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)

The neuronal calcium sensor (NCS) proteins (e.g. recoverin, neurocalcins, and frequenin) are expressed at highest levels in excitable cells, and some of them regulate desensitization of G protein-coupled receptors. Here we present NMR analysis and genetic functional studies of an NCS homolog in fission yeast (Ncs1p). Ncs1p binds three Ca2+ ions at saturation with an apparent affinity of 2 microm and Hill coefficient of 1.9. Analysis of NMR and fluorescence spectra of Ncs1p revealed significant Ca2+-induced protein conformational changes indicative of a Ca2+-myristoyl switch. The amino-terminal myristoyl group is sequestered inside a hydrophobic cavity of the Ca2+-free protein and becomes solvent-exposed in the Ca2+-bound protein. Subcellular fractionation experiments showed that myristoylation and Ca2+ binding by Ncs1p are essential for its translocation from cytoplasm to membranes. The ncs1 deletion mutant (ncs1Delta) showed two distinct phenotypes: nutrition-insensitive sexual development and a growth defect at high levels of extracellular Ca2+ (0.1 m CaCl(2)). Analysis of Ncs1p mutants lacking myristoylation (Ncs1p(G2A)) or deficient in Ca2+ binding (Ncs1p(E84Q/E120Q/E168Q)) revealed that Ca2+ binding was essential for both phenotypes, while myristoylation was less critical. Exogenous cAMP, a key regulator for sexual development, suppressed conjugation and sporulation of ncs1Delta, suggesting involvement of Ncs1p in the adenylate cyclase pathway turned on by the glucose-sensing G protein-coupled receptor Git3p. Starvation-independent sexual development of ncs1Delta was also complemented by retinal recoverin, which controls Ca2+-regulated desensitization of rhodopsin. In contrast, the Ca2+-intolerance of ncs1Delta was not affected by cAMP or recoverin, suggesting that the two ncs1Delta phenotypes are mechanistically independent. We propose that Schizosaccharomyces pombe Ncs1p negatively regulates sporulation perhaps by controlling Ca2+-dependent desensitization of Git3p.
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PMID:Fission yeast homolog of neuronal calcium sensor-1 (Ncs1p) regulates sporulation and confers calcium tolerance. 1472 91

The identification of endogenous or surrogate ligands for orphan G protein-coupled receptors (GPCRs) represents one of the most important tasks in GPCR biology and pharmacology. The challenge lies in choosing an appropriate assay in the absence of ways to activate the receptor of interest. We investigated the signaling pathway for an orphan GPCR referred to here as vasopressin receptor-related receptor 1 (VRR1) by generating a chimeric receptor, V1a/VRR1. The engineered construct contained vasopressin V1a receptor with all three intracellular loops and C terminus replaced by those of VRR1. The chimera behaved like a typical GPCR when transiently and stably expressed in mammalian cell lines based on radioligand binding and receptor internalization studies. Upon arginine vasopressin stimulation, this chimeric receptor induced robust calcium mobilization and increase of adenylate cyclase activity. The observed signaling activities are through the activation of the chimera instead of endogenously expressed receptors, as single amino acid changes in the second transmembrane regions of the chimera drastically reduced receptor efficacy and potency. Our results suggest that VRR1 has dual signaling properties in coupling to both G(q) and G(S) pathways. Analysis of native VRR1 receptor signaling pathway by using a recently identified ligand for VRR1 confirmed this conclusion and therefore validated the utility of the chimeric receptor approach for signaling pathway identification.
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PMID:Elucidation of signaling properties of vasopressin receptor-related receptor 1 by using the chimeric receptor approach. 1475 15

