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)

Thrombin is thought to stimulate responsive cells by cleaving cell-surface receptors coupled to intracellular second-messenger-generating enzymes via G-proteins. In order to understand this process better, we have examined the regulation of adenylate cyclase by thrombin in the megakaryoblastic HEL cell line and compared it with platelets. A notable difference was found. In HEL-cell membrane preparations, thrombin inhibited cyclic AMP (cAMP) formation by a pertussis-toxin-sensitive mechanism comparable with that observed in platelets. In contrast, when added to intact HEL cells, thrombin activated adenylate cyclase and caused an increase in cAMP formation synergistic with that produced by forskolin and prostaglandin I2. This increase, which was not seen with platelets, was accompanied by an increase in cAMP metabolism by phosphodiesterase. Like other responses to thrombin, the increase in cAMP formation required proteolytically active thrombin and was subject to homologous desensitization. An equivalent response could be evoked by the addition of a polypeptide, derived from the N-terminus of the thrombin receptor, that has been shown to activate the receptor. The effects of thrombin could not, however, be reproduced by the addition of phorbol ester and the Ca2+ ionophore, A23187, nor be prevented with inhibitors of arachidonate metabolism. Preincubation of the cells with adrenaline, which inhibited Gs-mediated activation of adenylate cyclase, or pertussis toxin, which inhibited phospholipase C activation, had no effect on thrombin-induced cAMP formation. These results suggest that thrombin can regulate cAMP formation by two different mechanisms. First, thrombin can inhibit adenylate cyclase in a Gi-dependent manner. This effect predominates in HEL-cell membrane preparations, as it does in platelets, but is not detectable when thrombin is added to intact HEL cells. Instead, in intact HEL cells thrombin activates adenylate cyclase. Although clearly receptor-mediated, this response does not appear to involve Gi, Gs, protein kinase C, eicosanoid formation or changes in the cytosolic Ca2+ concentration.
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PMID:Dual regulation of cyclic AMP formation by thrombin in HEL cells, a leukaemic cell line with megakaryocytic properties. 131 10

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.
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PMID:Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis. 131 81

Thrombin-induced platelet aggregation is associated with an increase in intracellular calcium. Epinephrine provokes aggregation in the absence of a rise in intracellular calcium. Adenosine has been postulated as an endogenous inhibitor of platelet aggregation. In this study, the authors examine the effect of adenosine on the rise in intracellular calcium and on platelet aggregation, and the role of cyclic AMP (cAMP) in these actions. Human platelets were obtained from citrated plasma containing 5 micrograms/mL of indomethacin. Intracellular calcium was determined by fura-2 fluorescent dye. Adenosine inhibited thrombin-induced platelet aggregation and the rise in intracellular calcium in a dose-dependent manner. At a concentration of 100 mumol/L, adenosine completely inhibited thrombin-induced aggregation, but only partly inhibited the rise in intracellular calcium (55%). Adenosine also partially inhibited the rise in calcium produced by thrombin in both calcium-containing and calcium-free media, suggesting that adenosine inhibits both calcium influx and calcium mobilization. The effects of adenosine on intracellular calcium, as in the case of platelet aggregation, appear to be linked to adenylate cyclase, since they were prevented by the adenylate cyclase inhibitor 2',5'-dideoxyadenosine (1-mmol/L) and were potentiated by phospho-diesterase inhibition with papaverine (1 mumol/L). Adenosine and dibutyryl-cAMP also inhibited epinephrine-stimulated platelet aggregation in a dose-dependent manner. Thus, it appears that adenosine may inhibit platelet aggregation independently of its ability to decrease cytosolic free calcium.
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PMID:Role of cyclic AMP in adenosine inhibition of intracellular calcium rise in human platelets. Comparison of adenosine effects on thrombin- and epinephrine-induced platelet stimulation. 132 39

