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)

In this review, the signal events regulated by angiotensin II (AngII) in vascular smooth muscle are analyzed based on activation of specific tyrosine kinases. AngII has been shown to play a critical role in the pathogenesis of hypertension, inflammation, atherosclerosis, and congestive heart failure. The expanding role of AngII indicates that multiple signal transduction pathways are likely to be activated in a tissue-specific manner. Although at least three AngII receptors have been characterized, it seems that the AngII type I receptor (AT1R) is physiologically most important since pharmacologic inhibitors of the AT1R block most AngII signal events and have beneficial effects on cardiovascular disease. The AT1R is a seven transmembrane-spanning G protein-coupled receptor that regulates intracellular signal events by activation of Gq and Gi. However, many recent data indicate that activation of tyrosine kinases by several different mechanisms contributes to AngII effects in target tissues. Tyrosine kinases activated by AngII include c-Src, focal adhesion kinase (FAK), Pyk2 (CADTK), Janus kinases (JAK2 and TYK2), and the receptor tyrosine kinases Ax1, epidermal growth factor, and platelet-derived growth factor. Finally, unknown tyrosine kinases may mediate tyrosine phosphorylation of paxillin, Shc, Raf, and phospholipase C-gamma after AngII stimulation. These AngII-regulated tyrosine kinases seem to be required for AngII effects such as vasoconstriction, proto-oncogene expression, and protein synthesis based on studies with tyrosine kinase inhibitors. Thus, understanding AngII-stimulated signaling events, especially those related to tyrosine kinase activity, may form the basis for the development of new therapies for cardiovascular diseases.
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PMID:Angiotensin II signal transduction in vascular smooth muscle: pathways activated by specific tyrosine kinases. 989 42

A calcium-sensing receptor (CaR) has functionally been described in the cortical thick ascending limb of Henle's loop (CTAL) of rat and mouse. This G protein-coupled receptor activates phospholipase C and increases the intracellular Ca2+ concentration. We observed that in the mouse CTAL cAMP formation, induced by 10(-8) mol/l AVP, was inhibited by more than 90% when the extracellular Ca2+ concentration ([Ca2+]e) was increased from 0.5 to 3 mmol/l. Measurements of transepithelial potential difference (PDte) in rat and mouse CTAL and medullary thick ascending limb (mTAL) segments and of transepithelial ion net fluxes in the mouse CTAL (isotonic perfusion conditions: 150 mmol/l NaCl in the lumen and bath) showed that an increase in the [Ca2+]e had no effect on basal and arginine vasopressin (AVP, 10(-10) mol/l)-stimulated transepithelial PDte, NaCl and Mg2+ transport. However, Ca2+ reabsorption was strongly inhibited by increased [Ca2+]e. Addition of AVP reversed this inhibitory effect of increased [Ca2+]e. Under hypotonic perfusion conditions (lumen 50 mmol/l NaCl; bath 150 mmol/l NaCl), a high [Ca2+]e induced a 50% decrease in Mg2+ reabsorption which was restored by AVP. Under these conditions, the effects on Ca2+ transport described above were still observed. In conclusion, activation of the CaR in the mouse TAL has no effect on basal and AVP-stimulated transepithelial NaCl reabsorption despite its large inhibitory effect on cAMP synthesis. The CaR, however, could play a role in the regulation of transepithelial Ca2+ and Mg2+ reabsorption.
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PMID:Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle's loop. 993 24

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.
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PMID:Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets. 1002 22

The P2Y1 receptor is a membrane-bound G protein-coupled receptor stimulated by adenine nucleotides. Using alanine scanning mutagenesis, the role in receptor activation of charged amino acids (Asp, Glu, Lys, and Arg) and cysteines in the extracellular loops (EL) of the human P2Y1 receptor has been investigated. The mutant receptors were expressed in COS-7 cells and measured for stimulation of phospholipase C induced by the potent agonist 2-methylthioadenosine-5'-diphosphate (2-MeSADP). In addition to single point mutations, all receptors carried the hemagglutinin epitope at the N- terminus for detection of cell-surface expression. The C124A and C202A mutations, located near the exofacial end of transmembrane helix 3 and in EL2, respectively, ablated phospholipase C stimulation by </=100 microM 2-MeSADP. Surface enzyme-linked immunosorbent assay detection of both mutant receptors showed <10% expression, suggesting that a critical disulfide bridge between EL2 and the upper part of transmembrane 3, as found in many other G protein-coupled receptors, is required for proper trafficking of the P2Y1 receptor to the cell surface. In contrast, the C42A and C296A mutant receptors (located in the N-terminal domain and EL3) were activated by 2-MeSADP, but the EC50 values were >1000-fold greater than for the wild-type receptor. The double mutant receptor C42A/C296A exhibited no additive shift in the concentration-response curve for 2-MeSADP. These data suggest that Cys42 and Cys296 form another disulfide bridge in the extracellular region, which is critical for activation. Replacement of charged amino acids produced only minor changes in receptor activation, with two remarkable exceptions. The E209A mutant receptor (EL2) exhibited a >1000-fold shift in EC50. However, if Glu209 were substituted with amino acids capable of hydrogen bonding (Asp, Gln, or Arg), the mutant receptors responded like the wild-type receptor. Arg287 in EL3 was impaired similarly to Glu209 when substituted by alanine. Substitution of Arg287 by lysine, another positively charged residue, failed to fully restore wild-type activity.
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PMID:The role of amino acids in extracellular loops of the human P2Y1 receptor in surface expression and activation processes. 1032 57