Secretion of GH by pituitary somatotropes is primarily stimulated by the hypothalamic GHRH through the activation of a specific G protein-coupled receptor, GHRH receptor (GHRH-R). GH is also released in response to ghrelin, a peptide produced in the stomach, hypothalamus, and pituitary that activates somatotropes via a distinct G protein-coupled receptor, referred to as the GH secretagogue receptor (GHS-R). Here, we have analyzed the expression of both GHRH-R and GHS-R (by multiplex RT-PCR) in porcine pituitary cell cultures, after acute (4 h) treatment with GHRH or ghrelin as well as with other regulators of somatotropes (somatostatin, dexamethasone). Exposure of cultures to GHRH decreased GHRH-R mRNA content and also diminished GHS-R transcript levels. Likewise, ghrelin down-regulated both GHS-R and GHRH-R expression. Interestingly, administration of the activator of adenylate cyclase, forskolin, decreased GHRH-R mRNA levels but had no effect on GHS-R, thus suggesting a distinct contribution of the various intracellular signals operating in somatotropes to the regulation of the expression of these receptors. Accordingly, an atypical activator of adenylate cyclase in the pig somatotrope is low-dose (10(-13) m) somatostatin, which also suppressed GHRH-R mRNA levels without altering GHS-R expression. Finally, dexamethasone did not modify GHRH-R or GHS-R expression. In summary, our data show for the first time that ghrelin, as well as GHRH, mediates homologous and heterologous down-regulation of their own receptor synthesis. However, our results also indicate that the expression of porcine GHRH-R and GHS-R is regulated by distinct signals that may differ from those reported in other mammalian species.
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PMID:Homologous and heterologous regulation of pituitary receptors for ghrelin and growth hormone-releasing hormone. 1504 57

Communication between receptor tyrosine kinase and G protein-coupled receptor (GPCR)-mediated signaling is recognized as a common integrator linking diverse aspects of intracellular signaling systems. Here, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation of salivary phospholipid release occurs with the involvement of epidermal growth factor receptor (EGFR). Using sublingual gland acinar cells, we show that prosecretory effect of isoproterenol on phospholipid release was subjected to suppression by EGFR kinase inhibitor, PD153035, and wortmannin, an inhibitor of PI3K, but not by PD98059, an inhibitor of extracellular signal regulated kinase (ERK). Furthermore, wortmannin, but not the ERK inhibitor, caused the reduction in the acinar cell secretory responses to beta-adrenergic agonist-generated cAMP as well as adenyl cyclase activator, forskolin. The acinar cell phospholipid secretory responses to isoproterenol, moreover, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation. Taken together, our data are the first to demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of salivary phospholipid secretion in response to beta-adrenergic GPCR activation.
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PMID:Salivary phospholipid secretion in response to beta-adrenergic stimulation is mediated by Src kinase-dependent epidermal growth factor receptor transactivation. 1511 Jul 80

Efficient signaling requires accurate spatial and temporal compartmentalization of proteins. RACK1 is a scaffolding protein that fulfils this role through interaction of binding partners with one of its seven WD40 domains. We recently identified the kinase Fyn and the NR2B subunit of the N-methyl-D-Aspartate receptor (NMDAR) as binding partners of RACK1. Scaffolding of Fyn near its substrate NR2B by RACK1 inhibits Fyn phosphorylation of NR2B and thereby negatively regulates channel function. We found that Fyn and NR2B share the same binding site on RACK1; however, their binding to RACK1 is not mutually exclusive (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). We therefore tested the hypothesis that RACK1 forms a homodimer that allows the simultaneous binding of Fyn and NR2B. We found that RACK1 binds to itself both in vitro and in the brain. Deletion analyses identified a RACK1-RACK1 dimer-binding site within the 4th WD40 repeat, and application of the 4th WD40 repeat or a peptide derivative to hippocampal slices inhibited NMDAR activity. We further found that in hippocampal slices, both RACK1 and NR2B associated with another WD40 protein, the beta-subunit of G protein (Gbeta), previously shown to heterodimerize with RACK1 in vitro (Dell, E. J., Connor, J., Chen, S., Stebbins, E. G., Skiba, N. P., Mochly-Rosen, D., and Hamm, H. E. (2002) J. Biol. Chem. 277, 49888-49895). However, activation of the pituitary adenylate cyclase polypeptide (1-38) G protein-coupled receptor, previously found to induce the dissociation of RACK1 from the NMDAR complex (Yaka, R., He, D. Y., Phamluong, K., and Ron, D. (2003) J. Biol. Chem. 278, 9630-9638), attenuated the association of Gbeta with RACK1 and NR2B. Based on these results, we propose that WD40-mediated homo- and heterodimerization of RACK1 mediate the formation of a transient signaling complex that includes the NMDAR, a G protein and Fyn.
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PMID:Spatial and temporal regulation of RACK1 function and N-methyl-D-aspartate receptor activity through WD40 motif-mediated dimerization. 1514 Aug 93

