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:4.6.1.2 (guanylate cyclase)
8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Folic acid and cAMP are chemoattractants in Dictyostelium discoideum, which bind to different surface receptors. The signal is transduced from the receptors via different G proteins into a common pathway which includes guanylyl cyclase and acto-myosin. To investigate this common pathway, ten mutants which do not react chemotactically to both cAMP and folic acid were isolated with a simple new chemotactic assay. Genetic analysis shows that one of these mutants (KI-10) was dominant; the other nine mutants were recessive, and comprise nine complementation groups. In wild-type cells, the chemoattractants activate adenylyl cyclase, phospholipase C, and guanylyl cyclase in a transient manner. In mutant cells the formation of cAMP and IP3 were generally normal, whereas the cGMP response was altered in most of the ten mutants. Particularly, mutant KI-8 has strongly reduced basal guanylyl cyclase activity; the enzyme is present in mutant KI-10, but can not be activated by cAMP or folic acid. The cGMP response of five other mutants is altered in either magnitude, dose dependency, or kinetics. These observations suggest that the second messenger cGMP plays a key role in chemotaxis in Dictyostelium.
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PMID:Non-chemotactic Dictyostelium discoideum mutants with altered cGMP signal transduction. 790 39

In Dictyostelium discoideum extracellular cyclic AMP (cAMP), as shown by previous studies, induces a transient accumulation of intracellular cyclic guanosine-5'-monophosphate (cGMP), which peaks at 10 s and recovers basal levels at 30 s after stimulation, even with persistent cAMP stimulation. Additional investigations have shown that the cAMP-mediated cGMP response is built up from surface cAMP receptor-mediated activation of guanylyl cyclase and hydrolysis of cGMP by phosphodiesterase. The regulation of these activities was measured in detail on a seconds time-scale, demonstrating complex adaptation of the receptor, allosteric activation of cGMP-phosphodiesterase by cGMP, and potent inhibition of guanylyl cyclase by Ca2+. In this paper we present a computer model that combines all experimental data on the cGMP response. The model is used to investigate the contribution of each structural and regulatory component in the final cGMP response. Four models for the activation and adaptation of the receptor are compared with experimental observations. Only one model describes the magnitude and kinetics of the response accurately. The effect of Ca2+ on the cGMP response is simulated by changing the Ca2+ concentrations outside the cell (Ca2+ influx) and in stores (IP3-mediated release) and changing phospholipase C activity. The simulations show that Ca2+ mainly determines the magnitude of the cGMP accumulation; simulations are in good agreement with experiments on the effect of Ca2+ in electropermeabilized cells. Finally, when cGMP-phosphodiesterase activity is deleted from the model, the simulated cGMP response is elevated and prolonged, which is in close agreement with the experimental observations in mutant stmF that lacks this enzyme activity. We conclude that the computer model provides a good description of the observed response, suggesting that the main structural and regulatory components have been identified.
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PMID:A model for cAMP-mediated cGMP response in Dictyostelium discoideum. 791 38

Previous results have shown that the G alpha protein subunit G alpha 2 is required for aggregation in Dictyostelium discoideum and is essential for coupling cell-surface cAMP receptors to downstream effectors in vivo during this stage of development. G alpha 2 expresses at least four distinct transcripts that are differentially regulated during development; two of the transcripts are expressed exclusively in the multicellular stages and their expression is restricted to prestalk cells. We partially dissected the G alpha 2 promoter and identified a component that is expressed exclusively during the multicellular stages using luciferase gene fusions. When this promoter region is coupled to lacZ, beta-gal expression is restricted to the multicellular stages and localized in prestalk cells with a pattern similar to that of the ecmA prestalk-specific promoter. We show that expression in wild-type cells of the G alpha 2 mutant protein [G alpha 2(G206T)] during the early stages of development blocks aggregation and cAMP-mediated activation of adenylyl cyclase and guanylyl cyclase, suggesting it functions as a dominant negatively active G alpha subunit. When this mutant G alpha protein is expressed from the ecmA prestalk-specific promoter, abnormal stalk differentiation during culmination is observed. Expression of the mutant G alpha 2 from the SP60 prespore promoter or wild-type G alpha 2 from either the ecmA or the SP60 promoter results in no detectable phenotype. The results suggest that G alpha 2 plays an essential role during the culmination stage in prestalk cells and may mediate cAMP receptor activation of these processes during multicellular development.
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PMID:Spatial and temporal expression of the Dictyostelium discoideum G alpha protein subunit G alpha 2: expression of a dominant negative protein inhibits proper prestalk to stalk differentiation. 818 66

