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)

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.
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PMID:A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity. 173 35

The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP-binding sites (down regulation) and modification of the receptor (presumably via phosphorylation) correlated with adaptation. These processes occur simultaneously, but differ in the cAMP dose dependency and reversibility. In this study we investigated the mechanism of adaptation; cells were incubated with a cAMP analog to induce desensitization mediated by adaptation. The cells were then washed, lysed and the interaction between cAMP, receptor, guanine-nucleotide-binding regulatory proteins (G proteins) and GTP was investigated. (1) cAMP receptors that are phosphorylated in vivo remain phosphorylated for at least 45 min after lysis. (2) Desensitization did not alter basal cAMP binding to the receptor nor the inhibitory effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on this binding. (3) The stimulatory effect of cAMP on GTP[S] binding was also unchanged, while basal GTP[S] binding and the kinetics of binding were only slightly different. (4) Basal high-affinity GTPase activity was not altered but cAMP stimulation was reduced from 43 +/- 7% in control lysates to 14 +/- 4% in lysates from desensitized cells. (5) cAMP stimulation of GTPase was decreased by pretreatment of cells with pertussis toxin from 43 +/- 7% to 17 +/- 8% but this was not further altered in lysates from desensitized pertussis-toxin-treated cells. These observations indicate that during desensitization the phosphorylated receptor can still interact with G proteins. Furthermore, desensitization did not affect cAMP stimulation of GTP[S] binding but strongly reduced cAMP stimulation of GTPase, suggesting that a G protein becomes activated. This G protein is pertussis toxin sensitive and may be the inhibitor G protein (Gi). This would imply that adenylate cyclase desensitizes because Gi becomes activated.
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PMID:Activation of a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein during desensitization of Dictyostelium discoideum cells to chemotactic signals. 184 68

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.
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PMID:Involvement of cyclic AMP cell surface receptors and G-proteins in signal transduction during slug migration of Dictyostelium discoideum. 185 Mar 66

The thromboxane A2 (TXA2) receptor in human platelets is coupled to a pertussis toxin-insensitive G protein whose identity has remained unknown. Candidates for this role include the atypical G protein known as Gz and members of a recently discovered G protein family known as Gq. Because of the proven utility of antibodies directed against the C terminus of G protein alpha subunits as functional probes, we prepared an antibody against a synthetic decapeptide corresponding to the C-terminal sequence shared by alpha 11 and alpha q, two members of the new family. This antibody (QL) does not recognize known alpha subunits but selectively binds to a 42-kDa protein in a variety of tissues, including human platelet membranes. QL and two other C-terminal antibodies, QN and AS, known to recognize alpha z and alpha i2, respectively, were tested for their ability to block agonist-stimulated GTPase activity in human platelet membranes. Pretreatment of platelet membranes with AS has previously been shown to interfere with alpha 2 adrenergic receptor-mediated inhibition of adenylylcyclase. As expected, only AS antibody produced inhibition of alpha 2 receptor-stimulated GTPase. Pretreatment of membranes with QL, but not QN or AS, caused marked inhibition of TXA2 receptor-stimulated GTPase. This identifies the G protein coupled to human platelet TXA2 receptors as a member of the novel Gq family.
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PMID:The G protein coupled to the thromboxane A2 receptor in human platelets is a member of the novel Gq family. 185 Nov 74

Several domains of guanine nucleotide-binding proteins are conserved and form the guanine nucleotide-binding pocket. Mutations in these domains in EF-Tu, ras, and Gas have been shown to result in informative phenotypes. We made several analogous changes in SCG1, which encodes the alpha subunit of the G protein involved in pheromone response in yeast. The scg1Lys388 and scg1Ala391 mutations resulted in severe growth and cell morphology defects; this phenotype is similar to the null phenotype and results from constitutive activation of the pheromone response pathway. On the basis of the model for the action of the yeast G protein, the effect of these mutations is consistent with the effect of analogous mutations in ras, which result in a transforming phenotype. The SCG1Ala322 mutation resulted in pheromone response and mating defects. This effect is similar to the effect of the analogous G alpha s mutation, which results in a defect in stimulation of adenylate cyclase. The scg1Val50 mutation, which is analogous to the transforming mutation rasVal12, resulted in multiple effects, including defects in growth, cell morphology, and mating. Some of our results and interpretations are different from previously published results of others for the same mutation in SCG1; specifically, our gene replacement of this mutation resulted in high basal activation of the pheromone response pathway, consistent with a GTPase defect, which was not seen previously with scg1Val50 on a low-copy plasmid. Implications of these phenotypes are discussed.
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PMID:Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway. 190 Apr 95

The GTPase activity of a G protein alpha subunit functions as a timer to control the lifetime of the activated conformation of the protein. Expression of the GTPase-deficient Gi2 alpha subunit oncogene, gip2 (alpha i2Q205L), in Chinese hamster ovary cells inhibited the stimulation of adenylylcyclase and altered the calcium regulation of the Gi2-phospholipase A2 (PLA2) effector complex. The phenotypic consequence of the activated alpha i2 mutant on hormonal stimulation of PLA2 varied depending on the cytoplasmic calcium transient elicited by different Gi2-linked receptors. The stimulation of PLA2 by thrombin, which mobilized calcium only from internal stores, was markedly attenuated in gip2-expressing cells. In contrast, the attenuation of the PLA2 response to ATP, a purinergic agonist which mobilizes calcium from both extracellular space and internal stores, was significantly less than that observed for thrombin. Ionomycin, a calcium ionophore, stimulated PLA2 activity in clones which expressed gip2 to a level similar to that observed in wild-type Chinese hamster ovary cells. Thus, the dominant GTPase-deficient gip2 polypeptide will constitutively inhibit adenylylcyclase but differentially modulate enzymes regulated by calcium and coupled to Gi2.
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PMID:GTPase-deficient G alpha i2 oncogene gip2 inhibits adenylylcyclase and attenuates receptor-stimulated phospholipase A2 activity. 190 71

