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)

Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied beta-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by beta-adrenergic receptor kinase ( beta-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for beta-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C-linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser345-Leu463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30-60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparin-Sepharose. The purified protein kinase represented an approximately 18,600-fold purification and was a single polypeptide with a molecular weight of approximately 40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, beta-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.
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PMID:Identification of a novel receptor kinase that phosphorylates a phospholipase C-linked muscarinic receptor. 863 12

1. Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores. 2. Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor-activated [Ca2+]i increase. 3. Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). 4. Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-alpha q/alpha 11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine-induced Ca2+ release. 5. Intracellular applications of either an anti-beta com antibody or a peptide corresponding to the G beta gamma binding domain of the beta-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release. 6. Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol-specific phospholipase C.
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PMID:Specific Gq protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes. 917 86

Transgenic mice were generated with cardiac-specific overexpression of the wild-type (WT) alpha1B-adrenergic receptor (AR) using the murine alpha-myosin heavy chain gene promoter. Previously, we described transgenic mice with alpha-myosin heavy chain-directed expression of a constitutively active mutant alpha1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113). In animals with >40-fold WT alpha1-AR overexpression, basal myocardial diacylglycerol content was significantly increased, indicating enhanced alpha1-adrenergic signaling and phospholipase C activity. In contrast to the mice overexpressing constitutively active mutant alpha1B-ARs, the hearts of these mice did not develop cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic factor. In vivo physiology was studied in anesthetized intact animals and showed left ventricular contractility in response to the beta-agonist isoproterenol to be significantly depressed in animals overexpressing WT alpha1B-ARs. Membranes purified from the hearts of WT alpha1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with isoproterenol, norepinephrine, or phenylephrine. Interestingly, these in vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high levels of WT alpha1B-ARs can lead to coupling to pertussis toxin-sensitive G proteins. Another potential contributor to the observed decreased myocardial signaling and function could be enhanced beta-AR desensitization as beta-adrenergic receptor kinase (betaARK1) activity was found to be significantly elevated (>3-fold) in myocardial extracts isolated from WT alpha1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart failure where betaARK1 levels are elevated and beta-ARs are down-regulated, leading to a higher percentage of cardiac alpha1-ARs. Thus, these mice serve as a unique experimental model to study the in vivo interactions between alpha- and beta-ARs in the heart.
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PMID:Transgenic mice with cardiac overexpression of alpha1B-adrenergic receptors. In vivo alpha1-adrenergic receptor-mediated regulation of beta-adrenergic signaling. 926 Nov 35

In transfected Chinese hamster ovary (CHO-A1) cells the human adenosine A1 receptor directly stimulates pertussis toxin-sensitive increases in inositol phosphate production and potentiates (synergistically) the inositol phosphate responses mediated by Gq-coupled P2Y2 purinoceptor and CCK(A) receptors. In the present study we have investigated the role of Gbetagamma subunits in mediating adenosine A1 receptor effects on phospholipase C activation (both direct and synergistic) by transiently transfecting CHO-A1 cells with a scavenger of Gbetagamma subunits: the C-terminus of beta-adrenoceptor kinase 1 (beta ark1 residues 495-689). [3H]inositol phosphate responses to the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA; 1 microM) were inhibited (41 +/- 1%) in CHO-A1 cells transiently transfected with the Gbetagamma scavenger, beta ark1 (495-689). Expression of beta ark1 (495-689) protein was confirmed by Western blotting. In contrast, adenosine A1 receptor-mediated inhibition of forskolin stimulated [3H]cyclic AMP accumulation was unaffected by transient expression of beta ark1 (495-689). Beta ark1 (495-689) expression had no significant effect on the [3H]inositol phosphate responses produced by activation of the endogenous P2Y2 purinoceptor (100 microM UTP; 92 +/- 0.8% of control). [3H]inositol phosphate accumulation in response to adenosine A receptor activation was also attenuated in CHO-K1 cells co-transfected with the beta ark1 (495-689) minigene (59 +/- 4% inhibition of control response to 1 microM CPA). Finally, transient expression of beta ark1 (495-689) in CHO-A1 cells inhibited the augmentation of [3H]inositol phosphate responses resulting from co-activation of adenosine A1 receptors and P2Y2 purinoceptors. These experiments indicate that Gbetagamma subunits are involved in the direct coupling the adenosine A1 receptor to phospholipase C and that they also participate in the augmentation of P2Y2 purinoceptor-mediated [3H]inositol phosphate responses by the adenosine A1 receptor.
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PMID:Involvement of G-protein betagamma subunits in coupling the adenosine A1 receptor to phospholipase C in transfected CHO cells. 975 42

