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1
ROELOFS, Jeroen, Helena SNIPPE, Reinhard G. KLEINEIDAM, and Peter J. M. Van HAASTERT. "Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase." Biochemical Journal 354, no. 3 (March 8, 2001): 697–706. http://dx.doi.org/10.1042/bj3540697.
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The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyosteliumdiscoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase.
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Dessauer, Carmen W., Bruce A. Posner, and Alfred G. Gilman. "Visualizing Signal Transduction: Receptors, G-Proteins, and Adenylate Cyclases." Clinical Science 91, no. 5 (November 1, 1996): 527–37. http://dx.doi.org/10.1042/cs0910527.
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1. The first glimpses of heterotrimeric G-proteins came with the discoveries of the ubiquitous adenylate cyclase activator, Gs, and the specialized retinal cGMP phosphodiesterase activator, Gt or transducin. The model that evolved for regulation of adenylate cyclase activity by G-proteins soon proved to be a general paradigm for a large number of signalling pathways. Although many different G-proteins interact with a diverse array of receptors and effectors, each is composed of a guanine-nucleotide-binding α-subunit and a tightly associated complex of a β- and a γ-subunit. 2. Receptors catalyse the activation of G-proteins by promoting exchange of GDP for GTP, while G-proteins catalyse their own deactivation as a result of their intrinsic GTPase activity. Crystallographic analysis has described several of the various conformational states that G-proteins undergo as they are activated and deactivated and has provided great insight into the kinetic models of G-protein-mediated signal transduction. 3. The regulation of adenylate cyclase has proven to be intriguing and complex. Gsα activates all forms of mammalian adenylate cyclase; other G-proteins (Gi, Go and Gz) inhibit certain isoforms of the enzyme. The discovery of new isoforms of adenylate cyclase has revealed synergistic and conditional mechanisms of regulation. These include activation or inhibition by the G-protein βγ-subunit complex, activation by Ca2+-calmodulin, and phosphorylation by protein kinases. The large number of receptors, G-proteins and adenylate cyclases provides a complex signalling network that integrates and interprets a multitude of convergent inputs.
3
SHENOY, Avinash R., Nandini P. SREENATH, Mohana MAHALINGAM, and Sandhya S. VISWESWARIAH. "Characterization of phylogenetically distant members of the adenylate cyclase family from mycobacteria: Rv1647 from Mycobacterium tuberculosis and its orthologue ML1399 from M. leprae." Biochemical Journal 387, no. 2 (April 5, 2005): 541–51. http://dx.doi.org/10.1042/bj20041040.
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Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylate and guanylate cyclases. Rv1647 was predicted to be an active adenylate cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, the cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the previously characterized Rv1625c adenylate cyclase. Adenylate cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate-specifying residues to those present in guanylate cyclases failed to convert the enzyme into a guanylate cyclase, and did not alter its oligomeric status. Orthologues of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The orthologue from M. leprae (ML1399) was cloned, and the protein was expressed, purified and shown biochemically to be an adenylate cyclase, thus representing the first adenylate cyclase to be described from M. leprae. Importantly, Western-blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that the Rv1647 and ML1399 gene products respectively were expressed in these bacteria. Additionally, M. tuberculosis was also found to express the Rv1625c adenylate cyclase, suggesting that multiple adenylate cyclase proteins may be expressed simultaneously in this organism. These results suggest that class III cyclase-like gene products probably have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.
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Nocero, M., T. Isshiki, M. Yamamoto, and C. S. Hoffman. "Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8)." Genetics 138, no. 1 (September 1, 1994): 39–45. http://dx.doi.org/10.1093/genetics/138.1.39.
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Abstract In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, adenylate cyclase enzymatic activity is regulated by heterotrimeric guanine nucleotide-binding proteins (G proteins). In the budding yeast Saccharomyces cerevisiae, adenylate cyclase enzymatic activity is regulated by monomeric, guanine nucleotide-binding Ras proteins. We show here that git8 is identical to the gpa2 gene that encodes a protein homologous to the alpha subunit of a G protein. Mutations in two additional genes, git3 and git5 are suppressed by gpa2+ in high copy number. Furthermore, a mutation in either git3 or git5 has an additive effect in strains deleted for gpa2 (git8), as it significantly increases expression of an fbp1-lacZ reporter gene. Therefore, git3 and git5 appear to act either in concert with or independently from gpa2 (git8) to regulate adenylate cyclase activity.
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Mitts, M. R., J. Bradshaw-Rouse, and W. Heideman. "Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1." Molecular and Cellular Biology 11, no. 9 (September 1991): 4591–98. http://dx.doi.org/10.1128/mcb.11.9.4591.
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The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.
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Mitts, M. R., J. Bradshaw-Rouse, and W. Heideman. "Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1." Molecular and Cellular Biology 11, no. 9 (September 1991): 4591–98. http://dx.doi.org/10.1128/mcb.11.9.4591-4598.1991.
Full textAbstract:
The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.
7
Ros, M., J. K. Northup, and C. C. Malbon. "Adipocyte G-proteins and adenylate cyclase. Effects of adrenalectomy." Biochemical Journal 257, no. 3 (February 1, 1989): 737–44. http://dx.doi.org/10.1042/bj2570737.
