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)

The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta-arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.
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PMID:Bradykinin-induced internalization of the human B2 receptor requires phosphorylation of three serine and two threonine residues at its carboxyl tail. 1021 57

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.
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PMID:Protein kinase C antagonizes pertussis-toxin-sensitive coupling of the calcitonin receptor to adenylyl cyclase. 1023 69

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is, at present, the only G-protein-coupled receptor that activates phospholipase C and lacks a C-terminal tail. We have previously demonstrated that this unique structural feature is associated with resistance to rapid desensitization of phosphoinositide signaling in COS-7 and HEK-293 cells (Heding, A., Vrecl, M., Bogerd, J., McGregor, A., Sellar, R., Taylor, P. L., and Eidne, K. A. (1998) J. Biol. Chem. 273, 11472-11477). Using receptors tagged with a nonapeptide of the influenza hemagglutinin protein to enable immunoprecipitation, we now demonstrate that the mammalian GnRH-R is not phosphorylated in an agonist-dependent manner. In contrast, the mammalian thyrotropin-releasing hormone receptor and the African catfish GnRH-R, both of which have a C-terminal tail, are phosphorylated in response to agonist challenge. Furthermore, chimeras of the mammalian GnRH-R with the C-terminal tail of either the mammalian thyrotropin-releasing hormone receptor or the catfish GnRH-R are also phosphorylated in an agonist-dependent manner. Only those receptors having C-terminal tails showed desensitization of phosphoinositide responses within 5-10 min of agonist challenge. We also show that the internalization of all these receptors when expressed transiently in COS-7 cells is similar. This dissociates receptor internalization from rapid desensitization and demonstrates that the lack of a C-terminal tail in the mammalian GnRH-R results in an inability of the receptor to undergo agonist-dependent phosphorylation and that this results directly in a resistance to rapid desensitization.
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PMID:Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization. 1051 4

Contribution of sphingosine kinase (SPK)-catalyzed production of sphingosine-1-phosphate (SPP), in comparison to phospholipase C (PLC), to Ca(2+) signalling by epidermal growth factor (EGF) was studied in two HEK-293 cell clones (HEK2 and HEK3), expressing functional EGF receptors and exhibiting release of stored Ca(2+) by intracellular SPP. In HEK3 cells, EGF increased [Ca(2+)](i) and stimulated both, SPK and PLC. [Ca(2+)](i) increase, but not PLC stimulation, was strongly reduced by SPK inhibition. In HEK2 cells, EGF similarly stimulated PLC, but did not increase [Ca(2+)](i) or stimulate SPK, suggesting that intracellular SPP production plays a major role for Ca(2+) signalling by EGF in HEK-293 cells.
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PMID:Role of sphingosine kinase in Ca(2+) signalling by epidermal growth factor receptor. 1056

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.
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PMID:Homologous and heterologous phosphorylation of the vasopressin V1a receptor. 1057 29

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 scorpion alpha-toxins Lqh II, Lqh III, and Lqh alphaIT from Leiurus quinquestriatus hebraeus are representatives of typical alpha-toxins, specific for either mammals (Lqh II) or insects (Lqh alphaIT), and alpha-like toxins (Lqh III) which act on both mammals and insects. For a comparative study of the effects of these toxins on mammalian sodium channels we stably expressed rat skeletal muscle sodium channel alpha subunits (microI) in HEK 293 cells and measured Na+ currents in the whole-cell patch-clamp mode. The alpha- and alpha-like toxins strongly slowed down channel inactivation with a half-maximal effect at 1.4 nM (Lqh II), 5.4 nM (Lqh III), and 0.5 nM (Lqh alphaIT). The recovery from fast inactivation was accelerated by all toxins with the potency sequence: Lqh II>Lqh alphaIT>Lqh III. The voltage dependence of inactivation and recovery from inactivation were reduced while the threshold for activation was only slightly shifted by approximately equal to 10 mV without altering the slope factors, suggesting uncoupling of the impaired inactivation from the activation. The toxins induced an increase in peak inward current, which was accounted for by an increased maximal open-channel probability. Although all three toxins induced similar modifications of the channel properties, their kinetics of association and dissociation were very different. Between -140 and -80 mV toxin association was not voltage dependent. In 100 nM toxin the association time constants were: 1.3 s (Lqh II), 20 s (Lqh III), and 3.8 s (Lqh alphaIT). At positive voltages the toxin dissociated from the channel; at +100 mV the dissociation time constants were 30, 321, and 135 ms, respectively. In contrast to the association, dissociation was voltage dependent with a similar slope of about 12 mV per e-fold change for all three toxins. The strong differences in the association and dissociation kinetics of these toxins may identify them as members of different scorpion alpha-toxin subgroups.
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PMID:Modulation of cloned skeletal muscle sodium channels by the scorpion toxins Lqh II, Lqh III, and Lqh alphaIT. 1067 38

