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 cellular basis of down-regulation and desensitization in phospholipase C-linked receptors is unclear. Recent studies with some receptors suggest that elements in the carboxyl terminus of the receptor are important in mediating these processes. Three mutant gastrin-releasing peptide receptors (GRP-R) were studied: one whose last 37 carboxyl-terminal amino acids were eliminated (construct MGT346); one that replaced all of the carboxyl-terminal Ser and Thr eliminated in MGT346 with Ala, Asn, or Gly (construct JF1); and one that selectively replaced the Ser and Thr of the protein kinase C consensus sequence (PKC-CS) located within the same region with alanine (construct TS360AA). Desensitization was assessed by measuring the ability to activate phospholipase C and increase cellular [3H]inositol phosphates, or increase [Ca2+]i, after pre-exposure to 3 nM bombesin for 24 h. Wild-type GRP-R was maximally desensitized and down-regulated after a 24-h exposure to 3 nM bombesin, and removal of the PKC-CS alone markedly attenuated each process. Elimination of additional serines and threonines by truncation (MGT346) or replacement (JF1) did not decrease down-regulation or desensitization further. To confirm the necessity of second messenger activation in mediating down-regulation, we further investigated two additional mutant GRP-R that bound agonist with high affinity but fail to activate phospholipase C (constructs R139G and A263E). Neither construct underwent significant down-regulation. Removal of all GRP-R carboxyl-terminal Ser or Thr, either by MGT346 or JF1, reduced internalization by > 80%, whereas elimination of the PKC-CS in TS360AA only attenuated internalization by 21 +/- 2%. These data suggest that activation of the distal carboxyl-terminal PKC-CS is essential for chronic desensitization and down-regulation of the GRP-R, and provide no evidence for involvement of second messenger-independent processes. In contrast, internalization is equally regulated by both second messenger-dependent and independent processes.
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PMID:Chronic desensitization and down-regulation of the gastrin-releasing peptide receptor are mediated by a protein kinase C-dependent mechanism. 785 20

4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K+ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4 beta-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] mimics the action of 4 beta-phorbol dibutyrate, but only in the presence of 2 microM arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1S,3R)-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca2+ channel or Ca(2+)-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1S,3R)-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1S,3R)-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.
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PMID:Glutamate exocytosis and MARCKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically activated by diacylglycerol and arachidonic acid. 793 Dec 82

Consequent to agonist exposure, many G protein-coupled receptors undergo sequestration or internalization. Results with receptors linked to adenylate cyclase, such as the beta 2-adrenergic receptor, or receptors linked to phospholipase C (PLC) have provided conflicting results regarding the role of second messenger-dependent (i.e., protein kinase A or C) and -independent (i.e., beta-adrenergic receptor kinase) kinases in mediating this process. Recent results for truncated and mutated gastrin-releasing peptide (GRP) receptors (GRP-R), as well as muscarinic cholinergic receptors, suggest that activation of protein kinase C may be needed for full receptor internalization. Nearly all G protein-coupled receptors studied to date, including the GRP-R, possess two highly conserved amino acids that are important in mediating receptor-G protein coupling to second messengers, i.e., arginine in the proximal second intracellular loop and alanine in the distal third intracellular loop. We selectively mutated each of these residues in the GRP-R to determine their importance for activation of PLC. Site-directed mutagenesis was performed to change arginine at position 139 to glycine (R139G mutant) and alanine at position 263 to glutamate (A263E mutant), with stable cell lines being created by transfection of the wild-type or mutated receptor cDNA into BALB/3T3 fibroblasts. Both R139G (Kd = 12.0 +/- 1.6 nM) and A263E (Kd = 12.2 +/- 1.7 nM) had a lower affinity for bombesin than did wild-type GRP-R (Kd = 1.4 +/- 0.4 nM); however, characteristic stoichiometries for the binding of agonists to this receptor were maintained equally in all three cell lines (bombesin > GRP >> neuromedin B). The wild-type GRP-R exposed to bombesin increased [3H]inositol phosphates (a measure of PLC activation) approximately 4-fold, with an EC50 of 5.1 +/- 2.2 nM. In contrast, [3H]inositol phosphates were not significantly increased in cells expressing R139G or A263E receptors, demonstrating that Arg139 and Ala263 are required for GRP-R activation of PLC. However, when receptor internalization at 37 degrees was assessed by ligand acid-stripping studies, 53 +/- 2% of A263E receptors were internalized at 90 min, compared with 85 +/- 5% of wild-type GRP-R, whereas only 10 +/- 3% of R139G receptors were internalized. Preincubation of either mutant cell line with 100 nM 12-O-tetradecanoylphorbol-13-acetate markedly increased internalization rates, such that at 90 min 62 +/- 2% of R139G receptors and 82 +/- 1% of A263E receptors were internalized.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes. 793 30

