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

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

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and purification from bovine brain of a guanine-nucleotide-binding protein distinct from Gs, Gi and Go.

A guanine-nucleotide-binding protein (G-protein) was purified from cholate extracts of bovine brain membranes by sequential DEAE-Sephacel, Ultrogel AcA-34, heptylamine-Sepharose and Sephadex G-150 chromatography. Guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])-binding activity copurified with a 25,000 Da peptide and a 35,000-36,000 Da protein doublet. Neither pertussis toxin nor cholera toxin catalysed the ADP-ribosylation of a protein associated with the GTP[35S]-binding activity. Photoaffinity labelling of the purified protein with 8-azido[gamma-32P]GTP indicated that the GTP-binding site resides on the 25,000 Da protein. The 35,000-36,000 Da protein doublet was electrophoretically indistinguishable from the beta-subunits of other GTP-binding proteins, and the 36,000 Da protein was recognized by antiserum to oligomeric Gt. The purified protein specifically bound 17.2 nmol of GTP[35S]/mg of protein. The Kd of the binding site for radioligand was approx. 15 nM. The brain GTP-binding protein co-migrated during SDS/polyacrylamide-gel electrophoresis with a GTP-binding protein, named Gp, purified from human placenta [Evans, Brown, Fraser & Northup (1986) J. Biol. Chem. 261, 7052-7059], and cross-reacted with antiserum raised against the placental protein, but not with antiserum raised to brain Go. SDS/polyacrylamide-gel electrophoresis of the brain and placental GTP-binding proteins in the presence of Staphylococcus aureus V8 protease yielded identical peptide maps.     

The disturbance of the transduction of adenylyl cyclase inhibiting hormonal signal in myocardium and brain of rats with experimental type II diabetes

At present, the data obtained by us and other authors give evidence that disturbances in hormonal signaling systems are the main causes of development of pathological changes and complications under the diabetes. However, the molecular mechanisms of these disturbances remain obscure, especially in the case of insulin-independent type II diabetes. Using neonatal streptozotocin model of 80- and 180-days type II diabetes the changes in functional activity of hormone-regulated adenylyl cyclase (AC) signaling systems components in the myocardium and the brain striatum of diabetic rats in comparison with the control animals were found. The transduction of AC inhibitory hormonal signal meditated through Gi proteins was shown to by disturbed under diabetes. This was manifested in both the decrease of hormone inhibitory effect on AC activity and weakening of hormone stimulation of G-protein GTP-binding activity. In the case of noradrenaline (myocardium) the inhibitory pathway of AC regulation by the hormone was vanished and the stimulation pathway, in contrary, was protected. Prolongation of diabetes from 80 up to 180 days led to some weakening of Gi-protein-mediated hormonal signal transduction. Stimulating effect of biogenic amines and relaxin on the AC activity and GTP-binding in the myocardium and brain of diabetic rats were weakly changed in the case of both 80- and 180-days diabetes. To sum up, the experimental type II diabetes caused disturbances mainly in Gi-coupled signaling cascades participating in hormone inhibition of AC activity.     

High molecular weight forms of GTP-binding regulatory proteins from the bovine cerebellum

A new form of a low Km GTPase belonging to the family of regulatory GTP-binding G-proteins has been identified in bovine cerebellum. The molecular weight of this G-protein is several times as high as that of other G-proteins known to be alpha beta gamma heterotrimers: i. e., Gs, Gi, Go, transducin and a new G-protein which had recently been isolated in our laboratory from bovine cerebellum. The high molecular weight G-protein is stable against dissociation; its molecular mass does not change after treatment with DTT, colchicine and NaF. Using antibodies against the alpha-subunit of the formerly isolated cerebellar G-protein and the transducin beta-subunit, it was demonstrated that the both immunoreactive subunits are present in the high molecular weight G-protein. The two forms of the cerebellar G-proteins, i. e., "high" and "low molecular weight" ones, differ drastically in terms of the Mg2+ effect on their GTPase activity. Whereas at submicromolar concentrations of Mg2+ the GTPase activity of the former is virtually absent, the GTPase activity of the latter is more elevated in the presence of EDTA than in the presence of Mg2+.     

