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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.
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PMID:Adenylyl cyclases and the interaction between calcium and cAMP signalling. 770 Mar 50

A single point mutation that encodes an aspartic acid (Asp578) to glycine substitution in the LH/CG receptor (LH/CGR) gene, D578G, was recently found in American patients with familial male-limited precocious puberty and in a Japanese patient with a sporadic form of the disorder. Transfection of the mutant, compared to the wild-type, LH/CGR complementary DNA into COS-7 cells results in higher basal cAMP production, but a normal agonist-induced response; the mutation is, therefore, proposed to constitutively activate Leydig cells and elevate serum testosterone, despite low levels of gonadotropin. In the current study we examined two additional Japanese patients with male-limited precocious puberty without a family history of the disease. We describe a heterozygous cytosine (C) to thymine (T) transition at nucleotide 1715 in both; the mutation encodes an alanine to valine substitution in codon 572 of transmembrane helix 6, A572V. Transfected into COS-7 cells, the A572V mutant exhibited the same constitutively high basal cAMP levels and normal agonist-induced cAMP response as the D578G mutant. We conclude that the constitutively higher cAMP levels caused by the A572V mutation led to Leydig cell activation and male-limited precocious puberty, as in the previously described D578G mutation. As the mother of one of the two patients had the same heterozygous mutation, this patient represents the first recognized case of inherited male-limited precocious puberty in the Japanese population. The previously described D578G mutant did not increase basal or agonist-induced inositol phosphate production in transfected COS-7 cells, or the number of LH/CGRs or their affinity for LH/CG. In contrast, transfection of the A572V mutation in COS-7 cells exhibited significantly higher inositol phosphate levels basally and at 10(-11) mol/L hCG, but significantly lower inositol phosphate levels at 10(-7) mol/L hCG. These data suggest that the A572V mutation of the LH/CGR may have effects on the guanine nucleotide binding protein which activates phospholipase C (Gq) coupling and phospholipase-C activation in addition to its effects on Gs coupling and activation of adenylyl cyclase. A572V-transfected cells also exhibited a higher affinity, despite an apparent decrease in the number of binding sites, for [125I]hCG, compared to transfectants with the wild-type LH/CGR. We hypothesize that these differences between the A572V and D578G mutations reflect a greater impact of the A572V mutation on receptor conformation.
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PMID:A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty. 771 85

The G protein-linked receptor for neurokinin A (NKA) couples to stimulation of phospholipase C and, in some cells, adenylyl cyclase. We have examined the function of the C-terminal cytoplasmic domain in receptor signaling and desensitization. We constructed C-terminal deletion mutants of the human NK-2 receptor (epitope tagged) to remove potential Ser/Thr phosphorylation sites, and expressed them in both mammalian and insect cells. When activated, truncated receptors mediate stronger and more prolonged phosphoinositide hydrolysis than wild-type receptor; however, the amplitude and kinetics of the NKA-induced rise in cytosolic Ca2+ remain unaltered. Protein kinase C (PKC)-activating phorbol ester abolishes wild-type receptor signaling but not mutant receptor signaling. Mutant receptors also mediate enhanced and prolonged cAMP generation, at least in part via PKC activation. When expressed in COS cells or Sf9 insect cells, the wild-type receptor is phosphorylated; receptor phosphorylation increases after addition of either NKA or phorbol ester. In contrast, mutant receptors are not phosphorylated by either treatment. Our results suggest that C-terminal Ser/Thr phosphorylation sites in the NK-2 receptor have a critical role in both homologous and heterologous desensitization. Removal of these phosphorylation sites results in a receptor that mediates sustained activation of signaling pathways and is insensitive to inhibition by PKC.
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PMID:C-terminal truncation of the neurokinin-2 receptor causes enhanced and sustained agonist-induced signaling. Role of receptor phosphorylation in signal attenuation. 772 3

