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 alpha subunits of Gq family G proteins, GL1 alpha and GL2 alpha, are bovine homologues of mouse G14 alpha and G11 alpha, respectively, and are closely related to each other. When expressed in Xenopus oocytes together with metabotropic glutamate receptors, GL2 alpha activates endogenous phospholipase C (PLCx) in response to glutamate stimulation, whereas GL1 alpha inhibits the activation of PLCx. By examining the properties of 10 chimeras between GL1 alpha and GL2 alpha and their mutants, we tried to identify the regions on the G alpha proteins that are important for the activation of PLCx. The results indicated that a necessary (but not sufficient) condition for a chimeric G alpha protein to be able to clearly activate PLCx was that its N-terminal quarter portion should be derived from GL2 alpha. No correlation was found between the origin (GL1 alpha or GL2 alpha) of C-terminal regions of the chimeras and the ability of chimeras to activate PLCx. One of the chimeras is different from GL2 alpha at only four amino acid residues in the N-terminal region, and yet it could not activate PLCx. When one of the four residues, Ser-59, in the chimera was mutated back to Ala as in the original GL2 alpha, the resulting mutant became capable of activating PLCx. This residue is localized in the midst of the N-terminal linker connecting the two major domains in the G alpha proteins. These results indicate that Ala-59 is critical for the activation of PLCx, and that the linker may play important roles in determining functions of G alpha proteins.
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PMID:Importance of N-terminal regions of G protein alpha subunits for the activation of phospholipase C in Xenopus oocytes. 898 68

A preliminary model of the rat AT1A angiotensin II (AII) receptor (Joseph, M. P., Maigret, B., Bonnafous J.-C., Marie, J., and Scheraga, H. A. (1995) J. Protein Chem. 14, 381-398) has predicted an interaction between Asn111 located in transmembrane domain (TM) III and Tyr292 (TM VII) in the nonactivated receptor; a disruption of this interaction upon AII activation would allow Tyr292 to interact with the conserved Asp74 (TM II). The previous verification that Tyr292 is essential for receptor coupling to phospholipase C (Marie, J., Maigret, B., Joseph, M. P., Larguier, R., Nouet, S., Lombard, C., and Bonnafous, J.-C. (1994) J. Biol. Chem. 269, 20815-20818) prompted us to check the possible alterations in receptor properties upon Asn111 --> Ala mutation. The mutated receptor (N111A) displayed: (i) strong constitutive activity, with amplification of the maximal phospholipase C response to AII; (ii) agonist behavior of the AT2-specific ligand CGP 42112A, [Sar1, Ile8]AII, and [Sar1,Ala8]AII, antagonists of the wild-type receptor; (iii) inverse agonism behavior of the non-peptide ligands DuP 753, LF 7-0156, and LF 8-0129. The results are discussed in the light of the allosteric ternary complex models and other described examples of constitutive activation of G protein-coupled receptors.
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PMID:Mutation of Asn111 in the third transmembrane domain of the AT1A angiotensin II receptor induces its constitutive activation. 899 67

A series of single alanine substituted analogs of amyloid beta peptide (25-35) were tested for their ability to activate the phospholipases of cultured LA-N-2 cells. Substitution of alanine for the amino acids 29-34 prevented the activation of phospholipases A2 and D. In addition substitution of alanine at 28 prevented phospholipase D but not phospholipase A2 activation. All the alanine substitutions, except for positions 33 and 35, blunted phospholipase C activations. There were no activations by scrambled amyloid beta peptide.
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PMID:Activation of LA-N-2 cell phospholipases by single alanine substitution analogs of amyloid beta peptide (25-35). 909 25

1. An alanine residue at the C-terminal tail of the third intracellular loop is highly conserved among various Gq protein-coupled receptors including rat cholecystokininB (CCKB) and neurotensin receptors. To investigate the functional significance of the conserved alanine in the activation of Gq proteins and phospholipase C (PLC) by CCKB and neurotensin receptors, the alanine residue was mutated in the present study. Subsequently, the ability of resulting mutant receptors to activate PLC was investigated by measuring the formation of inositol phosphates (IP) in COS-7 cells and recording Ca(2+)-activated chloride currents from Xenopus oocytes. 2. Site-directed mutagenesis was performed to mutate alanine at position 332 of rat CCKB receptor to glutamate. When the (A332E) mutant receptor was expressed in COS-7 cells and Xenopus oocytes, the efficacy and the potency of sulphated cholecystokinin octapeptide (CCK-8) to stimulate polyphosphoinositide hydrolysis in COS-7 cells and evoke calcium-dependent Cl- currents in oocytes were not significantly affected. 3. Alanine residue at position 302 of rat neurotensin receptor was also mutated to glutamate. When expressed in COS-7 cells and Xenopus oocytes, the resulting (A302E) mutant receptor was strongly defective in stimulating phosphatidylinositol turnover in COS-7 cells and evoking Ca(2+)-dependent chloride currents in oocytes. 4. In summary, the present study demonstrates that alanine residue at the C-terminus of third cytoplasmic domain is required for the full activation of Gq proteins and PLC by neurotensin receptors. However, in contrast to other Gq protein-coupled receptors, alanine at the distal third intracellular loop does not play a significant role in CCKB receptor activation of PLC.
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PMID:A site-directed mutagenesis study on the conserved alanine residue in the distal third intracellular loops of cholecystokininB and neurotensin receptors. 915 42