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Steroid hormones modulate the ability of cells to respond to hormones that act via cyclic AMP. In adipocytes of adrenalectomized rats, cyclic AMP accumulation and lipolysis in response to adrenaline are attenuated. However, the mechanism(s) of these effects are poorly understood. The effects of altered glucocorticoid status in vivo on the steady-state amounts of components of the hormone-sensitive adenylate cyclase were analysed in rat adipocytes. beta-Adrenergic receptors were analysed by using radioligand binding and immunoblotting with an anti-receptor antiserum. Neither the amount of radioligand binding nor the amount of beta-adrenergic-receptor peptide (Mr 67,000) was altered by adrenalectomy, whereas treatment of adrenalectomized rats with dexamethasone was found to increase both parameters by more than 25% with respect to the control. Forskolin-stimulated adenylated cyclase activity was unchanged in membranes isolated from adipocytes of adrenalectomized rats, but was decreased (50%) in those from dexamethasone-treated rats. The alpha-subunit of Gs was probed by using cholera-toxin-catalysed ADP-ribosylation. Immunoblotting was used to analyse the steady-state amounts of G-protein beta-subunits (beta-G35/36). Adrenalectomy was associated with decreases in the steady-state amounts of alpha-Gs (30%) and beta-G35/36 (50%). Dexamethasone treatment of adrenalectomized animals partially restored the lipolytic response of adipocytes to adrenaline and the amounts of alpha-Gs, increased the amounts of beta-G35/36 subunits from 50% to 150% of control values, increased beta-adrenergic receptors by more than 25% and decreased adenylate cyclase activity (50%). These results suggest that the steady-state amounts of components of hormone-sensitive adenylate cyclase are differentially regulated by glucocorticoids.
8
Inhorn, L., JW Fleming, D. Klingberg, TG Gabig, and HS Boswell. "Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase." Blood 71, no. 4 (April 1, 1988): 1003–11. http://dx.doi.org/10.1182/blood.v71.4.1003.1003.
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Abstract Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3- independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the “differentiation” agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G- proteins appear to play a secondary role.
9
Inhorn, L., JW Fleming, D. Klingberg, TG Gabig, and HS Boswell. "Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase." Blood 71, no. 4 (April 1, 1988): 1003–11. http://dx.doi.org/10.1182/blood.v71.4.1003.bloodjournal7141003.
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Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3- independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the “differentiation” agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G- proteins appear to play a secondary role.
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Marshall, M. S., J. B. Gibbs, E. M. Scolnick, and I. S. Sigal. "An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations." Molecular and Cellular Biology 8, no. 1 (January 1988): 52–61. http://dx.doi.org/10.1128/mcb.8.1.52.
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Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.
11
Marshall, M. S., J. B. Gibbs, E. M. Scolnick, and I. S. Sigal. "An adenylate cyclase from Saccharomyces cerevisiae that is stimulated by RAS proteins with effector mutations." Molecular and Cellular Biology 8, no. 1 (January 1988): 52–61. http://dx.doi.org/10.1128/mcb.8.1.52-61.1988.
Full textAbstract:
Conservative amino acid substitutions were introduced into the proposed effector regions of both mammalian Ha-ras (residues 32 to 40) and Saccharomyces cerevisiae RAS2 (residues 39 to 47) proteins. The RAS2[Ser 42] protein had reduced biological function in the yeast S. cerevisiae. A S. cerevisiae strain with a second-site suppressor mutation, SSR2-1, was isolated which could grow on nonfermentable carbon sources when the endogenous RAS2 protein was replaced by the RAS2[Ser 42] protein. The SSR2-1 mutation was mapped to the structural gene for adenylate cyclase (CYR1), and the gene containing SSR2-1 was cloned and sequenced. SSR2-1 corresponded to a point mutation that would create an amino acid substitution of a tyrosine residue for an aspartate residue at position 1547. The SSR2-1 gene encodes an adenylate cyclase that is dependent on ras proteins for activity, but is stimulated by Ha-ras and RAS2 mutant proteins that are unable to stimulate wild-type adenylate cyclase.
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McKenzie, F. R., and G. Milligan. "δ-opioid-receptor-mediated inhibition of adenylate cyclase is transduced specifically by the guanine-nucleotide-binding protein Gi2." Biochemical Journal 267, no. 2 (April 15, 1990): 391–98. http://dx.doi.org/10.1042/bj2670391.
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Mouse neuroblastoma x rat glioma hybrid cells (NG108-15) express an opioid receptor of the delta subclass which both stimulates high-affinity GTPase activity and inhibits adenylate cyclase by interacting with a pertussis-toxin-sensitive guanine-nucleotide-binding protein(s) (G-protein). Four such G-proteins have now been identified without photoreceptor-containing tissues. We have generated anti-peptide antisera against synthetic peptides which correspond to the C-terminal decapeptides of the alpha-subunit of each of these G-proteins and also to the stimulatory G-protein of the adenylate cyclase cascade (Gs). Using these antisera, we demonstrate the expression of three pertussis-toxin-sensitive G-proteins in these cells, which correspond to the products of the Gi2, Gi3 and Go genes, as well as Gs. Gi1, however, is not expressed in detectable amounts. IgG fractions from each of these antisera and from normal rabbit serum were used to attempt to interfere with the interaction of the opioid receptor with the G-protein system by assessing ligand stimulation of high-affinity GTPase activity, inhibition of adenylate cyclase activity and conversion of the receptor to a state which displays reduced affinity for agonists. The IgG fraction from the antiserum (AS7) which specifically identifies Gi2 in these cells attenuated the effects of the opioid receptor. This effect was complete and was not mimicked by any of the other antisera. We conclude that the delta-opioid receptor of these cells interacts directly and specifically with Gi2 to cause inhibition of adenylate cyclase, and that Gi2 represents the true Gi of the adenylate cyclase cascade. The ability to measure alterations in agonist affinity for receptors following the use of specific antisera against a range of G-proteins implies that such techniques should be applicable to investigations of the molecular identity of the G-protein(s) which interacts with any receptor.
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Dixon, B. S., E. Sutherland, A. Alexander, D. Nibel, and F. R. Simon. "Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes." American Journal of Physiology-Gastrointestinal and Liver Physiology 265, no. 4 (October 1, 1993): G686—G698. http://dx.doi.org/10.1152/ajpgi.1993.265.4.g686.
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Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.
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Cooper, Dermot M. F., and Valentina G. Tabbasum. "Adenylate cyclase-centred microdomains." Biochemical Journal 462, no. 2 (August 7, 2014): 199–213. http://dx.doi.org/10.1042/bj20140560.