A-protein catalyzes the ADP-ribosylation of G-protein from cow rod outer segments

A 20-kilodalton adenosine nucleotide-binding protein (A-protein) extracted from rod outer segments is shown to catalyze the cholera toxin-mediated ADP-ribosylation of GTP-binding protein (G-protein) from the outer segment. Radiolabel from [adenylate-32P] NAD+ was associated specifically with both the alpha-subunit of G-protein and with A-protein in the presence of activated cholera toxin. In the absence of added A-protein, G-protein appears to undergo ADP-ribosylation at a slower rate. In the absence of G-protein, A-protein was found to be labeled following incubation with [adenylate-32P]NAD+ and cholera toxin. In the presence of G-protein, a light-dependent component of A-protein labeling was observed. A-protein is a labile component of rod outer segments and has an affinity for ADP. The findings suggest that A-protein may act as an ADP-ribosyltransferase in the cholera toxin-mediated ADP-ribosylation of G-protein.     

Physical and functional association of cytosolic inositolphospholipid-specific phospholipase C of calf thymocytes with a GTP-binding protein

A soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5,800-fold from the cytosolic fraction of calf thymocytes. The purification was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTP gamma S-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTP gamma S-binding activities in the partially purified enzyme preparation could be separated by the column chromatography on Sephadex G-100 only in the presence of 1% sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTP gamma S (10 microM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTP gamma S. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.     

Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

Prostaglandin E2 (PGE2) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet (Negishi, M., Ito, S., Tanaka, T., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1987) J. Biol. Chem. 262, 12077-12084). In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, we purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its alpha subunit from two known pertussis toxin substrate G-proteins (Gi and Go) purified from bovine brain. The molecular weight of the alpha subunit was 40,000, which is between those of Gi and Go. The purified protein was also distinguished immunologically from Gi and Go and was referred to as Gam. PGE receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and freed from G-proteins by wheat germ agglutinin column chromatography. Reconstitution of the PGE receptor with pure Gam, Gi, or Go in phospholipid vesicles resulted in a remarkable restoration of [3H]PGE2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. The displacement of [3H]PGE2 binding was specific for PGE1 and PGE2. Furthermore, addition of PGE2 stimulated the GTPase activity of the G-proteins in reconstituted vesicles. These results indicate that the PGE receptor can couple functionally with Gam, Gi, or Go in phospholipid vesicles and suggest that Gam may be involved in signal transduction of the PGE receptor in bovine adrenal medulla.     

The adipocyte Go alpha-immunoreactive polypeptide is different from the alpha subunit of the brain Go protein.

Rat adipose tissue possesses two Bordetella pertussis toxin (PTX) substrates and, in the same 39-41 kDa molecular mass range, positive immunoreactivity has also been reported with antibodies against the alpha subunit of Go, the major brain GTP-binding protein (G-protein). In this study, the presence of the brain Go alpha subunit at 39 kDa in adipocytes was reassessed, since direct correspondence between PTX substrates and Go alpha immunoreactivity has not yet been clearly established. On resolutive SDS/polyacrylamide-gel electrophoresis, the PTX substrates of human adipocytes were compared with the three PTX substrates found in brain. No ADP-ribosylated substrate at the level of the 39 kDa brain Go alpha could be detected in adipocyte membranes. Immunoblotting of human adipocyte membranes stained with our anti-Go alpha antibodies confirmed the presence of a positive immunoreactivity in this tissue, but the apparent molecular mass of the immunoreactive polypeptide in adipocytes was higher than that found in nervous tissues. Taken together, these results indicate that the brain Go alpha subunit is not present in adipose tissue. They also suggest the existence of a G-protein in adipocytes which is immunologically related to Go alpha but having a slightly higher molecular mass.     

Activation of human neutrophils by a synthetic anti-microbial peptide, KLKLLLLLKLK-NH2, via cell surface calreticulin.

We previously reported that a synthetic anti-bacterial peptide, KLKLLLLLKLK-NH2 (L5), showed significant chemotherapeutic activity in methicillin-resistant Staphylococcus aureus-infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP-binding protein (G-protein) participates in this process. We isolated an L5-binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti-calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5-bound calreticulin transmits a signal into cells via G-protein to activate neutrophils to generate superoxide anion.     