In membranes of rat striatum, phorbol 12-myristate 13-acetate (PMA), a potent activator of Ca2+/phospholipid-dependent protein kinase, enhanced adenylate cyclase activity by counteracting the inhibition elicited by GTP. Exposure to pertussis toxin caused a similar alteration of the GTP-regulation of the enzyme activity and largely prevented the PMA effects. PMA treatment increased by threefold the GTP requirement of acetylcholine-induced inhibition of adenylate cyclase activity but did not affect the GTP-dependence of the enzyme stimulation by dopamine. The hydrolysis of GTP by membrane-bound high affinity GTPase was significantly inhibited by PMA (IC 50 10 nM) in a Ca2(+)-dependent manner. Like PMA, phorbol 12,13-dibutyrate inhibited the GTPase activity, whereas the biologically inactive 4-beta phorbol 13-acetate and 4-beta phorbol were without effect. These results suggest that activation of Ca2+/phospholipid-dependent protein kinase by PMA stimulates adenylate cyclase activity by impairing the activity of the GTP-dependent inhibitory protein, possibly through a reduction of the GTP-GDP exchange.
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PMID:Alteration of the GTP-dependent inhibitory pathway of rat striatal adenylate cyclase by phorbol esters. 208 70

The properties of RAS2Gly19----Val and RAS2Thr152----Ile, two mutants suppressing the CDC25 requirement for the activation of adenylate cyclase in Saccharomyces cerevisiae, were compared with the properties of wild-type RAS2. We examined (a) the guanine nucleotide interaction, (b) the intrinsic GTPase (EC 3.6.1-) activity, and (c) the ability to activate adenylate cyclase in vitro. The low GTPase of RAS2Val19 is associated with an increased stability of the GTP complex. By contrast, RAS2Ile152 shows a strong destabilization of the GDP complex (the dissociation rate constants of the RAS2Ile152.GDP complex is enhanced almost 50 times) and an increased GTPase activity. Remarkably, all the parameters of the interaction with GDP and GTP as well as the catalytic activity are modified by the two mutations in an opposite manner. Our kinetic results show that the functional modifications of RAS2 compensating for the CDC25 inactivation can not only be associated with the presence of a long-lived RAS2.GTP complex, but also with a rapid GDP to GTP exchange reaction. As a striking result, the functional modifications induced by Thr152----Ile activate the adenylate cyclase in vitro much more efficiently than those induced by Gly19----Val. This stresses the importance of a rapid regeneration of the RAS2.GTP complex for the activation of the adenylate cyclase pathway.
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PMID:Different kinetic properties of the two mutants, RAS2Ile152 and RAS2Val19, that suppress the CDC25 requirement in RAS/adenylate cyclase pathway in Saccharomyces cerevisiae. 210 46

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.
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PMID:Functional reconstruction of purified Gi and Go with mu-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide. 215 51

Treatment of NG108-15 neuroblastoma x glioma cells (24 h) with cholera toxin (0.1-10 micrograms/ml) resulted in a concentration-dependent reduction of the membrane levels of subunits of GTP-binding regulatory proteins (G proteins), as determined by quantitative immunoblot procedures. The extent of reduction differed for different types of subunits: the levels of Go alpha and G beta 1 were reduced by 40-50%, whereas those of G alpha common immunoreactivity and Gi2 alpha were only reduced by 10-20% following treatment with 10 micrograms/ml cholera toxin. This effect of the toxin could not be mimicked by incubation with the resolved B oligomer of cholera toxin, nor by exposure of cells to agents able to raise the intracellular levels of cAMP. Basal adenylate cyclase was stimulated in a biphasic manner by cholera toxin, being stimulated at low concentrations (0.01-10 ng/ml) and then decreased at high (0.1-10 micrograms/ml) concentrations. Thus, the down regulation of G-protein subunits produced by cholera toxin requires its (ADP-ribosyl)transferase activity but does not result from a cAMP-mediated mechanism. The toxin-mediated decrease of Go alpha in the membrane was correlated with a diminution of opioid-receptor-mediated stimulation of high-affinity GTPase activity, suggesting that opioid receptors interact with Go in native membranes of NG108-15 cells. Northern-blot analysis of cytoplasmic RNA prepared from cells treated with cholera toxin showed that the levels of mRNA coding for G beta 1 did not change. Thus, the cholera-toxin-induced decrease of G-protein subunits may not result from an alteration in mRNA levels, but may involve a direct effect of the toxin on the process of insertion and/or clearance of G proteins into and/or from the membrane. These data indicate that cholera toxin, besides catalyzing the ADP-ribosylation of Gs and Gi/Go types of G proteins, can also reduce the steady state levels of Go alpha and G beta 1 subunits in the membrane and thus alter by an additional mechanism the function of inhibitory receptor systems.
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PMID:Cholera toxin differentially decreases membrane levels of alpha and beta subunits of G proteins in NG108-15 cells. 215 84


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