In neuroblastoma x glioma hybrid NG108-15 cells, ATP induced a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), accompanied by inositol phosphate formation. Under the same conditions, we found a marked increase in cAMP levels produced by ATP at concentrations similar to those required to increase [Ca2+]i. The Ca2+ ionophore A23187 or bradykinin, which evoked inositol phosphate formation and increases in [Ca2+]i, did not increase, and instead slightly decreased, cAMP content, indicating that ATP-induced cAMP accumulation was not due to activation of Ca(2+)-sensitive adenylyl cyclase. The effect of ATP on cAMP production was not dependent on generation of adenosine caused by ATP hydrolysis. Among several P2 purinoceptor agonists, adenosine-5'-O-(3-thio)triphosphate, 5'-adenylylimidodiphosphate, and adenosine-5'-O-(2-thio)diphosphate evoked both cAMP accumulation and Ca2+ mobilization. In contrast, beta,gamma-methylene-ATP selectively elicited cAMP accumulation, whereas 2-methylthio-ATP and UTP induced only Ca2+ mobilization, without affecting cAMP levels. The potent P2x purinoceptor agonist alpha,beta-methylene-ATP did not induce cAMP accumulation or Ca2+ mobilization. The cAMP accumulation induced by ATP was not affected by the P2 receptor antagonist suramin but was inhibited by P1 receptor antagonists such as 8-(p-sulfophenyl)theophylline, 3-isobutyl-1-methylxanthine, and xanthine amine congener. However, the ATP-induced increase in [Ca2+]i was not affected by suramin or xanthine amine congener. Taken together, these results indicate that ATP activates two distinct purinoceptors that are coupled to different signal transduction systems, one being adenylyl cyclase and the other phospholipase C, in NG108-15 cells. Furthermore, pharmacological profiles of the adenylyl cyclase-coupled receptor were quite different from those of any known purinoceptor subtypes, especially in the unusual sensitivity of the receptor to P1 and P2 receptor agonists and antagonists. It is therefore suggested that ATP-induced cAMP accumulation may be mediated by a novel subtype of purinoceptor in NG108-15 cells.
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PMID:Extracellular ATP stimulates adenylyl cyclase and phospholipase C through distinct purinoceptors in NG108-15 cells. 772 48

To identify the mechanisms of action of isoforms angiotensin II receptors (AT1A, AT1B, and AT2) and to overcome the difficulties encountered in attempts to purify the receptors, we have expression-cloned their cDNAs from bovine and rat sources and isolated human cDNA and rat and human genomic DNA. The AT1A and AT1B cDNAs were found to encode respective receptor proteins with 359 amino acid residues, whereas, AT2 encodes a 363 amino acid residue receptor protein. Both AT1 and AT2 were found to conform with the seven transmembrane receptor structural motif, but showed only 32% amino acid residue identity to each other. The AT1 receptor was shown to be coupled to, at least, three different G proteins activating phospholipase C, inhibiting adenylyl cyclase and opening an L-type Ca(2+)-channel, whereas, AT2 was found to inhibit a phosphotyrosine phosphatase activity without affecting guanylyl cyclase by a pertussis-toxin-sensitive, presumably G-protein-mediated mechanism.
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PMID:Angiotensin II receptors: cloning and expression. 774 65

Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP3) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca2+ ([Ca2+]i) with an EC50 value of 35 microM. In addition, carbachol produced a 1.3-3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E2 (PGE2). The M3 antagonist p-fluorohexahydrosiladifenidol (IC50 = 0.5-0.8 microM) inhibited the increases in [Ca2+]i, IP3, and cAMP more effectively than the M1 antagonist pirenzepine (IC50 = 5-9 microM) and the M2 antagonist methoctramine (IC50 = 20-30 microM). The involvements of [Ca2+]i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 microM) inhibited significantly the synergistic effects of carbachol and PGE2 on the production of cAMP, whereas the Ca2+ ionophore ionomycin (1 microM) clearly enhanced PGE2-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE2-stimulated cAMP production. These data suggest that phospholipase C-linked M3 receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca(2+)-dependent system in the neuronal cells.
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PMID:Stimulation of adenylyl cyclase mediated by phospholipase C-linked M3 muscarinic receptor in human neuroblastoma SK-N-BE (2) C cells. 776 29