ATPase and GTPase activities copurifying with GTP-binding proteins in E. coli

Intrinsic GTPase activity of GTP-binding proteins plays the vital role in regulating the downstream activation pathway. We examined the GTP and ATP hydrolyzing (NTPase) abilities of various bacterial and human GTP-binding proteins under different metabolic conditions. Two metabolic components, acetate and 3-phosphoglyceric acid (3-PG), have shown significant stimulatory action on NTPase activity of G-protein preparations. Acetyl phosphate and 2,3-bisphosphoglyceric acid (2,3-BPG) blocked these stimulations. From gel filtration analyses, we have determined two fractions containing metabolite-inducible NTPase activities which are independent of GTP-binding protein enzymatic actions. Therefore, one should be cautious when NTPase activity is examined in a buffer containing acetate often used for NTPase assay.     

Interaction of GTP-binding regulatory proteins with chemosensory receptors

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.     

Characterization of calcium/calmodulin-dependent protein kinase II activity in the nervous system of the lobster,Panulirus interruptus

Nervous system tissue fromPanulirus interruptus has an enzyme activity that behaves like calcium/calmodulin-dependent protein kinase II (CaM KII). This activity phosphorylates known targets of CaM KII, such as synapsin I and autocamtide 3. It is inhibited by a CaM KII-specific autoinhibitory domain peptide. In addition, this lobster brain activity displays calcium-independent activity after autophosphorylation, another characteristic of CaM KII. A cDNA from the lobster nervous system was amplified using polymerase chain reaction. The fragment was cloned and found to be structurally similar to CaM KII. Serum from rabbits immunized with a fusion protein containing part of this sequence immunoprecipitated a CaM KII enzyme activity and a family of phosphoproteins of the appropriate size for CaM KII subunits. Lobster CaM KII activity is found in the brain and stomatogastric nervous system including the commissural ganglia, commissures, stomatogastric ganglion and stomatogastric nerve. Immunoblot analysis of these same regions also identifies bands at an apparent molecular weight characteristic of CaM KII.     

Investigation of rhodopsin catalyzed G-protein GTP-binding using [35S] GTP gamma S--effects of regeneration and hydroxylamine

A simple reconstitution system for studying rhodopsin-catalyzed G-protein GTP-binding is described. Purified rhodopsin is recombined with a G-protein containing extract in the presence of [35S]GTP gamma S and after incubation the reaction is stopped by rapid cooling and filtration. The system has been used to study the effects of 11-cis-retinal and hydroxylamine on R*-catalyzed G-protein GTP-binding. Since both these compounds greatly reduced binding, our results provide further evidence that it is the rhodopsin intermediate metarhodopsin II which interacts with G-protein.     

RGS1 is expressed in monocytes and acts as a GTPase-activating protein for G-protein-coupled chemoattractant receptors.

The leukocyte response to chemoattractants is transduced by the interaction of transmembrane receptors with GTP-binding regulatory proteins (G-proteins). RGS1 is a member of a protein family constituting a newly appreciated and large group of proteins that act as deactivators of G-protein signaling pathways by accelerating the GTPase activity of G-protein alpha subunits. We demonstrate here that RGS1 is expressed in human monocytes; by immunofluorescence and subcellular fractionation RGS1 was localized to the plasma membrane. By using a mixture of RGS1 and plasma membranes, we were able to demonstrate GAP activity of RGS1 on receptor-activated G-proteins; RGS1 did not affect ligand-stimulated GDP-GTP exchange. We found that RGS1 desensitizes a variety of chemotactic receptors including receptors for N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and C5a. Interaction of RGS proteins and ligand-induced G-protein signaling can be demonstrated by determining GTPase activity using purified RGS proteins and plasma membranes.     