In Dictyostelium discoideum extracellular cAMP induces chemotaxis via a transmembrane signal transduction cascade consisting of surface cAMP receptors, G-proteins and effector enzymes including adenylyl cyclase, guanylyl cyclase and phospholipase C. Previously it was demonstrated that some cAMP derivatives such as 3'-deoxy-3'-aminoadenosine 3':5'-monophosphate (3'NH-cAMP) bind to the receptor and induce normal activation of adenylyl cyclase and guanylyl cyclase. However these analogues do not induce chemotaxis, probably because the signal is transduced in an inappropriate manner. We have now studied the regulation of phospholipase C by cAMP and these chemotactic antagonists. cAMP induced the two-fold activation of phospholipase C leading to a transient increase of Ins(1,4,5)P3 levels. In contrast, the analogues induced a rapid decrease of intracellular Ins(1,4,5)P3 levels, due to the inhibition of phospholipase C activity. In a transformed cell-line lacking the G-protein that mediates phospholipase C inhibition, 3'NH-cAMP did not decrease phospholipase C activity and was no longer an antagonist of chemotaxis. These results suggest that inhibition of phospholipase C leads to aberrant chemotaxis.
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PMID:Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C. 838 94

During development, Dictyostelium discoideum cells produce platelet activating factor (PAF). When cells are stimulated with external cAMP pulses, PAF is transiently synthesized. To determine whether PAF is involved in signal transduction, we have tested the effect of PAF on some cellular responses which are regulated by cAMP, such as spontaneous light-scattering oscillations of suspended cells, cAMP relay, transient increases of cGMP level, and extracellular calcium uptake. Our results show that PAF specifically interferes with spontaneous spike-shaped oscillations, without affecting sinusoidal ones. PAF increases the amplitude of a spike, but has no effects on its phase or frequency. When cells fail to oscillate spontaneously, PAF does not induce spikes; however, if administered together with cAMP, it amplifies the light-scattering response to cAMP. Amplification of light-scattering changes is accompanied by a threefold increase in the concentration levels of both cellular cAMP and cGMP. Extracellular Ca2+ uptake is also stimulated by PAF. This latter response is independent of endogenous or exogenously added cAMP. All these effects are specific for the naturally occurring R-enantiomer of PAF, the S-enantiomer and lyso-PAF being inactive. These results suggest that PAF modulates signal transduction in Dictyostelium, probably by interacting with an intracellular acceptor, which is involved in the pathways regulating membrane Ca2+ channels, adenylate and guanylate cyclase.
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PMID:Platelet activating factor modulates signal transduction in Dictyostelium. 838 95

Aggregating Dictyostelium cells secrete cAMP during cell aggregation. cAMP induces two fast responses, the production of more cAMP (relay) and directed cell locomotion (chemotaxis). Extracellular cAMP binds to G-protein-coupled receptors leading to the activation of second messenger pathways, including the activation of adenylyl cyclase, guanylyl cyclase, phospholipase C and the opening of plasma membrane Ca2+ channels. Many genes encoding these sensory transduction proteins have been cloned and null mutants of nearly all components have been characterized in detail. Undoubtedly, activation of adenylyl cyclase is the most complex, involving G-proteins, a soluble protein called CRAC and components of the MAP kinase pathway. Null mutants in this pathway do not aggregate, but can exhibit chemotaxis and develop normally when supplied with exogenous cAMP. The pathways leading to the activation of phospholipase C were identified, but unexpectedly, deletion of the phospholipase C gene has no effect on chemotaxis and development, nor on intracellular Ins(1,4,5)P3 levels; the metabolism of this second messenger will be discussed in some detail. Activation of guanylyl cyclase is G-protein-dependent and essential for chemotaxis. Analysis of a collection of chemotactic mutants reveals that most mutants are defective in either the production or intracellular detection of cGMP, thereby placing this second messenger at the center of chemotactic signal transduction. Analysis of the cAMP-mediated opening of plasma membrane calcium channels in signal transduction mutants suggests that it has two components, one that depends on G-proteins and intracellular cGMP and one that is G-protein-independent.
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PMID:Transduction of the chemotactic cAMP signal across the plasma membrane of Dictyostelium cells. 853 2