A plasma membrane rich fraction was prepared from olfactory rosettes of Atlantic salmon and used to study binding of L-glutamic acid and activation of phospholipase C (PLC). Glutamate binding was saturable, high affinity, and inhibited by aspartic acid and taurocholate but not by alanine and lysine. Binding of glutamate was potently inhibited by various ligands for rat brain metabotropic glutamate receptors (mGluR) and also by kainate and N-methyl-D-aspartate. Glutamate stimulated phosphatidylinositol 4,5-bisphosphate breakdown consistent with G protein-dependent activation of PLC. Northern blot analyses demonstrated the presence of olfactory rosette RNA that hybridizes with cDNA probes for mGluR1 and mGluR4 under low stringency conditions. The results indicate the salmon olfactory system includes a subtype of the metabotropic glutamate receptor family.
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PMID:A subtype of the metabotropic glutamate receptor family in the olfactory system of Atlantic salmon. 795 44

The platelet-activating factor (PAF) receptor couples with multiple signaling pathways such as activation of phospholipase C, phospholipase A2, and mitogen-activated protein kinase and the inhibition of adenylate cyclase. The PAF-induced signals are attenuated by repetitive or long standing applications of the agonist (homologous desensitization). To investigate mechanisms underlying the agonist-induced desensitization, we constructed mutant forms of the cloned guinea pig PAF receptor and stably expressed them in Chinese hamster ovary cells. The cells expressing the wild type receptor transiently activated phospholipase C in response to PAF. Intracellular inositol 1,4,5-trisphosphate level and intracellular Ca2+ concentration reached the maximal levels within 20 s and returned to the basal levels in several minutes, even in the continuous presence of the ligand. In contrast, a truncated PAF receptor lacking the carboxyl-terminal cytoplasmic tail induced sustained elevations of inositol 1,4,5-trisphosphate and intracellular Ca2+ concentrations. Similar findings were noted in another mutant, in which the Ser/Thr residues in the carboxyl-terminal tail were substituted with Ala. Both mutant PAF receptors more potently activated the other signals (mitogen-activated protein kinase kinase, arachidonate release, and inhibition of adenylate cyclase) than did the wild type receptor. Thus, while the carboxyl-terminal cytoplasmic tail of the PAF receptor is not required for the forward activation of multiple signals, it does have a critical role for signal attenuation induced by the agonist through phosphate accepters. We also noted that the synthetic peptide of the PAF receptor carboxyl-terminal tail was strongly phosphorylated by the recombinant beta-adrenergic receptor kinase 1, suggesting that it or its relatives might be involved in PAF receptor phosphorylation and homologous desensitization.
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PMID:Role of cytoplasmic tail phosphorylation sites of platelet-activating factor receptor in agonist-induced desensitization. 807 75

Receptor-mediated activation of phospholipase C (PLC) which releases diacylglycerol and inositol trisphosphate has been implicated in the action of gonadotropin-releasing hormone (GnRH) on gonadotrophs. Previously we demonstrated that the synthetic diacylglycerol, phorbol 12-myristate 13-acetate (PMA) and PLC mimic the stimulatory effects of GnRH on both luteinizing hormone (LH) glycosylation and release. In this study we further investigated how PMA or PLC interact with GnRH to control LH release versus glycosylation. Cultured pituitary cells were incubated in the presence of radiolabeled precursors and GnRH (0, 1, or 100 nM), with or without PMA (10 nM) or PLC (0.24 U/ml) for 4 h. LH translation and glycosylation were monitored by measuring incorporation of [14C]alanine and [3H]glucosamine, respectively, into total (cell and medium) immunoprecipitable LH. Immunoreactive LH (IRLH) was measured by radioimmunoassay. Both PMA and PLC increased (p < 0.01) basal IRLH release, and IRLH release stimulated by 1 nM GnRH. Neither PMA nor PLC exerted an additive effect on IRLH release stimulated by 100 nM GnRH. The interactions between PMA or PLC and GnRH on IRLH release were significant (p < 0.01). Both PMA and PLC elevated (p < 0.01) total [3H]glucosamine-LH, but had no additive effect with 1 nM GnRH; PLC depressed (p < 0.05) the stimulatory effect of 100 nM GnRH, whereas PMA had no effect. The interactions between PMA or PLC and GnRH on LH glycosylation were significant (p < 0.01). PMA, PLC or GnRH alone did not affect total [14C]alanine-LH. In the presence of 1 or 100 nM GnRH, PLC, but not PMA, decreased (p < 0.05) total [14C]alanine-LH.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential actions of phospholipase C on gonadotropin-releasing-hormone-stimulated release and glycosylation of luteinizing hormone in rat anterior pituitary cells. 809 Feb 83