gamma-Aminobutyric acid (GABA)(B) receptor-mediated modulation of spontaneous GABA release onto Purkinje cells was investigated in cerebellar slices from 3- to 5-week-old mice. The GABA(B) receptor agonists baclofen and CGP 44533 each reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs), with no significant effect on mIPSC amplitude; together, consistent with a presynaptic site of action. The GABA(B) receptor antagonist CGP 55845 blocked baclofen-induced inhibition. The sulphydryl alkylating agent N-ethylmaleimide occluded baclofen effects, implicating G(i/o) subunits in mediating a GABA(B) G protein-coupled receptor pathway. Baclofen-induced inhibition persisted in the presence of Ba(2+), a blocker of K(+) channels, and Cd(2+), a blocker of Ca(2+) channel-mediated GABA release. Application of nominally Ca(2+)-free extracellular solutions reduced mIPSC frequency and amplitude; however, baclofen produced a significant inhibition in mIPSC frequency, further suggesting that this pathway was independent of Ca(2+) influx. Spontaneous GABA release was increased by the adenylate cyclase activator, forskolin, and the phorbol ester, phorbol 12,13-dibutyrate. However, baclofen-induced inhibition was not significantly changed in either condition. Baclofen action was also not affected by the adenylate cyclase inhibitor SQ 22536 or the protein kinase C inhibitor chelerythrine chloride. Baclofen still reduced mIPSC frequency in the presence of the polyvalent cation ruthenium red, which acts as a secretagogue here; however, baclofen-induced inhibition was reduced significantly. Furthermore, baclofen produced no clear inhibition during high-frequency mIPSCs bursts induced by the potent secretagogue alpha-Latrotoxin. Together, these results suggest that GABA(B) inhibition occurs downstream of Ca(2+) influx and may be mediated, in part, by an inhibition of the vesicular release mechanism.
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PMID:Mechanism of GABA receptor-mediated inhibition of spontaneous GABA release onto cerebellar Purkinje cells. 1525 79

Adenosine A2a receptor, a member of the G protein-coupled receptor superfamily, has been demonstrated to be an important pharmacological target. It couples to stimulatory G protein and activates adenylate cyclase upon agonist stimulation. Here we attempted to stably transfect Chinese hamster ovary (CHO-K1) cells, which lack any known subtypes of adenosine receptors, with recombinant human adenosine A2a receptors (hA2aR). Rapid down-regulation of hA2aR in a clonal cell line, CHOA2a-2, was observed over a short period of time in culture. This is consistent with other groups' findings of low expression and poor G protein coupling of this receptor in several cell systems. To facilitate pharmacological profiling for hA2aR ligand, we introduced a cyclic AMP response element (CRE)-linked beta-galactosidase reporter gene into CHOA2a-2 cells to generate a stable cell line, CHOA2a-2CREbetagal#26. Robust cyclic AMP signal amplification was obtained using a colorimetric assay measuring beta-galactosidase activity. The EC(50) of 5'-N-ethylcarboxamidoadenosine (NECA), a potent A2a agonist, for inducing beta-galactosidase activity was 23.3 +/- 3.5 nM, similar to 22.7 +/- 3.9 nM, which was the NECA EC(50) in the direct measurement of cyclic AMP of CHOA2a-2 cells in early culture. Subsequently we validated this assay for high throughput screening for hA2aR agonists. The Z' factor for robotic assay performance was 0.79 +/- 0.03, the ratio of signal/noise was 157 +/- 36, and the ratio of signal/background was 10.6 +/- 1.2, demonstrating that this assay is well suitable for quality high throughput screening. High throughput screening of Johnson & Johnson libraries uncovered a couple of distinct series of nonadenosine small molecules, in addition to adenosine analogues, as potential hA2aR agonists with EC(50) values of 2-6 microM. Preliminary characterization of those compounds was presented.
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PMID:Development of a sensitive and HTS-compatible reporter gene assay for functional analysis of human adenosine A2a receptors in CHO-K1 cells. 1528 9

