Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.
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PMID:Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells. 282 23

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.
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PMID:Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system. 282 45

We investigated the mechanisms of receptor-mediated stimulation of high-affinity GTPase activity in response to opioid peptides and to foetal-calf serum in membranes of the neuroblastoma X glioma hybrid cell line NG108-15. Increases in GTPase activity in response to both of these ligands was abolished by prior exposure of the cells to pertussis toxin. Pertussis toxin in the presence of [32P]NAD+ catalysed incorporation of radioactivity into a broad band of approx. 40 kDa in membranes prepared from untreated, but not from pertussis-toxin-pretreated, cells. Additivity studies indicated that the responses to opioid peptides and to foetal-calf serum were mediated by separate guanine-nucleotide-binding proteins (G-proteins). Whereas opioid peptides produced an inhibition of adenylate cyclase in membranes of untreated cells, foetal-calf serum did not. Affinity-purified antibodies which recognize the C-terminus of the inhibitory G-protein identified a 40 kDa polypeptide in membranes of NG108-15 cells. These antibodies attenuated opioid-stimulated high-affinity GTPase activity, but did not markedly affect the response to foetal-calf serum. We conclude that receptors for the opioid peptides function via the inhibitory G-protein (Gi), whereas foetal-calf serum activates a second pertussis-toxin-sensitive G-protein, which has a C-terminal sequence significantly different from that of Gi.
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PMID:Antibodies which recognize the C-terminus of the inhibitory guanine-nucleotide-binding protein (Gi) demonstrate that opioid peptides and foetal-calf serum stimulate the high-affinity GTPase activity of two separate pertussis-toxin substrates. 283 23

Opiates act through a specific receptor to inhibit the striatal adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4,6.1.1] and stimulate a high-affinity GTPase (EC 3.6.1). The present study analyzes the functions of the striatal adenylate cyclase complex following chronic morphine treatment in the rat. The inhibitory effects of GTP on basal adenylate cyclase activity, between 10(-6) and 10(-4) M, were reduced. Moreover, the half-maximal inhibitory concentration of the opiate receptor agonist (D-Ala2-Met5)-enkephalinamide (DAME) on striatal adenylate cyclase activity was increased by about four times, whereas the maximal effect was reduced in membranes from treated rats. In parallel, the half-maximal stimulatory concentration of DAME on GTPase was increased by two times, and the maximal stimulation was reduced from 60 to 25%. Binding studies performed with [3,5-3H]DAME (saturation curves) and with [3H]naloxone (competition curves) did not show any change in opiate receptor numbers and affinity. Moreover, the kinetics of the activation of the inhibitory GTP binding protein (Gi) which transduces the opiate receptor effect on adenylate cyclase showed a small but significant delay. Therefore, hypofunction of Gi can be, at least in part, responsible for the observed desensitization by morphine of the opiate-dependent GTPase and adenylate cyclase.
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PMID:Effects of the desensitization by morphine of the opiate-dependent adenylate cyclase system in the rat striatum on the activity of the inhibitory regulatory G protein. 283 70

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
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PMID:Cloning of bovine GAP and its interaction with oncogenic ras p21. 284 90

Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.
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PMID:Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. 284 1

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.
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PMID:Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein. 284 45

The vast majority of extracellular signals alters cell function by activating cell surface receptors. The transmembranous signalling process initiated by an activated receptor leads to the generation of an intracellular signal and eventually to a cellular response. In contrast to receptors that are permanently coupled to an enzyme or an ion channel representing the effector, a large number of surface receptors for hormones, neurotransmitters and receptors for exogenous chemical or physical stimuli reversibly interacts with membranous signal transduction components which, in turn, regulate intracellular messenger-generating effectors. The transducer molecules isolated so far form a family of guanine nucleotide-binding proteins (G- or N-proteins). All isolated G-proteins are composed of three different subunits (alpha, beta, gamma). The alpha-subunit, which is specific for the individual G-protein, binds and hydrolyzes GTP and is target of ADP-ribosylating bacterial toxins. Hormone-induced activation of a receptor causes interaction with the alpha-subunit of a G-protein and the exchange of bound GDP with GTP. The GTP-bound form of the alpha-subunit represents the active form of the G-protein, which is capable of stimulating or inhibiting the respective effector. The active state of the alpha-subunit is terminated by its inherent GTPase activity causing hydrolysis of bound GTP. The beta gamma-complexes of G-proteins are structurally very similar and functionally interchangeable; they appear to dissociate from the alpha-subunits during receptor activation of the G-protein. Possible functions of the beta gamma-complex are to anchor the non-activated G-protein in the membrane, to facilitate G-protein-receptor interaction, and to promote the inactive state of the alpha-subunit. G-protein-regulated effectors include enzymes, ion channels and probably transporters. The best studied G-protein-regulated enzyme is the retinal cyclic GMP-phosphodiesterase which is activated by bleached rhodopsin via the tissue-specific G-protein, termed transducin. The ubiquitously occurring membrane-bound adenylate cyclase is under dual control by families of stimulatory and inhibitory receptors, acting via G-proteins called Gs and Gi, respectively. Moreover, the receptor control of phospholipases A2 and C and probably of phospholipase D most likely involves G-proteins which have not yet been identified. Finally, the activity of NADPH oxidase of neutrophils and that of cyclic AMP phosphodiesterases in liver and fat cells may be regulated via G-proteins. Modulations of non-enzymatic effectors are reviewed elsewhere.
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PMID:[Guanidine nucleotide binding proteins as membrane signal transduction components and regulators of enzymatic effectors]. 284 11