1. Barrier function and cytosolic free calcium content [Ca2+]i was measured in monolayers of bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC). 2. Thrombin (1 u ml-1) increased albumin transfer across monolayers of BPAEC but not BAEC, yet induced biphasic increases in [Ca2+]i in both endothelial cell types, consisting of a rapid, initial phasic component which decayed to a lower, more sustained plateau phase. 3. 4 beta-Phorbol 12-myristate 13-acetate (PMA; 0.3-3000 nM) increased albumin transfer across monolayers of BPAEC and BAEC, but had no effect on basal levels of [Ca2+]i in either endothelial cell type. 4. Treatment of BPAEC and BAEC with forskolin (30 microM), an activator of adenylate cyclase, had no effect on resting transfer of albumin, but inhibited that stimulated by PMA (600 nM). It also inhibited the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but enhanced the plateau phase of the associated increase in [Ca2+]i. 5. Treatment of BPAEC and BAEC with either atriopeptin II (100 nM), an activator of particulate guanylate cyclase, or 8 bromo cyclic GMP (30 microM) had no effect on resting or PMA (600 nM)-stimulated transfer of albumin. Both agents did, however, inhibit the thrombin (1 u ml-1)-induced increase in albumin transfer across monolayers of BPAEC, but had no effect on the associated increase in [Ca2+]i. 6. These data suggest a dissociation between the ability of agents that increase or decrease albumin transfer and their effects on [Ca2+]i. Consequently, activation of protein kinase C may be the major stimulus for trans-endothelial transfer of macromolecular solutes. Endothelial barrier function is enhanced by elevation of either cyclic AMP or cyclic GMP content. Cyclic AMP appears to act by inhibiting the actions of protein kinase C, while cyclic GMP may act to inhibit a key step proximal to activation of this enzyme.
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PMID:Modulation of barrier function of bovine aortic and pulmonary artery endothelial cells: dissociation from cytosolic calcium content. 133 54

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.
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PMID:Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase. 137 79

Human parathyroid hormone, hPTH, an 84 amino acid polypeptide, was produced intracellularly in Escherichia coli as a fusion protein, linked to the C-terminus of a 15 kD IgG-binding protein. Approximately 100 mg fusion protein was obtained per liter fermentation medium. To test the efficiency of two alternative enzymatic cleavage methods, two fusion proteins differing only in the linker region were constructed. Cleavage of a Phe-Phe-Pro-Arg linker was obtained with bovine thrombin and cleavage of a Phe-Ala-His-Tyr linker with recombinant H64A subtilisin. Both enzymes yielded the correct N-terminus and cleaved their respective linkers quantitatively, although additional internal cleavage sites in hPTH were detected and characterized. The linker cleavage conditions were optimized and hPTH was purified to homogeneity. Thrombin cleavage resulted in a final yield of 5 mg hPTH/L, while H64A subtilisin cleavage was more specific and gave 8 mg/L. The purified recombinant product was identical to native hPTH and exhibited full biological activity in an adenylate cyclase assay.
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PMID:Thrombin and H64A subtilisin cleavage of fusion proteins for preparation of human recombinant parathyroid hormone. 179 10

The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have used the U937 human monocytic cell line to explore the signal transduction mechanisms utilised by thrombin in these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP3) and the mobilisation of intracellular Ca2+ [( Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B2 synthesis in the differentiated cells. In contrast, the chemotactic peptide N-formyl-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxB2 synthesis under conditions where it evoked increases in IP3 formation and [Ca2+]i mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thrombin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. These results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB2 synthesis or cause Gi-mediated inhibition of adenylate cyclase in U937 cells contrasts with its effects in human platelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin receptor or receptor-effector coupling mechanism(s) present in other thrombin-responsive cells.
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PMID:Thrombin signalling in U937 human monocytic cells is coupled to inositol phosphate formation but not to thromboxane B2 synthesis nor to inhibition of adenylate cyclase: distinct differences in thrombin signalling between U937 cells and platelets. 180 Jan 26

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.
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PMID:Receptor and G protein-mediated responses to thrombin in HEL cells. 184 99

Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.
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PMID:Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation. 193 86

ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin.
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PMID:Aggregin: a platelet ADP receptor that mediates activation. 240 87


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