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Recent advances in the AC (adenylate cyclase)/cAMP field reveal overarching roles for the ACs. Whereas few processes are unaffected by cAMP in eukaryotes, ranging from the rapid modulation of ion channel kinetics to the slowest developmental effects, the large number of cellular processes modulated by only three intermediaries, i.e. PKA (protein kinase A), Epacs (exchange proteins directly activated by cAMP) and CNG (cyclic nucleotide-gated) channels, poses the question of how selectivity and fine control is achieved by cAMP. One answer rests on the number of differently regulated and distinctly expressed AC species. Specific ACs are implicated in processes such as insulin secretion, immunological responses, sino-atrial node pulsatility and memory formation, and specific ACs are linked with particular diseased conditions or predispositions, such as cystic fibrosis, Type 2 diabetes and dysrhythmias. However, much of the selectivity and control exerted by cAMP lies in the sophisticated properties of individual ACs, in terms of their coincident responsiveness, dynamic protein scaffolding and organization of cellular microassemblies. The ACs appear to be the centre of highly organized microdomains, where both cAMP and Ca2+, the other major influence on ACs, change in patterns quite discrete from the broad cellular milieu. How these microdomains are organized is beginning to become clear, so that ACs may now be viewed as fundamental signalling centres, whose properties exceed their production of cAMP. In the present review, we summarize how ACs are multiply regulated and the steps that are put in place to ensure discrimination in their signalling. This includes scaffolding of targets and modulators by the ACs and assembling of signalling nexuses in discrete cellular domains. We also stress how these assemblies are cell-specific, context-specific and dynamic, and may be best addressed by targeted biosensors. These perspectives on the organization of ACs uncover new strategies for intervention in systems mediated by cAMP, which promise far more informed specificity than traditional approaches.
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Shimada, N., M. Tsubokura, and N. Kimura. "Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets." Thrombosis and Haemostasis 55, no. 02 (1986): 178–83. http://dx.doi.org/10.1055/s-0038-1661517.
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SummaryIsolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.
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Smiley, B. L., A. W. Stadnyk, P. J. Myler, and K. Stuart. "The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins." Molecular and Cellular Biology 10, no. 12 (December 1990): 6436–44. http://dx.doi.org/10.1128/mcb.10.12.6436.
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We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.
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Smiley, B. L., A. W. Stadnyk, P. J. Myler, and K. Stuart. "The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins." Molecular and Cellular Biology 10, no. 12 (December 1990): 6436–44. http://dx.doi.org/10.1128/mcb.10.12.6436-6444.1990.
Full textAbstract:
We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.
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Field, J., D. Broek, T. Kataoka, and M. Wigler. "Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 6 (June 1987): 2128–33. http://dx.doi.org/10.1128/mcb.7.6.2128.
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In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.
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Field, J., D. Broek, T. Kataoka, and M. Wigler. "Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 6 (June 1987): 2128–33. http://dx.doi.org/10.1128/mcb.7.6.2128-2133.1987.
Full textAbstract:
In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.
20
Banga, H. S., R. K. Walker, L. K. Winberry, and S. E. Rittenhouse. "Platelet adenylate cyclase and phospholipase C are affected differentially by ADP-ribosylation. Effects on thrombin-mediated responses." Biochemical Journal 252, no. 1 (May 15, 1988): 297–300. http://dx.doi.org/10.1042/bj2520297.
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Thrombin stimulates phospholipase C and inhibits adenylate cyclase in human platelets. We have studied the effect of purified S1 monomer, the ADP-ribosylating subunit of pertussis toxin, on these receptor-coupled G-protein-dependent activities. ADP-ribosylation of a 41 kDa protein is associated with a marked decrease in the ability of thrombin to inhibit cyclic AMP formation, but has little effect on phospholipase C. Therefore adenylate cyclase and phospholipase C appear to be modulated by different G-proteins.
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Horiuchi, H., K. Kaibuchi, M. Kawamura, Y. Matsuura, N. Suzuki, Y. Kuroda, T. Kataoka, and Y. Takai. "The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase." Molecular and Cellular Biology 12, no. 10 (October 1992): 4515–20. http://dx.doi.org/10.1128/mcb.12.10.4515.
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Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
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Horiuchi, H., K. Kaibuchi, M. Kawamura, Y. Matsuura, N. Suzuki, Y. Kuroda, T. Kataoka, and Y. Takai. "The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase." Molecular and Cellular Biology 12, no. 10 (October 1992): 4515–20. http://dx.doi.org/10.1128/mcb.12.10.4515-4520.1992.
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Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
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Bookbinder, L. H., G. W. Moy, and V. D. Vacquier. "Identification of sea urchin sperm adenylate cyclase." Journal of Cell Biology 111, no. 5 (November 1, 1990): 1859–66. http://dx.doi.org/10.1083/jcb.111.5.1859.
Full textAbstract:
Calmodulin (CaM) affinity chromatography of a detergent extract of sea urchin sperm yielded approximately 20 major proteins. One of these proteins, of Mr 190,000, was purified and used to immunize rabbits. After absorption with living sperm, the serum reacted monospecifically on one- and two-dimensional Western immunoblots with the Mr 190,000 protein. The anti-190-kD serum inhibited 94% of the adenylate cyclase (AC) activity of the CaM eluate. An immunoaffinity column removed 95% of the AC activity, and the purified (but inactive) Mr 190,000 protein was eluted from the column. The antiserum also inhibited 23% of the activity of bovine brain CaM-sensitive AC and 90% of the activity of horse sperm CaM-sensitive AC. These data support the hypothesis that the Mr 190,000 protein is sea urchin sperm AC. Although this AC bound to CaM, it was not possible to demonstrate directly a Ca2+ or CaM sensitivity. However, two CaM antagonists, calmidazolium and chlorpromazine, both inhibited AC activity, and the inhibition was released by added CaM, suggesting the possibility of regulation of this AC by CaM. Indirect immunofluorescence showed the Mr 190,000 protein to be highly concentrated on only the proximal half of the sea urchin sperm flagellum. This asymmetric localization of AC may be important to its function in flagellar motility. This is the first report of the identification of an AC from animal spermatozoa.
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Gilman, Alfred G. "Guanine Nucleotide-Binding Regulatory Proteins and Adenylate Cyclase." Japanese Journal of Pharmacology 40 (1986): 7. http://dx.doi.org/10.1016/s0021-5198(19)58890-4.