A new substrate analogue, (2R)-1,2-dipalmitoyloxypropanethiophospho-1-D-myo-inositol (DPsPI), has been used in a new, continuous assay for phosphatidylinositol-specific phospholipase C (PI-PLC). DPsPI is superior to other substrate analogs that have been used for assaying PI-PLC since it is synthesized as a pure diastereomer and maintains both acyl chains of the natural substrate, dipalmitoylphosphatidylinositol (DPPI). The assay that has been developed using this new analogue has allowed us to elucidate detailed kinetic data so far lacking in the field. In addition, several mutants of PI-PLC were constructed and assayed. The results show that Arg-69 is essential for catalysis, since mutations at this position led to a 10(3)- 10(4)-fold decrease in activity with respect that of to the wild-type (WT) enzyme. An alanine mutant of Asp-67, a residue also found at the active site, displays activity similar to that of WT. We have also used nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy to analyze the structural integrity and conformational stability of the mutants. The results show that the overall global conformation of the enzyme is not perturbed by the mutants. The 15N-1H HSQC NMR spectrum of WT PI-PLC is also reported at 600 MHz. The stereoselectivity of the reaction toward the stereoisomers of another analogue, 1,2-dipalmitoyl-sn-glycero-3-thiophospho-1-myo-inositol (DPPsI), was used to probe whether Arg-69 interacts with the phosphate moiety of the substrate. We have calculated that the WT enzyme shows a stereoselectivity ratio of 160000:1 in favor of the Rp isomer versus the Sp isomer. The R69K mutant displayed a significant 10(4)-fold relaxation of stereoselectivity. Our data support the role of Arg-69 in stabilizing the negative charge on the pentacoordinate phosphate in the transition state during catalysis.
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PMID:Phosphatidylinositol-specific phospholipase C: kinetic and stereochemical evidence for an interaction between arginine-69 and the phosphate group of phosphatidylinositol. 918 43

Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk- and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displayed similar agonist affinities as the wild-type receptor. In Ltk- cells, the T149A 5-HT1A receptor inhibited cAMP accumulation by 30% compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) was observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor failed to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibited basal cAMP and partially inhibited Gs-stimulated cAMP accumulation compared with wild-type receptor (57 +/- 14% versus 86 +/- 2%). In contrast, the T149A 5-HT1A receptor mutant failed to block the influx of calcium induced by calcium channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibited the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by betagamma subunits of Gi2) and to inhibition of calcium channel activation (mediated by betagamma subunits of Go) but plays a minor role in coupling to alpha1-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors.
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PMID:A conserved threonine residue in the second intracellular loop of the 5-hydroxytryptamine 1A receptor directs signaling specificity. 922 26