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR.CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.
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PMID:Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site interact with the penultimate aspartic acid and the C-terminal amide of cholecystokinin. 1040 Jun 73

The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Metabolic labeling of human embryonic kidney (HEK) 293 cells stably overexpressing the hemagluttinin epitope-tagged IP in the presence of [(3)H]mevalonolactone established that the mouse IP is isoprenylated. Studies involving in vitro assays confirmed that recombinant forms of the human and mouse IP are modified by carbon 15 farnesyl isoprenoids. Disruption of isoprenylation, by site-directed mutagenesis of Cys(414) to Ser(414), within the CAAX motif, abolished isoprenylation of IP(SSLC) both in vitro and in transfected cells. Scatchard analysis of the wild type (IP) and mutant (IP(SSLC)) receptor confirmed that each receptor exhibited high and low affinity binding sites for [(3)H]iloprost, which were not influenced by receptor isoprenylation. Whereas stable cell lines overexpressing IP generated significant agonist (iloprost and cicaprost)-mediated increases in cAMP relative to nontransfected cells, cAMP generation by IP(SSLC) cells was not significantly different from the control, nontransfected HEK 293 cells. Moreover, co-expression of the alpha (alpha) subunit of Gs generated significant augmentations in cAMP by IP but not by IP(SSLC) cells. Whereas IP also demonstrated significant, dose-dependent increases in [Ca(2+)](i) in response to iloprost or cicaprost compared with the nontransfected HEK 293 cells, mobilization of [Ca(2+)](i) by IP(SSLC) was significantly impaired. Co-transfection of cells with either Galpha(q) or Galpha(11) resulted in significant augmentation of agonist-mediated [Ca(2+)](i) mobilization by IP cells but not by IP(SSLC) cells or by the control, HEK 293 cells. In addition, inhibition of isoprenylation by lovastatin treatment significantly reduced agonist-mediated cAMP generation by IP in comparison to the nonisoprenylated beta(2) adrenergic receptor or nontreated cells. Hence, isoprenylation of IP does not influence ligand binding but is required for efficient coupling to the effectors adenylyl cyclase and phospholipase C.
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PMID:The prostacyclin receptor is isoprenylated. Isoprenylation is required for efficient receptor-effector coupling. 1044 29

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

As reports on G protein-coupled receptor signal transduction mechanisms continue to emphasize potential differences in signaling due to relative receptor levels and cell type specificities, the need to study endogenously expressed receptors in appropriate model systems becomes increasingly important. Here we examine signal transduction mechanisms mediated by endogenous kappa-opioid receptors in C6 glioma cells, an astrocytic model system. We find that the kappa-opioid receptor-selective agonist U69,593 stimulates phospholipase C activity, extracellular signal-regulated kinase 1/2 phosphorylation, PYK2 phosphorylation, and DNA synthesis. U69,593-stimulated extracellular signal-regulated kinase 1/2 phosphorylation is shown to be upstream of DNA synthesis as inhibition of signaling components such as pertussis toxin-sensitive G proteins, L-type Ca2+ channels, phospholipase C, intracellular Ca2+ release, protein kinase C, and mitogen-activated protein or extracellular signal-regulated kinase kinase blocks both of these downstream events. In addition, by overexpressing dominant-negative or sequestering mutants, we provide evidence that extracellular signal-regulated kinase 1/2 phosphorylation is Ras-dependent and transduced by Gbetagamma subunits. In summary, we have delineated major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa-opioid receptor in C6 glioma cells.
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PMID:Mitogenic signaling via endogenous kappa-opioid receptors in C6 glioma cells: evidence for the involvement of protein kinase C and the mitogen-activated protein kinase signaling cascade. 1064 7

In order to investigate the molecular mechanism of calcium signaling pathways common to the vertebrate gustatory systems, we have analyzed the expression of their molecular components. We first identified a phospholipase C (PLC) beta subtype expressed in the taste buds of pond loach (Misgurnus anguillicaudatus), designated DPLCbeta2, which is closely related to mammalian PLCbeta2 shown recently to be expressed in rat taste buds. The taste bud-specific expression of PLCbeta2 in a fish species as well as rat strongly suggests that PLCbeta2 mediates the tastant-induced second messenger response in taste buds, which is common to vertebrates. Next, we examined the correlation of gene expression of the candidate components leading to PLCbeta2 activation in rat circumvallate papillae, including G proteins, G(i2) and gustducin, and a G protein-coupled receptor, TR2. As a result, it was shown that the mRNAs for PLCbeta2 and G(i2) co-exist in the same cells, and PLCbeta2- and G(i2)-positive cells include both gustducin-positive cells and TR2-positive cells. However, no correlation was found between the expressions of TR2 and gustducin as reported previously. Our results thus indicate that a taste transduction pathway comprising TR2, G(i2) and PLCbeta2 occurs in a subset of taste cells.
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PMID:Co-expression of calcium signaling components in vertebrate taste bud cells. 1072 34


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