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RASENICK, MARK M. "Regulation of Neuronal Adenylate Cyclase by Microtubule Proteins." Annals of the New York Academy of Sciences 466, no. 1 Dynamic Aspec (June 1986): 794–97. http://dx.doi.org/10.1111/j.1749-6632.1986.tb38462.x.
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Stadel, J. M., R. Rebar, and S. T. Crooke. "Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled β-adrenergic receptors in avian erythrocyte membranes." Biochemical Journal 252, no. 3 (June 15, 1988): 771–76. http://dx.doi.org/10.1042/bj2520771.
Full textAbstract:
Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.
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Milde, Markus, Ruth C. Werthmann, Kathrin von Hayn, and Moritz Bünemann. "Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins." Biochemical Society Transactions 42, no. 2 (March 20, 2014): 239–43. http://dx.doi.org/10.1042/bst20130280.
Full textAbstract:
A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas Gq-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)–cAMPS (adenosine 3′,5′-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via Gi-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled Gi-proteins and AC5. FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity. We found the dissociation of Gαi1 subunits and AC5 to occur slower than the Gi-protein deactivation. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.
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Anand-Srivastava, M. B. "Enhanced expression of inhibitory guanine nucleotide regulatory protein in spontaneously hypertensive rats. Relationship to adenylate cyclase inhibition." Biochemical Journal 288, no. 1 (November 15, 1992): 79–85. http://dx.doi.org/10.1042/bj2880079.
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We have previously shown that the stimulatory effects of guanine nucleotides, N-ethylcarboxamide-adenosine and other agonists on adenylate cyclase activity were diminished in aorta and heart sarcolemma of spontaneously hypertensive rats (SHR) [Anand-Srivastava (1988) Biochem. Pharmacol. 37, 3017-3022]. In the present studies, we have examined whether the decreased response of these agonists is due to the defective GTP-binding proteins (G-proteins) which couple the receptors to adenylate cyclase, and have therefore measured the levels of G-proteins in aorta and heart from SHR and their respective Wistar-Kyoto (WKY) controls by using pertussis toxin (PT)- and cholera toxin (CT)-catalysed ADP-ribosylations and immunoblotting techniques using specific antibodies against G-proteins. The labelling with [32P]NAD+ and PT identified a 40/41 kDa protein in heart and aorta from WKY and SHR and was significantly increased in the hearts (approximately 100%) and aorta (approximately 30-40%), from SHR as compared with WKY. Immunoblotting revealed an increase in the levels of the G-protein alpha-subunits Gi alpha-2 and Gi alpha-3 in heart and Gi alpha-2 in aorta, whereas no change in Go alpha was observed in heart from SHR and WKY. On the other hand, no differences were observed in CT labelling or immunoblotting of stimulatory G-protein (Gs) in heart and aorta from WKY and SHR. In addition, CT stimulated the adenylate cyclase activity in heart sarcolemma from WKY and SHR to a similar extent. These results were correlated with adenylate cyclase inhibition and stimulation by various hormones. Angiotensin II (AII), atrial natriuretic factor (ANF) and oxotremorine-mediated inhibition was found to be greater in SHR as compared with WKY, whereas the stimulatory effects of adrenaline, isoprenaline, dopamine and forskolin were diminished in SHR aorta as compared to WKY. These results indicate that regulatory protein G(i) is more expressed in SHR, which may be associated with the decreased responsiveness of stimulatory hormones and increased sensitivity of inhibitory hormones to stimulate/inhibit adenylate cyclase activity. It may thus be suggested that the enhanced G(i) activity may be one of the mechanisms responsible for the diminished vascular tone and impaired myocardial functions in hypertension.
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Moullet, O., and J. L. Dreyer. "Selective inhibition of adenylate cyclase in bovine cortex by quinones: a novel cellular substrate for quinone cytotoxicity." Biochemical Journal 300, no. 1 (May 15, 1994): 99–106. http://dx.doi.org/10.1042/bj3000099.
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Quinones are widely distributed substances of often potential toxicological significance. On the other hand, cyclic AMP is known to promote a cell-survival response and to retard apoptosis [Berridge, Tan and Hilton (1993) Exp. Hematol. 21, 269-276]. Therefore the effects of quinones on adenylate cyclase were tested. Adenylate cyclase is rapidly inhibited by quinones, with IC50 values of 40-45 microM for p-benzoquinone (BQ) or 200 microM for dichlorophenol-indophenol (DCIP), with 2-substituted quinones being inactive. Membrane solubilization decreases the IC50 values for BQ and DCIP to 18 microM and 40 microM respectively. The inhibition is not affected by GTP, GDP or analogues, or by cholera and pertussis toxins; therefore it is not mediated by a G-protein or the activation of a defined receptor. Further, the inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of quinone and forskolin restoring the enzyme activity to its basal value. Reduction of BQ with sodium dithionite stoichiometrically prevents the inhibition of adenylate cyclase; in turn, oxidation of hydroquinone with ferricyanide fully restores it, indicating that the oxidized state of the quinone is required for inhibition. In addition, BQ is cytotoxic in vivo on HepG2, a human hepatocellular carcinoma cell line, but the effect can be prevented with forskolin. In plasma membranes, BQ tightly binds only one major and two minor proteins; these BQ-binding proteins were purified by means of labelling with [14C]BQ followed by PAGE under native conditions. Together these observations indicate that the action of quinone can be traced to targeting a limited number of proteins at the plasma membrane in a highly selective way and to affecting key enzymes such as adenylate cyclase.
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Bahouth, S. W. "Thyroid hormone regulation of transmembrane signalling in neonatal rat ventricular myocytes by selective alteration of the expression and coupling of G-protein α-subunits." Biochemical Journal 307, no. 3 (May 1, 1995): 831–41. http://dx.doi.org/10.1042/bj3070831.