5-HT1a receptors in the hippocampus play a critical role in modulating limbic system output. The activity and level of 5-HT1a receptors are modulated by glucocorticoid levels. The present study was undertaken to test the hypothesis that glucocorticoids attenuate the transcriptional activity of the 5-HT1a receptor gene. Using in situ hybridization and RNase protection assays, we observed a substantial increase in 5-HT1a mRNA expression after adrenalectomy in the same hippocampal regions in which 5-HT1a binding sites are increased. This increase in 5-HT1a mRNA expression occurs as early as 1 h after adrenalectomy and precedes the increase in receptor binding sites. Further in situ hybridization analysis showed that 5-HT1a mRNA is increased within individual hippocampal cells after adrenalectomy. Administration of dexamethasone completely prevents the adrenalectomy-induced elevation in hippocampal 5-HT1a receptor mRNA. Nuclear run-on assays showed that the rate of transcription of 5-HT1a mRNA after adrenalectomy increased 70% above the rate from control preparations and could be reduced to basal levels by the administration of dexamethasone. Adrenalectomy did not cause an increase in functional coupling of 5-HT1a receptors to adenylyl cyclase or phospholipase C. These results suggest that transcription of hippocampal 5-HT1a receptor mRNA is under negative regulation by corticosteroid hormones.
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PMID:Transcriptional regulation of hippocampal 5-HT1a receptors by corticosteroid hormones. 776 98

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.
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PMID:A density-sensing factor regulates signal transduction in Dictyostelium. 777 72

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1
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PMID:Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. 778 10

Adenylyl cyclase exists as a family of closely related subtypes which differ in their tissue distribution and regulatory properties. Submicromolar rises in [Ca2+]i produced via activation of phospholipase C (PLC) or Ca2+ channel opening, provide a mechanism by which Ca2+/calmodulin (CaM) or protein kinase C (PKC)-sensitive isoforms of adenylyl cyclase can be regulated. In this study we have examined, in detail, the muscarinic (M3) regulation of adenylyl cyclase in SH-SY5Y cells and report a role for both [Ca2+]e and [Ca2+]i. Carbachol (1 mM) and potassium (100 mM) caused a time (T1/2 = 3 and 4 min, respectively) and dose (EC50 = 6.95 microM and 34.7 mM respectively) related increase in cAMP formation. This amounted to an approximate two-fold increase over basal levels. Carbachol and potassium also caused a biphasic increase in [Ca2+]i with basal, peak and plateau values of 118.4 nM, 697.6 nM, 253.0 nM and 104.0 nM, 351.6 nM, 181.5 nM, respectively. Calcium channel blockade with nickel (2.5 mM) abolished potassium-stimulated cAMP formation and rises in [Ca2+]i. However, carbachol-stimulated cAMP formation was significantly decreased only at the later time points, where rises in [Ca2+]i were also essentially abolished. Further evidence for a role for [Ca2+]e and [Ca2+]i is provided by the stimulation of cAMP formation by carbachol in the absence of added Ca2+, followed by a further increase on its re-addition. Carbachol- and potassium-stimulated cAMP formation were inhibited by the CaM antagonist trifluoperazine (100 microM). The mu-opiate agonists, morphine and fentanyl also inhibited carbachol-stimulated cAMP formation. In addition, cAMP formation in SH-SY5Y cell membranes was significantly increased in the presence of Ca2+ (1.46 microM), CaM (200 nM) and forskolin (1 microM). PKC inhibition with Ro 31 8220 did not affect carbachol-stimulated cAMP formation. Taken collectively, these data suggest that SH-SY5Y cells express type 1, and possibly type 8 isoforms of adenylyl cyclase, which can be regulated by intra- and extracellular Ca2+.
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PMID:Adenylyl cyclase in SH-SY5Y human neuroblastoma cells is regulated by intra- and extracellular calcium. 778 4


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