HIKE is a highly conserved sequence motif that selectively occurs in proteins candidate to bind PH domains, e.g., the beta subunit of heterotrimeric G proteins, kinases, ankyrin and kinesin. Thus, the HIKE region has been predicted to be a protein docking site for PH domains. This work evidentiates recent experimental evidence that unambiguously defines the functional role of HIKE in Gbeta as a multiple effector docking site and as a major regulatory region of G protein's function. Indeed, the Gbeta HIKE interacts with the beta-adrenergic receptor kinase, Galpha, Ggamma, adenylyl cyclase 2, phospholipase C beta2, inward rectifier K channels, calcium channel alpha1B, calmodulin, phosducin, ste20. Quite interestingly, HIKE is located in the Gbeta region that faces the cell membrane. Thus, HIKE also interacts with the cell membrane and may dynamically regulate membrane vs effector binding of the Galphabetagamma trimer. These findings fulfill a major prediction of the HIKE model, i.e., that HIKE is a regulatory region for protein-protein interactions. A role of HIKE as a proteic binding site for PH domains is supported by the profound influence of HIKE mutations on the largely PH-mediated binding of beta-ARK to Gbeta.
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PMID:HIKE, a candidate protein binding site for PH domains, is a major regulatory region of Gbeta proteins. 1032 71

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of these receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinases (GRK). When expressed in rat basophilic leukemia cells and exposed to C3a, the C3aR underwent rapid (t(1/2) approximately 15 s), dose-dependent (EC50 approximately 10 nM) and reversible phosphorylation by a kinase refractory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed that the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5 - 1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 and GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells. GRK overexpression in cells which co-expressed C5aR and were exposed to C5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphorylation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) which contain significant intracellular levels of GRK2 and GRK3.
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PMID:Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases. 1050 78

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
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PMID:Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. 1056 30

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.
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PMID:Pasteurella multocida toxin stimulates mitogen-activated protein kinase via G(q/11)-dependent transactivation of the epidermal growth factor receptor. 1063 31

The present studies were carried out to evaluate the mechanisms by which PTH/PTHrP receptor (PTHR) activation influences cell viability. In 293 cells expressing recombinant PTHRs, PTH treatment markedly reduced the number of viable cells. This effect was associated with a marked apoptotic response including DNA fragmentation and the appearance of apoptotic nuclei. Similar effects were evidenced in response to serum withdrawal or to the addition of tumor necrosis factor (TNFalpha). Addition of caspase inhibitors or overexpression of bcl-2 partially abrogated apoptosis induced by serum withdrawal. Caspase inhibitors also protected cells from PTH-induced apoptosis, but overexpression of bcl-2 did not. The effects of PTH on cell number and apoptosis were neither mimicked by activators of the cAMP pathway (forskolin, isoproterenol) nor blocked by an inhibitor (H-89). However, elevation of Ca(i)2+ by addition of thapsigargin induced rapid apoptosis, and suppression of Ca(i)2+ by overexpression of the calcium- binding protein, calbindin D28k, inhibited PTH-induced apoptosis. The protein kinase C inhibitor GF 109203X partially inhibited PTH-induced apoptosis. Regulator of G protein signaling 4 (RGS4) (an inhibitor of the activity of the alpha-subunit of Gq) suppressed apoptotic signaling by the PTHR, whereas the C-terminal fragment of GRK2 (an inhibitor of the activity of the beta(gamma)-subunits of G proteins) was without effect. Chemical mutagenesis allowed selection of a series of 293 cell lines resistant to the apoptotic actions of PTH; a subset of these were also resistant to TNFalpha. These results suggest that 1) apoptosis produced by PTHR and TNF receptor signaling involve converging pathways; and 2) Gq-mediated phospholipase C/Ca2+ signaling, rather than Gs-mediated cAMP signaling, is required for the apoptotic effects of PTHR activation.
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PMID:Apoptosis mediated by activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP). 1067 97

The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC(2)) receptor was shown to induce both [(3)H]inositol phosphate ([(3)H]InsP)and cAMP production in transfected COS7 cells and in GH(3) cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [(3)H]InsP response, suggesting independent coupling of the two pathways. The VPAC(2) receptor-mediated [(3)H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G beta gamma-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH(3) cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca(2+) influx pathways (Co(2+) and SKF 96365) also partially inhibited VPAC(2) receptor-mediated [(3)H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca(2+) channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC(2) receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G(q/11) and G(i/o), respectively) to generate [(3)H]InsP. In addition to G beta gamma, G(i/o) activation appears to require receptor-activated Ca(2+) entry. This is consistent with the possibility that not only G alpha(q/11)-responsive and G beta gamma-responsive isoforms of phospholipase C but also Ca(2+)-responsive forms may contribute to the overall [(3)H]InsP response.
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PMID:Mechanisms of phospholipase C activation by the vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 receptor. 1118 37


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