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Thyroid hormone exerts profound effects on the activity of the hormone-sensitive adenylate cyclase system in the heart. Distinct guanine nucleotide-binding regulatory proteins (G-proteins) mediate stimulatory and inhibitory influences on adenylate cyclase activity. To examine whether the effects of thyroid hormone on adenylate cyclase involve specific changes in G-protein subunit expression, the influence of tri-iodothyronine (T3) on the biosynthesis and activity of G-proteins in neonatal rat ventricular myocytes was determined. In myocytes challenged with T3 for 5 days, Gs alpha levels increased by 4 +/- 0.5-fold, whereas Gi2 alpha levels declined by more than 80%. T3 down-regulated Gi2 alpha mRNA by 60% within 3 days, but had no effect on Gs alpha mRNA. The basis for the decline in Gi2 alpha mRNA was the T3-mediated suppression of Gi2 alpha gene transcription by 80 +/- 9% within 4 h. The decline in Gi2 alpha mRNA in response to T3 produced a 2-fold decrease in relative rate of synthesis of Gi2 alpha but not in its half-life (46 +/- 7 h). Gs alpha synthesis was not altered by T3, but the half-life of Gs alpha increased from 50 +/- 6 h in control cells to 72 +/- 8 h in T3-treated cells. In addition, T3 provoked the translocation of Gs alpha from the cytoplasmic to the membranous compartment. Membranous Gs alpha increased from 30 +/- 6% to 61 +/- 7% of total cellular Gs alpha, whereas cytoplasmic Gs alpha declined from 68 +/- 6% to 33 +/- 8% within 1 day of exposure to T3. T3-mediated up-regulation of Gs alpha enhanced the activation of myocardial adenylate cyclase by the stimulatory pathway whereas the down-regulation of Gi2 alpha attenuated the deactivation of myocardial adenylate cyclase by the inhibitory pathway.
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Liang, Xiao, Yu-Ru Chen, Wei Gao, Xing-Pan Guo, De-Wen Ding, Asami Yoshida, Kiyoshi Osatomi, and Jin-Long Yang. "Effects on larval metamorphosis in the mussel Mytilus coruscus of compounds that act on downstream effectors of G-protein-coupled receptors." Journal of the Marine Biological Association of the United Kingdom 98, no. 2 (October 11, 2016): 333–39. http://dx.doi.org/10.1017/s0025315416001417.
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The metamorphic responses of mussel (Mytilus coruscus) larvae to pharmacological agents affecting G proteins and the adenylate cyclase/cyclic AMP (AC/cAMP) pathway were examined in the laboratory. The G protein activators guanosine 5′-[β,γ-imido]triphosphate trisodium salt hydrate and guanosine 5′-[γ-thio]triphosphate tetralithium salt only induced larval metamorphosis in continuous exposure assays, and the G protein inhibitor guanosine 5′-[β-thio]diphosphate trilithium salt did not exhibit inducing activity. The non-specific phosphodiesterase inhibitor theophylline and the cAMP-specific phosphodiesterase IV inhibitor 4-(3-Butoxy-4-methoxybenzyl)imidazolidin-2-one exhibited inducing activity, while the non-specific phosphodiesterase inhibitor 3-Isobutyl-1-methylxanthine only showed inducing activity at 10−4 M in continuous exposure assays. The cyclic nucleotide analogue N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt did not exhibit significant inducing activity. Both the adenylate cyclase activator forskolin and the adenylate cyclase inhibitor nitroimidazole exhibited inducing activity at 10−4 to 10−3 M concentrations in continuous exposure assays. Among these tested agents, the adenylate cyclase inhibitor (±)-miconazole nitrate salt showed the most promising inducing effect. The present results indicate that G protein-coupled receptors and signal transduction by AC/cAMP pathway could mediate metamorphosis of larvae in this species.
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Miller, BA, K. Foster, JD Robishaw, CF Whitfield, L. Bell, and JY Cheung. "Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin." Blood 77, no. 3 (February 1, 1991): 486–92. http://dx.doi.org/10.1182/blood.v77.3.486.486.
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Abstract Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat- inactivated PT. These observations provided strong evidence that a PT- sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.
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Miller, BA, K. Foster, JD Robishaw, CF Whitfield, L. Bell, and JY Cheung. "Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin." Blood 77, no. 3 (February 1, 1991): 486–92. http://dx.doi.org/10.1182/blood.v77.3.486.bloodjournal773486.
Full textAbstract:
Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat- inactivated PT. These observations provided strong evidence that a PT- sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.
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Cooper, D. M. F. "Compartmentalization of adenylate cyclase and cAMP signalling." Biochemical Society Transactions 33, no. 6 (October 26, 2005): 1319–22. http://dx.doi.org/10.1042/bst0331319.
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Concepts of cAMP signalling have changed dramatically from the linear cascades of just a few years ago, with the realization that numerous cellular processes affect this motif. These influences include other signalling pathways – most significantly Ca2+, scaffolding proteins (which are themselves variously regulated) to organize the elements of the pathway, and subcellular targeting of components. An obvious implication of this organization is that global measurements of cAMP may trivialize the complexity of the cAMP signals and obscure the regulation of targets. In this presentation, current developments on the targeting and assembly of ACs (adenylate cyclases) and their delivery to selected raft or non-raft domains of the plasma membrane will be discussed, along with the susceptibility of raft-targeted ACs to very discrete modes of increases in the intracellular Ca2+ concentration. Single-cell explorations of cAMP dynamics, as measured with cyclic nucleotide-gated channels, are also described in this paper, particularly as applied to cells in which the composition of AKAP (A-kinase anchoring protein)–PKA (protein kinase A)–PDE (phosphodiesterase) assemblies is probed by RNA interference ablation of defined AKAPs.
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Gilman, Alfred G. "G Proteins and Regulation of Adenylate Cyclase(Nobel Lecture)." Angewandte Chemie International Edition in English 34, no. 1314 (July 31, 1995): 1406–19. http://dx.doi.org/10.1002/anie.199514061.
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Levitzki, Alexander. "Regulation of adenylate cyclase by hormones and G-proteins." FEBS Letters 211, no. 2 (January 26, 1987): 113–18. http://dx.doi.org/10.1016/0014-5793(87)81419-9.