Recombinant SEC14-like proteins (TAP) possess GTPase activity

The three human SEC14-like proteins TAP1, TAP2, and TAP3 were expressed in Escherichia coli and purified by means of an amino-terminal His-tag. The recombinant TAP proteins bound alpha-, beta-, gamma-, and delta-tocopherol, certain phospholipids, and squalene. Intriguingly, the TAP proteins showed considerable GTPase activity that was comparable to that of small GTP-binding proteins of the Rab family. Although the TAP proteins contain important motifs to provide GTPase activity, the surrounding secondary structure markedly differed from common G-protein domains. However, these motifs are located in close proximity in the TAP structure and may therefore form an active site for GTP-binding and hydrolysis.     

Subcellular distribution of GTP-binding proteins, Go and Gi2, in rat cerebral cortex

The localization of GTP-binding protein (G-protein) subunits, Go alpha, Gi2 alpha and beta, in subcellular fractions of rat cerebral cortex was determined by means of immunoassays specific for the respective subunits. High concentrations of all three subunits were observed in both crude mitochondrial and microsomal fractions. Muscarinic cholinergic receptors were also densely localized in these fractions. Then the crude mitochondrial and microsomal fractions were subfractionated by sucrose density gradient centrifugation. Each fraction obtained was evaluated morphologically by electron microscopy and biochemically by determination of membrane markers. The crude mitochondrial fraction was subfractionated into myelin, synaptic plasma membrane, and mitochondrial fractions. All the G-protein subunits examined and muscarinic receptors were exclusively localized in the synaptic plasma membrane fraction. Among the submicrosomal fractions, the heavy smooth-surfaced microsomal fraction showed the highest concentrations of all G-protein subunits and receptors, while the rough-surfaced microsomal fraction contained low amounts of them. The heavy smooth-surfaced microsomal fraction also contained high specific activity of (Na(+)-K+)-ATPase, a marker of the plasma membrane. These results indicated that the Go alpha, Gi2 alpha and beta subunits are mainly localized in the plasma membrane in the brain.     

Recombinant SEC14-like proteins (TAP) possess GTPase activity

The three human SEC14-like proteins TAP1, TAP2, and TAP3 were expressed in Escherichia coli and purified by means of an amino-terminal His-tag. The recombinant TAP proteins bound α-, β-, γ-, and δ-tocopherol, certain phospholipids, and squalene. Intriguingly, the TAP proteins showed considerable GTPase activity that was comparable to that of small GTP-binding proteins of the Rab family. Although the TAP proteins contain important motifs to provide GTPase activity, the surrounding secondary structure markedly differed from common G-protein domains. However, these motifs are located in close proximity in the TAP structure and may therefore form an active site for GTP-binding and hydrolysis.     

DISTRIBUTION OF AMINO ACIDS AND OF EXO- AND ENDOPEPTIDASES ALONG VERTEBRATE AND INVERTEBRATE NERVES

Abstract— The proximo-distal gradients for representative peptidases, peptidylpeptide hydrolases, and amino acids were measured in segments of peripheral nerve from invertebrates and vertebrates and in the lobster brain and ventral cord.
Crustacean nerve was characterized by a large pool of free amino acids totaling 100–200 μmoles/g wet wt. In lobster nerve, the principal free amino acid was aspartic acid which comprised 55 per cent of the free pool, whereas in the rat sciatic nerve it comprised only 5 per cent. The principal free amino acid in rat sciatic nerve was taurine (32 per cent of the pool) and in lobster brain glycine comprised 30 per cent of the pool. No consistent patterns emerged for the gradients along the nerves for amino acids and hydrolytic enzymes. In the leg nerve of the lobster, concentrations of aspartic acid and arginine were higher in the proximal region, and concentrations of proline and alanine were higher in the distal region. Concentrations of most amino acids were higher in the proximal regions of crab nerve, of lobster brain and ventral cord, and of rat sciatic nerve.
Rat sciatic nerve exhibited a pronounced proximo-distal increase in activity of aminopeptidase (Leu-Gly-Gly). In lobster leg nerve, activity of neutral proteinase was higher in the proximal segment, whereas activity of acid proteinase was higher in the distal segment. The best examples of proximo-distal gradients were found in lobster brain and ventral cord; activities of endopeptidases, arylamidases (Leu- and Arg-βNA), and aminopeptidase were higher in the supra-esophageal ganglia or cephalothorax segments than in the distal regions.