The role of polymorphonuclear leukocytes (PMN) in stemming systemic infection is executed mainly by the utilization of molecular O2 leading to the production of reactive oxygen intermediates (ROI). PMN-derived ROI also serve as intra- and extracellular second messengers providing both positive and negative feedback on cellular autoregulation. We investigated the effect of endogenous ROI on two signal transducing pathways: the receptor (R)-G-protein-phospholipase D (PLD) and receptor (R)-G-protein-phospholipase C pathways responsible for the subsequent interleukin-8 (IL-8)-induced PMN respiratory burst. Purified human PMN were primed with LPS adhered to plastic surfaces and stimulated with IL-8 with or without the presence of each of five different selective ROI scavengers/antioxidants: DMSO, N(a)N3, L-alanine, catalase, or superoxide dismutase. Total IL-8 surface receptor expression was assessed by 125I-IL-8 and 125I-labeled mAbs against IL-8R type A and B binding assays; PLD activation was assessed by measuring formation of phosphatidyl ethanol (PEt) in the presence of ethanol; PLC activation was measured by quantitative conversion of [32P]ATP-labeled phosphatidic acid (PA) into diacylglycerol (DAG); expression of G alpha-inhibitory subunit was assessed by SDS-PAGE and immunoblotting with polyclonal Abs against this subunit. Production of O2-, H2O2, HClO, and myeloperoxidase (MPO) in the experimental model was confirmed in a separate set of experiments. The overall impact of antioxidants on each component of the transducing tripartite complex was stimulatory; however, N(a)N3 and SOD exhibited the most ubiquitous effect with consistent up-regulation by N(a)N3 of IL-8R expression, whereas even trace amounts of externally added authentic MPO significantly down-regulated the functional activity of both effector enzymes. These results demonstrate a multiple site-specific targeting of the signal-transducing complex by endogenous PMN-derived ROI and an overall protective effect of ROI scavengers/antioxidants.
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PMID:Endogenous PMN-derived reactive oxygen intermediates provide feedback regulation on respiratory burst signal transduction. 926 41

Functional analysis of the immunoreceptor tyrosine-based activation motif (ITAM) derived from the membrane-proximal ITAM of CD3zeta demonstrates that mutations at either the tyrosine or leucine residues in the N-terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C-terminal YxxL segment abrogate signals for interleukin (IL)-2 production but do not prevent tyrosine phosphorylation of the N-terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C-gamma1 or calcium mobilization. Cross-linking of chimeric receptors containing a C-terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL-2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL-2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N-terminal YxxL segment reduces the receptor cross-linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross-linking required to trigger tyrosine phosphorylation-dependent signaling events.
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PMID:Functional analysis of immunoreceptor tyrosine-based activation motif (ITAM)-mediated signal transduction: the two YxxL segments within a single CD3zeta-ITAM are functionally distinct. 929 38

Two active site histidine residues have been implicated in the catalysis of phosphatidylinositol-specific phospholipase C (PI-PLC). In this report, we present the first study of the pKa values of histidines of a PI-PLC. All six histidines of Bacillus cereus PI-PLC were studied by 2D NMR spectroscopy and site-directed mutagenesis. The protein was selectively labeled with 13C epsilon 1-histidine. A series of 1H-13C HSQC NMR spectra were acquired over a pH range of 4.0-9.0. Five of the six histidines have been individually substituted with alanine to aid the resonance assignments in the NMR spectra. Overall, the remaining histidines in the mutants show little chemical shift changes in the 1H-13C HSQC spectra, indicating that the alanine substitution has no effect on the tertiary structure of the protein. H32A and H82A mutants are inactive enzymes, while H92A and H61A are fully active, and H81A retains about 15% of the wild-type activity. The active site histidines, His32 and His82, display pKa values of 7.6 and 6.9, respectively. His92 and His227 exhibit pKa values of 5.4 and 6.9. His61 and His81 do not titrate over the pH range studied. These values are consistent with the crystal structure data, which shows that His92 and His227 are on the surface of the protein, whereas His61 and His81 are buried. The pKa value of 6.9 corroborates the hypothesis of His82 acting as a general acid in the catalysis. His32 is essential to enzyme activity, but its putative role as the general base is in question due to its relatively high pKa.
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PMID:Determination of pKa values of the histidine side chains of phosphatidylinositol-specific phospholipase C from Bacillus cereus by NMR spectroscopy and site-directed mutagenesis. 930 Apr 93

The involvement of basic residues of interleukin(IL)-8 receptors in coupling to the Gi and G16 proteins was investigated by using a series of IL-8 receptor mutants. Substitution of the basic amino acids in the third inner loop of the receptor does not alter the abilities of the receptor mutants to activate recombinant Galpha16 or phosphoinositide-specific phospholipase C (PLC) beta2 expressed in COS-7 cells. However, an IL-8 receptor mutant with double mutations at residues Lys158 and Arg159 of the second inner loop loses its abilities to activate Galpha16 but retains its ability to activate PLC beta2. The activation of PLC beta2 by an IL-8 receptor that is sensitive to pertussis toxin has been previously demonstrated to be mediated through Gbetagamma. Surprisingly, the IL-8 receptor mutants with substitution of Ala for either residue Lys158 or Arg159 can still activate Galpha16, which suggests that either of the two basic residues in the second inner loop of the IL-8 receptor is sufficient for Galpha16 coupling.
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PMID:Two basic amino acids in the second inner loop of the interleukin-8 receptor are essential for Galpha16 coupling. 931 98


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