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Thekkumkara, Thomas, and Stuart L. Linas. "Role of internalization in AT1A receptor function in proximal tubule epithelium." American Journal of Physiology-Renal Physiology 282, no. 4 (April 1, 2002): F623—F629. http://dx.doi.org/10.1152/ajprenal.00118.2001.
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Angiotensin II (ANG II), acting through angiotensin type I (AT1) receptors on apical and basolateral surfaces of proximal tubule epithelial cells, increases sodium reabsorption in proximal tubules. Apical and basolateral receptors internalize after exposure to ANG II, but the role of internalization in receptor signaling and transport is not well defined. To determine the role of receptor internalization in ANG II-mediated receptor signaling and sodium transport, we stably expressed full-length and truncated AT1A receptors in opossum kidney cells. After stimulation with ANG II, wild-type receptors on apical and basolateral surfaces rapidly internalized, inhibited adenylate cyclase, and increased transcellular sodium transport. Truncation of the cytoplasmic tail of the AT1Areceptor (TL314) resulted in receptors that were expressed on apical and basolateral surfaces but did not internalize, inhibit adenylate cyclase, or increase sodium transport. Because the cytoplasmic tail contains putative G protein coupling sites, mutant receptors that leave G protein interaction sites intact were designed. Cells expressing the truncation (TK333) or deletion (Del 315–329) also failed to internalize. When ANG II was added to basolateral surfaces of TK333 or Del 315–329, adenylate cyclase activity was inhibited and sodium transport was increased. In contrast, apical addition of ANG II was not associated with decreases in adenylate cyclase or increases in sodium transport. In conclusion, internalization pathways are important for AT1A receptor function in polarized proximal tubule epithelial cells. Apical AT1A receptors internalize before they interact with G proteins and signal cAMP. In contrast, basolateral AT1A receptors interact with G proteins and signal cAMP without internalizing.
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Mangels, L. A., R. R. Neubig, H. E. Hamm, and M. E. Gnegy. "Calmodulin binding distinguishes between βγ subunits of activated G proteins and transducin." Biochemical Journal 283, no. 3 (May 1, 1992): 683–90. http://dx.doi.org/10.1042/bj2830683.
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The interactions between guanine nucleotide regulatory proteins and the Ca(2+)-binding protein calmodulin were studied using calmodulin-Sepharose affinity chromatography. Purified bovine brain beta gamma subunits bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. On the contrary, beta gamma subunits produced in an activated Go/Gi preparation did not bind to calmodulin-Sepharose. The effect was independent of the type of bovine brain G protein (Go/Gi, Gs), method of activation and the presence of magnesium. To distinguish whether the binding of purified beta gamma subunits to calmodulin was unique to brain beta gamma or to the method of purification, similar experiments were performed using transducin. In contrast to bovine brain G proteins, both purified transducin beta gamma subunits and beta gamma released from rhodopsin-activated transducin bound to calmodulin-Sepharose in a Ca(2+)-dependent manner. To assess the functional significance of the binding of bovine brain beta gamma subunits to calmodulin, the ability of purified beta gamma and of beta gamma in unactivated and activated Go/Gi to inhibit partially purified calmodulin-sensitive adenylate cyclase was determined. Purified beta gamma was highly effective in inhibiting calmodulin-stimulated adenylate cyclase activity. However, unactivated Go/Gi and preactivated Go/Gi inhibited calmodulin-stimulated adenylate cyclase activity to the same extent. This Go/Gi-mediated inhibition also occurred in the presence of a 500-fold molar excess of calmodulin over added G protein. These results demonstrate: (1) that beta gamma subunits may not be completely released upon G protein activation, and (2) that inhibition of calmodulin-stimulated adenylate cyclase by beta gamma subunits does not appear to be mediated by a direct beta gamma-calmodulin interaction. Differences in the binding properties of activated bovine brain G proteins versus those of transducin could be explained by differences in the gamma subunit between the proteins, or by differences in affinities of the alpha and beta gamma subunits for each other and for calmodulin. The different functional properties of purified beta gamma subunits and beta gamma subunits produced in situ by activation of G proteins indicates that extrapolation from the effects of purified subunits to events occurring in membranes should be done with caution.
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Bominaar, A. A., F. Kesbeke, B. E. Snaar-Jagalska, D. J. Peters, P. Schaap, and P. J. Van Haastert. "Abberant chemotaxis and differentiation in Dictyostelium mutant fgdC with a defective regulation of receptor-stimulated phosphoinositidase C." Journal of Cell Science 100, no. 4 (December 1, 1991): 825–31. http://dx.doi.org/10.1242/jcs.100.4.825.
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Dictyostelium cells use extracellular cyclic AMP both as a chemoattractant and as a morphogen inducing cell-type-specific gene expression. Cyclic AMP binds to surface receptors, activates one or more G-proteins, and stimulates adenylate cyclase, guanylate cyclase and phosphoinositidase C. Mutant fgdC showed aberrant chemotaxis, and was devoid of cyclic AMP-induced gene expression and differentiation. Both the receptor- and G-protein-mediated stimulation of adenylate cyclase and guanylate cyclase were unaltered in mutant fgdC as compared to wild-type cells. In wild-type cells phosphoinositidase C was activated about twofold by the cyclic AMP receptor. In mutant fgdC cells, however, the enzyme was inhibited by about 60%. These results suggest that phosphoinositidase C is regulated by a receptor-operated activation/inhibition switch that is defective in mutant fgdC. We conclude that activation of phosphoinositidase C is essential for Dictyostelium development.
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Post, S. R., R. Hilal-Dandan, K. Urasawa, L. L. Brunton, and P. A. Insel. "Quantification of signalling components and amplification in the β-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes." Biochemical Journal 311, no. 1 (October 1, 1995): 75–80. http://dx.doi.org/10.1042/bj3110075.