Although M1-M4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells internalize on agonist stimulation, only M1, M3, and M4 but not M2 mAChRs recycle to the plasma membrane. To investigate the functional consequences of this phenomenon, we compared desensitization and resensitization of M2 versus M4 mAChRs. Treatment with 1 mM carbachol for 1 h at 37 degrees C reduced numbers of cell surface M2 and M4 mAChRs by 40-50% and M2 and M4 mAChR-mediated inhibition of adenylyl cyclase, intracellular Ca2+ concentration ([Ca2+]i) increases, and phospholipase C (PLC) activation by 60-70%. Receptor-mediated inhibition of adenylyl cyclase and [Ca2+]i increases significantly resensitized within 3 h. However, M4 but not M2 mAChR-mediated PLC activation resensitized. At 16 degrees C, M2 mAChR-mediated [Ca2+]i increases and PLC stimulation desensitized to a similar extent as at 37 degrees C. However, at 16 degrees C, where M2 mAChR internalization is negligible, both M2 mAChR responses resensitized, demonstrating that M2 mAChR resensitization proceeds at the plasma membrane. Examination of M2 mAChR responses following inactivation of cell surface mAChRs by quinuclidinyl benzilate revealed substantial receptor reserve for coupling to [Ca2+]i increases but not to PLC. We conclude that M2 mAChR internalization induces long-lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve.
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PMID:Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. 1073 31

Activation of stably expressed M(2) and M(3) muscarinic acetylcholine receptors (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiation of phospholipase C (PLC) stimulation by these and other G protein-coupled receptors (GPCRs). Here, we report that GPCRs can induce an up-regulation of PLC stimulation by receptor tyrosine kinases (RTKs) as well and provide essential mechanistic characteristics of this sensitization process. Pretreatment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophosphatidic acid, or ATP, followed by agonist washout, strongly increased (by 2-3-fold) maximal PLC stimulation (measured >/=40 min later) by epidermal growth factor and platelet-derived growth factor, but not insulin, and largely enhanced PLC sensitivity to these RTK agonists. The up-regulation of RTK-induced PLC stimulation was cycloheximide-insensitive and was observed for up to approximately 90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M(2) mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of Gbetagamma scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca(2+) suppressed the sensitization process, while overexpression of PKC-alpha, but not PKC-betaI, further enhanced the M(2) mAChR-induced sensitization of PLC stimulation. None of these treatments affected acute PLC stimulation by either GPCR or RTK agonists. Taken together, short term activation of GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G(i)-derived Gbetagammas as well as increases in intracellular Ca(2+) and activation of a PKC isoenzyme, most likely PKC-alpha.
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PMID:G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases. 1090 68

The M(3) muscarinic acetylcholine receptor (mAChR) expressed in HEK-293 cells couples to G(q) and G(12) proteins and stimulates phospholipase C (PLC) and phospholipase D (PLD) in a pertussis toxin-insensitive manner. To determine the type of G protein mediating M(3) mAChR-PLD coupling in comparison to M(3) mAChR-PLC coupling, we expressed various Galpha proteins and regulators of the G protein signaling (RGS), which act as GTPase-activating proteins for G(q)- or G(12)-type G proteins. PLD stimulation by the M(3) mAChR was enhanced by the overexpression of Galpha(12) and Galpha(13), whereas the overexpression of Galpha(q) strongly increased PLC activity without affecting PLD activity. Expression of the RGS homology domain of Lsc, which acts specifically on Galpha(12) and Galpha(13), blunted the M(3) mAChR-induced PLD stimulation without affecting PLC stimulation. On the other hand, overexpression of RGS4, which acts on Galpha(q)- but not Galpha(12)-type G proteins, suppressed the M(3) mAChR-induced PLC stimulation without altering PLD stimulation. We conclude that the M(3) mAChR in HEK-293 cells apparently signals to PLD via G(12)- but not G(q)-type G proteins and that G protein subtype-selective RGS proteins can be used as powerful tools to dissect the pertussis toxin-resistant G proteins and their role in receptor-effector coupling.
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PMID:The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins: regulators of G proteins as tools to dissect pertussis toxin-resistant G proteins in receptor-effector coupling. 1103 69


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