximately 66,000.  相似文献   

16.
The presence of specific guanine nucleotide-binding proteins in a zucchini (Cucurbita pepo L.) hypocotyl microsomal fraction was investigated. Polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of nitrocellulose blots with [alpha-32P]GTP and [gamma-32P]GTP indicated the presence of four specific and distinct GTP-binding proteins with molecular masses of approx. 23.4 kDa, 24.8 kDa, 26.6 kDa and 28.5 kDa. Binding of [alpha-32P]GTP could be completely prevented by 30 microM GDP or 10 microM guanosine 5'[gamma-thio]triphosphate. This report presents evidence for the presence in a microsomal fraction from zucchini hypocotyls of Gn-proteins as defined by Bhullar and Haslam (1987) Biochem.J. 245, 617-620. The four plant proteins resemble animal Gn-proteins when molecular weights and GTP-binding specificities are considered.  相似文献   

17.
The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5'-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.  相似文献   

18.
Antibody-induced antigenic modulation occurs after binding of antibodies to a variety of cell surface proteins. It is characterized by aggregation and subsequent loss of the molecules from the cell surface, usually by internalization. In this study we have investigated the effect of modulation of the T-cell antigen receptor complex (TCR) and the transferrin receptor (TFR) on the distribution of cholera toxin (CTx)- and pertussis toxin (PTx)-sensitive GTP binding proteins in human T-lymphocytes. Modulation of both the TCR and the TFR induced a selective shift of PTx-sensitive G-proteins from the plasma membrane to a high density membrane fraction enriched for lysosomal membranes. The distribution of CTx-sensitive G-proteins was unaffected. This shift was found in both the T-cell leukemia line Jurkat and in normal T-cells. The loss of PTx-sensitive G-proteins from the plasma membrane required approximately 15 h to be complete and was not inhibited by cycloheximide. It had no influence on T-cell triggering via anti-T-cell receptor antibodies and is unrelated to the inactivating effect of TCR-modulation on T-cell signalling. The loss of PTx-sensitive G-proteins was not accompanied by greater sensitivity to stimuli raising cAMP concentration. These results show that PTx-sensitive G-proteins can be selectively depleted from the plasma membrane by antibody treatment of T-cells.  相似文献   

19.
The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.  相似文献   

20.
The binding of G proteins to immobilized delipidated rhodopsin   总被引:1,自引:0,他引:1  
We have shown that delipidated rhodopsin immobilized on Concanavalin A-Sepharose is capable of binding transducin from crude bovine rod outer segment proteins and GIP-binding proteins (G proteins) of Go/Gi-type from solubilized bovine brain membrane as well. The binding is reversible in the presence of a solution containing 1.2% octyl-beta, D-glucopyranoside and 1 mM GTP. Also, alpha-subunits account for a large fraction of the G proteins which are bound to and then eluted from the immobilized rhodopsin. Concanavalin A-bound delipidated rhodopsin seems to be a useful model in isolating and purifying different G-proteins from crude cell lysates and solubilized membranes as well as for studying G-protein-receptor interaction.  相似文献   

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