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We have investigated the stoichiometric relationship of proteins involved in beta-adrenergic-receptor-mediated signal transduction in isolated rat cardiac myocytes. These cells contain about 2.1 x 10(5) beta-adrenergic receptors per cell, as determined by radio-ligand-binding assays. We have assessed the amount of Gs alpha present in myocyte membranes by immunoblotting using a purified glutathione S-transferase-Gs alpha fusion protein as a standard for quantification. By this method, we determined that cardiac myocytes contain about 35 x 10(6) and 12 x 10(6) molecules per cell of the 45 and 52 kDa forms of Gs alpha, respectively. [3H]Forskolin binding assays were used to assess the formation of high-affinity forskolin binding sites representing Gs alpha-adenylate cyclase complexes occurring in response to Gs alpha activation. Quantification of the adenylate cyclase complexes was facilitated by the permeabilization of cells with saponin. The addition of isoprenaline (isoproterenol) and guanosine 5′-[gamma-thio]trisphosphate to saponin-permeabilized myocytes results in the formation of 6 x 10(5) Gs alpha-adenylate cyclase complexes. Taken together, the data presented here demonstrate that, in a physiologically relevant setting, G-protein is present in large stoichiometric excess relative to both receptor and effector. In addition, we show that, overall, only modest signal amplification occurs between receptor and adenylate cyclase. Thus adenylate cyclase (rather than Gs) is the component distal to receptor that limits agonist-mediated increases in cyclic AMP production. Although limited data are as yet available for other G-protein-regulated effectors, we hypothesize that the stoichiometry of signalling components and the extent of signal amplification described for the beta-adrenergic response pathway will be applicable to other G-protein-coupled hormone receptor systems.
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Dasso, L. L. T., and C. W. Taylor. "Heparin and other polyanions uncouple α1-adrenoceptors from G-proteins." Biochemical Journal 280, no. 3 (December 15, 1991): 791–95. http://dx.doi.org/10.1042/bj2800791.
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Several polyanionic compounds antagonize the interaction between receptors and the G-proteins that regulate adenylate cyclase or K+ channels, possibly by binding to a basic stretch of the receptor that is proposed to mediate its interaction with the G-proteins. We have studied the effects of polyanions on the interaction between the liver alpha 1-adrenoceptor and the G-protein through which it stimulates polyphosphoinositide turnover. Heparin [concn. causing 50% of maximal effect (EC50) = 0.5 microM], Trypan Blue (EC50 7.1 microM) or suramin (EC50 2.1 microM) prevented formation of the high-affinity adrenaline-receptor-G-protein complex without affecting antagonist binding. After alkaline treatment of the membranes, previously reported to cause G-protein removal, binding of agonists was insensitive to both guanine nucleotides and heparin. We conclude that these polyanions uncouple the alpha 1-adrenoceptor from its G-protein, suggesting that similar coupling mechanisms may underlie receptor activation of the G-proteins that activate polyphosphoinositide hydrolysis and those that regulate adenylate cyclase. This action of heparin severely limits its utility as a selective antagonist of the Ins(1,4,5)P3 receptor in intact cells.
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Eppler, Tanja, Pieter Postma, Alexandra Schütz, Uwe Völker, and Winfried Boos. "Glycerol-3-Phosphate-Induced Catabolite Repression in Escherichia coli." Journal of Bacteriology 184, no. 11 (June 1, 2002): 3044–52. http://dx.doi.org/10.1128/jb.184.11.3044-3052.2002.
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ABSTRACT The formation of glycerol-3-phosphate (G3P) in cells growing on TB causes catabolite repression, as shown by the reduction in malT expression. For this repression to occur, the general proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular EIIAGlc, as well as the adenylate cyclase and the cyclic AMP-catabolite activator protein system, have to be present. We followed the level of EIIAGlc phosphorylation after the addition of glycerol or G3P. In contrast to glucose, which causes a dramatic shift to the dephosphorylated form, glycerol or G3P only slightly increased the amount of dephosphorylated EIIAGlc. Isopropyl-β-d-thiogalactopyranoside-induced overexpression of EIIAGlc did not prevent repression by G3P, excluding the possibility that G3P-mediated catabolite repression is due to the formation of unphosphorylated EIIAGlc. A mutant carrying a C-terminally truncated adenylate cyclase was no longer subject to G3P-mediated repression. We conclude that the stimulation of adenylate cyclase by phosphorylated EIIAGlc is controlled by G3P and other phosphorylated sugars such as d-glucose-6-phosphate and is the basis for catabolite repression by non-PTS compounds. Further metabolism of these compounds is not necessary for repression. Two-dimensional polyacrylamide gel electrophoresis was used to obtain an overview of proteins that are subject to catabolite repression by glycerol. Some of the prominently repressed proteins were identified by peptide mass fingerprinting. Among these were periplasmic binding proteins (glutamine and oligopeptide binding protein, for example), enzymes of the tricarboxylic acid cycle, aldehyde dehydrogenase, Dps (a stress-induced DNA binding protein), and d-tagatose-1,6-bisphosphate aldolase.
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Deckmyn, H., C. Van Geet, and J. Vermylen. "Dual Regulation of Phospolipase C Activity by G Proteins." Physiology 8, no. 2 (April 1, 1993): 61–63. http://dx.doi.org/10.1152/physiologyonline.1993.8.2.61.
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Some subtypes of phosphatidylinositide-specific phospholipase C (PLC) are activated via pertussis toxin-sensitive or -insensitive G proteins. However, a G protein-dependent PLC inhibitory pathway also may exist. The resultant picture is of dual regulation of PLC, showing a close parallelism with the dual regulation of adenylate cyclase.
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Cloutier, M. M., and L. Guernsey. "Tannin inhibits adenylate cyclase in airway epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 5 (May 1, 1995): L851—L855. http://dx.doi.org/10.1152/ajplung.1995.268.5.l851.
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Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.
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Stadel, J. M., and S. T. Crooke. "Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding." Biochemical Journal 254, no. 1 (August 15, 1988): 15–20. http://dx.doi.org/10.1042/bj2540015.
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Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.