The visual excitation system of the retinal rod outer segments and the hormone-sensitive adenylate cyclase complex are regulated through guanine nucleotide-binding proteins, transducin in the former and inhibitory and stimulatory regulatory components, Gi and Gs, in the latter. These proteins are functionally and structurally similar; all are heterotrimers composed of alpha, beta, and gamma subunits and exhibit guanosine triphosphatase activity stimulated by light-activated rhodopsin or the agonist-receptor complex. Adenylate cyclase can be stimulated by vanadate, which, like NaF, probably acts through Gs. Effects of vanadate on the function of a guanine nucleotide-binding protein were investigated in a reconstituted model system consisting of purified transducin subunits (T alpha, T beta gamma) and rhodopsin in phosphatidylcholine vesicles. Vanadate (decameric) inhibited [3H]GTP binding to T alpha and noncompetitively inhibited GTP hydrolysis in a concentration-dependent manner with maximal inhibition of approximately 90% at 3-5 mM. Vanadate also inhibited release of bound GDP but did not affect the rate of hydrolysis of bound GTP (single turnover rate), indicating that vanadate did not interfere with the intrinsic GTPase activity of T alpha. Binding of T alpha to rhodopsin and the ADP-ribosylation of T alpha by pertussis toxin, both of which are enhanced in the presence of T beta gamma, were inhibited by vanadate. These findings are consistent with the conclusion that vanadate can cause the dissociation of T alpha from T beta gamma, resulting in the inhibition of GDP-GTP exchange and thereby GTP hydrolysis. Adenylate cyclase activation could result from a similar effect of vanadate on Gs.
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PMID:Mechanism of inhibition of transducin guanosine triphosphatase activity by vanadate. 284 71

Opioid receptors in intact NG 108-15 cells were irreversibly inactivated with increasing concentrations of the alkylating antagonist beta-chlornaltrexamine (CNA). The consequence of the reduction in density of opioid binding sites (quantified by saturation analysis of opioid binding in membranes) was studied at two steps of opioid receptor-mediated responses, (a) stimulation of high affinity GTPase and (b) inhibition of basal adenylate cyclase. Both agonist-mediated stimulation of GTPase and inhibition of adenylate cyclase activities were progressively reduced as the concentration of CNA in the pretreatment was increased. However, the loss of responsiveness for the two enzymes differed in two aspects. First, the diminution of GTPase responsiveness was in agreement with the loss of binding sites and took place at concentrations of CNA that were lower than those necessary to reduce responsiveness of adenylate cyclase. Second, the loss of responsiveness of GTPase occurred simply as reduction of maximal stimulation, whereas that of adenylate cyclase involved an initial reduction of apparent agonist affinity (10-fold) that was followed by a decrease in maximal effect. We next examined the loss of responsiveness of both GTPase and adenylate cyclase in membranes prepared from cells that had been exposed to increasing concentrations of pertussis toxin (PTX) to inactivate PTX-sensitive G proteins in vivo. Also in this case, the extent of reduction in responsiveness was more pronounced for GTPase than for adenylate cyclase, especially in membranes treated with high concentrations of PTX. However, the pattern of loss was identical for the two enzymes and involved a main reduction in maximal effect of the agonist that was followed only after a large degree of inactivation (greater than 60%) by a diminished apparent affinity for the agonist. Opioid receptor-mediated inhibition of cAMP accumulation in intact cells exhibits an IC50 for the agonist that is 30-10 times lower than that measured in membranes for stimulation of GTPase or inhibition of cyclase, respectively. Treatment of cells with either CNA (1 microM) or various concentrations of PTX altered the concentration-response curves for agonist-mediated inhibition of cAMP accumulation in a manner similar to that observed for adenylate cyclase in membranes, inasmuch as both maximal inhibition and apparent affinities for the agonist were decreased. However, this decrease in affinity (5-fold) was not sufficient to eliminate the discrepancy in agonist potency between membranes and intact cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Opioid receptors are coupled tightly to G proteins but loosely to adenylate cyclase in NG108-15 cell membranes. 284 42


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