Recent advances in understanding the nature of cellular responses mediated by G protein-coupled receptor (GPCR) activation indicate that integration of the converging regulatory signals into functional cellular pathways requires epidermal growth factor receptor (EGFR) transactivation. In this study, we report that G protein-coupled beta-adrenergic receptor activation leading to stimulation in gastric mucus phospholipid secretion occurs with the involvement of EGFR. Using [14C]choline-labeled gastric mucosal cells in culture, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on phospholipid release was subject to a dose-dependent suppression by EGFR kinase inhibitor, PD153035, as well as wortmannin, a specific inhibitor of PI3K. Both inhibitors, moreover, caused the reduction in the gastric mucosal cell phospholipid secretory responses to beta-adrenergic agonist-generated second messenger, cAMP as well as adenyl cyclase activator, forskolin. The gastric mucosal phospholipid secretory responses to isoproterenol, furthermore, were inhibited by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR phosphorylation, but not by ERK inhibitor, PD98059. The inhibition of ERK, moreover, did not cause attenuation in phospholipid secretory responses to cAMP and forskolin. The findings underline the central role of EGFR in mediation of gastric mucosal secretory processes, and demonstrate the requirement for Src kinase-dependent EGFR transactivation in regulation of gastric mucosal phospholipid secretion in response to beta-adrenergic GPCR activation.
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PMID:Secretion of gastric mucus phospholipids in response to beta-adrenergic G protein-coupled receptor activation is mediated by SRC kinase-dependent epidermal growth factor receptor transactivation. 1538 32

The principal regulatory factors that control the flow and make-up of salivary secretion are neurotransmitters, released by parasympathetic and sympathetic innervation, that trigger activation of G protein-coupled receptors (GPCRs) on the acinar cells of salivary glands and stimulate the generation of soluble second messengers. In this study, we report that activation of GPCR by beta-adrenergic agonist leading to stimulation in salivary mucin secretion occurs with the involvement of epidermal growth factor receptor (EGFR). Using [(3)H]glucosamine-labeled mucous acinar cells of sublingual salivary gland in culture, we show that stimulatory effect of beta-adrenergic agonist, isoproterenol, on mucin secretion was inhibited in a concentration-dependent manner by EGFR kinase inhibitor, PD153035, as well as wortmannin, an inhibitor of PI3K. Moreover, both inhibitors caused the impedance in the acinar cell mucin secretory responses to beta-adrenergic agonist-generated second messenger, cAMP, as well as adenylate cyclase activator, forskolin. The acinar cell secretory responses to isoproterenol, furthermore, were blunted in a concentration-dependent fashion by PP2, a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR autophosphorylation. However, no significant alterations in the acinar cell mucin secretory responses to isoproterenol, cAMP or forskolin were attained with an inhibitor of the ERK pathway, PD98059. Our findings underline the role of EGFR as a convergence point in modulation of salivary mucin secretion triggered by beta-adrenergic agonist GPCR activation and demonstrate the importance of Src kinase in the EGFR transactivation process.
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PMID:Src-kinase-dependent epidermal growth factor receptor transactivation in salivary mucin secretion in response to beta-adrenergic G-protein-coupled receptor activation. 1552 48

Adenosine is an endogenous signaling molecule that is highly up-regulated in inflammatory states. Adenosine acts through the A2b receptor, a G protein-coupled receptor that couples positively to Galpha(s) and activates adenylate cyclase. This leads to cAMP-mediated electrogenic chloride secretion in intestinal epithelia. To better understand the regulation of the A2b receptor in intestinal epithelia, we studied the effects of interferon-gamma (IFN-gamma), a potent immunomodulatory cytokine, in the T84 cell line. Pretreatment of cells with 500 units/ml IFN-gamma for 12 h inhibited an adenosine-induced short circuit current (Isc) without affecting the transepithelial resistance. Under these conditions, IFN-gamma did not inhibit the protein expression or membrane recruitment of the A2b receptor, shown to be essential for its function. Interestingly, IFN-gamma inhibited cAMP levels as well as its downstream signaling pathway as shown by the inhibition of adenosine-induced phosphorylation of cAMP response element-binding protein and protein kinase A activity. Similar studies with forskolin, a direct activator of adenylate cyclase, also demonstrated inhibition of cAMP and its downstream response by IFN-gamma. However, IFN-gamma did not affect secretory responses to the calcium-dependent secretagogue carbachol or cAMP analog 8-bromo-cAMP, indicating that normal secretory responses to adequate second messengers in IFN-gamma-treated cells are achievable. Moreover, IFN-gamma inhibited the expression of adenylate cyclase isoforms 5 and 7. In conclusion, we demonstrate that IFN-gamma down-regulates adenosine-mediated signaling possibly through the direct inhibition of adenylate cyclase expression. We propose that IFN-gamma may acutely affect global cAMP-mediated responses in the intestinal epithelia, thereby decreasing secretory responses, which may consequently aggravate inflammatory processes.
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PMID:Interferon-gamma down-regulates adenosine 2b receptor-mediated signaling and short circuit current in the intestinal epithelia by inhibiting the expression of adenylate cyclase. 1555 Mar 90


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