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STRYJEK-KAMINSKA, Danuta, Albrecht PIIPER, and Stefan ZEUZEM. "Epidermal growth factor regulates adenylate cyclase activity via Gs and Gi1-2 proteins in pancreatic acinar membranes." Biochemical Journal 316, no. 1 (May 15, 1996): 87–91. http://dx.doi.org/10.1042/bj3160087.
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In the present study, Western-blot and radioreceptor analyses have revealed the presence of the epidermal growth factor (EGF) receptor in pancreatic acinar membranes. Isolated pancreatic acinar membranes, which allow access of functional antibodies to individual components of the signal transduction cascade, were used to examine EGF-induced regulation of adenylate cyclase activity. Forskolin, vasoactive intestinal peptide (VIP) and to a smaller extent EGF increased cAMP production in pancreatic acinar membranes. Preincubation of the membranes with anti-Gsα antibody abolished EGF- and VIP-induced cAMP production, but had no effect on forskolin-induced cAMP accumulation. In the presence of either VIP or forskolin, EGF inhibited the VIP- and forskolin-induced cAMP production with an IC50 of 5 nM. Anti-Gαi1–2 protein antibody, but not anti-Gαi3 antibody, increased basal cAMP production, indicating that Gi proteins exert an inhibitory influence on basal adenylate cyclase activity. Anti-Gαi1–2 antibody, but not anti-Gαi3 antibody, abolished the inhibitory effect of EGF on the forskolin- and VIP-induced cAMP accumulation. A peptide corresponding to the juxtamembrane region in the cytosolic domain of the rat EGF receptor increased cAMP production in pancreatic acinar membranes in an anti-Gαs antibody-sensitive fashion, whereas the EGF receptor peptide did not mimic the inhibitory effect of the native EGF receptor. The tyrosine kinase inhibitors genistein and pp60v-src (137–157) inhibited both the stimulatory and the inhibitory effects of EGF on cAMP production. Thus the data of the present study show that EGF regulates adenylate cyclase via activation of Gs and Gi proteins by a tyrosine phosphorylation-dependent mechanism in pancreatic acinar membranes. This leads to stimulation of basal and inhibition of forskolin- and VIP-induced adenylate cyclase activity respectively.
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Daly, John W., Dan Shi, William L. Padgett, Xiao-Duo Ji, and Kenneth A. Jacobson. "Riboflavin: Inhibitory effects on receptors, G-proteins, and adenylate cyclase." Drug Development Research 42, no. 2 (October 1997): 98–108. http://dx.doi.org/10.1002/(sici)1098-2299(199710)42:2<98::aid-ddr6>3.0.co;2-k.
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Toda, Takashi, Isao Uno, Tatsuo Ishikawa, Scott Powers, Tohru Kataoka, Daniel Broek, Scott Cameron, James Broach, Kunihiro Matsumoto, and Michael Wigler. "In yeast, RAS proteins are controlling elements of adenylate cyclase." Cell 40, no. 1 (January 1985): 27–36. http://dx.doi.org/10.1016/0092-8674(85)90305-8.
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James, S. R., C. Vaziri, T. R. Walker, G. Milligan, and C. P. Downes. "The turkey erythrocyte β-adrenergic receptor couples to both adenylate cyclase and phospholipase C via distinct G-protein α subunits." Biochemical Journal 304, no. 2 (December 1, 1994): 359–64. http://dx.doi.org/10.1042/bj3040359.
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By contrast with mammalian beta-adrenergic receptors, the avian isoform elicits two distinct effector responses, activation of adenylate cyclase and polyphosphoinositide-specific phospholipase C (PLC) leading to the accumulation of both cyclic adenosine monophosphate (cyclic AMP) and inositol phosphates. We have investigated the mechanisms of beta-adrenergic receptor signalling in turkey erythrocytes. Stimulation of adenylate cyclase by the beta-adrenergic-receptor agonist isoprenaline exhibits a 30-fold lower EC50 than that for PLC activation, which may indicate a marked receptor reserve for the former effector. Similar Ki values were obtained for the inhibition of both responses by four beta-adrenergic antagonists, arguing that a single receptor population is responsible for both effects. Antibodies raised against G-protein peptide sequences were used to show that the identity of the G-protein mediating the PLC response was an avian homologue of G11, the level of expression of which was very similar to that of the stimulatory G-protein of adenylate cyclase, Gs. Thus a single population of beta-adrenergic receptors apparently interacts with distinct G-proteins to activate different effectors. The stoichiometries of the receptor-G-protein-effector interactions are therefore similar for both second-messenger responses and the data are discussed in terms of the different efficacies observed for each response.
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Weingart, Christine L., Gina Broitman-Maduro, Gary Dean, Simon Newman, Mark Peppler, and Alison A. Weiss. "Fluorescent Labels Influence Phagocytosis ofBordetella pertussis by Human Neutrophils." Infection and Immunity 67, no. 8 (August 1, 1999): 4264–67. http://dx.doi.org/10.1128/iai.67.8.4264-4267.1999.
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ABSTRACT To explore the role of neutrophil phagocytosis in host defense against Bordetella pertussis, bacteria were labeled extrinsically with fluorescein isothiocyanate (FITC) or genetically with green fluorescent protein (GFP) and incubated with adherent human neutrophils in the presence or absence of heat-inactivated human immune serum. In the absence of antibodies, FITC-labeled bacteria were located primarily on the surface of the neutrophils with few bacteria ingested. However, after opsonization, about seven times more bacteria were located intracellularly, indicating that antibodies promoted phagocytosis. In contrast, bacteria labeled intrinsically with GFP were not efficiently phagocytosed even in the presence of opsonizing antibodies, suggesting that FITC interfered with a bacterial defense. Because FITC covalently modifies proteins and could affect their function, we tested the effect of FITC on adenylate cyclase toxin activity, an important extracellular virulence factor. FITC-labeled bacteria had fivefold-less adenylate cyclase toxin activity than did unlabeled wild-type bacteria or GFP-expressing bacteria, suggesting that FITC compromised adenylate cyclase toxin activity. These data demonstrated that at least one extracellular virulence factor was affected by FITC labeling and that GFP is a more appropriate label forB. pertussis.