The change in extracellular osmolarity from 0.07 osm to 0.38 osm caused rapid cell shrinkage and loss of pseudopodes in Dictyostelium discoideum amoebae and induced elevation of total (cellular + extracellular) cGMP with a 2.5-min lag. cGMP accumulation reached a peak at 10-15 min after the change, and then the total cGMP gradually decreased. cGMP first accumulated intracellularly and was then secreted. A roughly identical osmotic concentration was required for the accumulation when the effect of KCl and glucose was tested. The non-osmolytes, formamide and ethanol, did not induce the accumulation. We concluded that hypertonic stress induces cGMP accumulation in D. discoideum amoebae. The hypertonic stress-induced accumulation of cGMP was observed in a streamer F mutant (NP368) that lacks cGMP-specific phosphodiesterase. While Dictyostelium cells also have nonspecific phosphodiesterases that degrade both cGMP and cAMP, hypertonic stress induced only a small increase in cAMP in wild type and streamer F cells. These results suggest that hypertonic stress-induced accumulation of cGMP is due to the activation of guanylate cyclase rather than the inhibition of phosphodiesterases. Binding of folic acid to the specific receptors on the cell surface induces a rapid transient accumulation of cGMP that reaches a peak at 10 s. When cells were stimulated by folic acid after the addition of 0.31 M glucose, rapid transient cGMP accumulation was observed immediately after the stimulation by folic acid and prolonged cGMP accumulation was induced 2-3 min after the addition of glucose irrespective of the timing of folic acid stimulation. These results suggest that the hypertonic stress-induced and the receptor-mediated accumulation proceed independently of one another. 2,3-Dimercapto-1-propanol, a thiol-reducing reagent, induces prolonged cGMP accumulation similar to hypertonic stress. However, the hypertonic stress-induced cGMP accumulation was enhanced by EDTA and was not suppressed by folic acid and cAMP. These characteristics are distinct from the reducing reagent-induced accumulation that is suppressed by EDTA, folic acid, and cAMP. These findings show that hypertonic stress has a unique effect on the activation of guanylate cyclase.
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PMID:cGMP accumulation induced by hypertonic stress in Dictyostelium discoideum. 862 17

Chemoattractants transiently activate guanylyl cyclase in Dictyostelium discoideum cells. Mutant analysis demonstrates that the produced cGMP plays an essential role in chemotactic signal transduction, controlling the actomyosin-dependent motive force. Guanylyl cyclase activity is associated with the particulate fraction of a cell homogenate. The addition of the cytosol stimulates guanylyl cyclase activity, whereas the cytosol plus ATP/Mg2+ inhibits enzyme activity. We have analyzed the regulation of guanylyl cyclase in chemotactic mutants and present evidence that a cGMP-binding protein mediates both stimulation and ATP-dependent inhibition of guanylyl cyclase. Upon chromatography of cytosolic proteins, cGMP binding activity co-elutes with both guanylyl cyclase-stimulating and ATP-dependent-inhibiting activities. In addition, ATP-dependent inhibition of guanylyl cyclase activity is enhanced by the cGMP analogue 8-Br-cGMP, suggesting that a cGMP-binding protein regulates guanylyl cyclase activity. Mutant KI-4 has an aberrant cGMP binding activity with very low Kd and shows a very small chemoattractant-mediated cGMP response; the cytosol from this mutant does not stimulate guanylyl cyclase. In contrast to KI-4, the aberrant cGMP binding activity of mutant KI-7 has a very high Kd and chemoattractants induce a prolonged cGMP response. The cytosol of this mutant stimulates guanylyl cyclase activity, but ATP does not inhibit the enzyme. Thus, two previously isolated chemotactic mutants are defective in the activation and inhibition of guanylyl cyclase, respectively. The positive and negative regulation of guanylyl cyclase by its product cGMP may well explain how cells process the temporospatial information of chemotactic signals, which is necessary for sensing the direction of the chemoattractant.
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PMID:Regulation of guanylyl cyclase by a cGMP-binding protein during chemotaxis in Dictyostelium discoideum. 879 95

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTP gamma S), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t1/2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca2+ ions inhibited the enzyme (half-maximal effect at 0.3 microM), whereas InsP3 had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells.
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PMID:Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells. 886 16

The chemoattractant cAMP induces directed cell locomotion in Dictyostelium cells. Several second messenger pathways are activated upon binding of cAMP to G-protein-coupled receptors, including adenylyl cyclase, guanylyl cyclase, phospholipase C, and the opening of plasma membrane Ca2+ channels. These second messenger responses are unaltered in many chemotactic mutants, except for the cGMP response. Activation of guanylyl cyclase depends on G-proteins and is regulated by a cGMP-binding protein in a complex manner. This cGMP-binding protein also mediates intracellular functions of cGMP to activate a PKC-related kinase that phosphorylates myosin II heavy chain, thereby allowing myosin filaments to rearrange during cell movement.
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PMID:cGMP as second messenger during Dictyostelium chemotaxis. 924 16


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