The primary structure of toxin III of Leiurus quinquestriatus quinquestriatus (Lqq III) was elucidated by automatic Edman degradation of the reduced and S-carboxymethylated protein and derived tryptic peptides. Like other scorpion toxins that are active on sodium channels, Lqq III, consisting of 64 amino acids, is a 7 kDa single-chain polypeptide crosslinked by four disulfide bridges. It belongs to the alpha-toxin group, as judged by competition experiments with 125I AaH II for binding to rat brain synaptosomes (K0.5 = 7 x 10(-7) M). Lqq III is the first alpha-toxin to be characterized that is highly toxic to mice [LD50 = 50 micrograms (7.1 nmol)/kg body wt], by subcutaneous injection, insects Blatella germanica [LD50 = 60 ng (8.5 pmol)/g body wt.] and Musca domestica [LD50 = 120 ng (17 pmol)/g body wt]. When tested via the intracerebroventricular route, the toxicity for mice [55 micrograms (8 nmol)/kg] was of the same order as that found by subcutaneous injection, indicating that Lqq III has a higher affinity for peripheral sodium channels that for those of the central nervous system. There are three differences between the sequences of Lqq III and Lqh alpha IT, an alpha-toxin isolated from the venom of Leiurus quinquestriatus hebraeus. These substitutions are found at positions 20, 24, and 64 (Ser-->Ala,Asp-->Glu and His-->Arg, respectively). Surprisingly Lqh alpha IT is only weakly active in mice [LD50 = 5 mg (0.7 mumol)/kg], while in insects its toxicity is similar to that of Lqq III [140 ng (20 pmol)/g body wt blowfly larvae]. These observations are relevant to the definition of scorpion toxin structure-activity relationships.
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PMID:Characterization of toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammals and insects. 816 52

The effects of bradykinin (BK) and angiotensin II (ANG II) were compared in cultured rat mesenteric arterial smooth muscle cells. BK and ANG II activated a phosphoinositide-specific phospholipase C, leading to the rapid release of [3H]inositol phosphates, an increase in intracellular calcium, and formation of sn-1,2-diacylglycerol (DAG). DAG formation was biphasic with a transient peak at 5 s followed by a sustained increase from 60 to 600 s. The BK-mediated increases in inositol triphosphate and DAG were dose dependent with half-maximal increases at concentrations of 5 and 2 nM, respectively. Both hormones were found to activate protein kinase C (PKC) as assessed by phosphorylation of the 68- to 72-kDa intracellular PKC substrate myristoylated alanine-rich C kinase substrate. However, despite similar phosphorylation of this substrate, only ANG II produced a significant increase in membrane-bound PKC activity. The mechanism accounting for the inability of BK to increase membrane-bound PKC activity is unclear. Our studies excluded differential translocation of PKC to the nuclear membrane, production of an inhibitor of membrane-bound PKC activity, and expression of BK and ANG II receptors on different cells as the mechanism. Vascular smooth muscle cells were found to express at least four different PKC isozymes: alpha, delta, zeta, and a faint band for epsilon. All of the isozymes except zeta-PKC were translocated by treatment with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate. However, neither ANG II nor BK produced significant translocation of any measured isozyme; therefore, we could not exclude the possibility that ANG II and BK activate different isozymes of PKC. Both hormones were found to have a similar small and inconsistent effect in stimulating [3H]thymidine incorporation. These observations demonstrate that BK and ANG II have similar biochemical effects on vascular smooth muscle cells and imply that, in selected vessels, the vasodilatory effects of BK mediated by the endothelium may be partially counterbalanced by a vasoconstrictor effect on the underlying vascular smooth muscle cells.
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PMID:Bradykinin and angiotensin II: activation of protein kinase C in arterial smooth muscle. 820 4

The beta- but not the gamma- and delta-type isozymes of inositol phospholipid-specific phospholipase C (PLC) are activated by G protein alpha q and beta gamma subunits. The beta-type PLC isozymes differ from other isozymes in that they contain a long carboxyl-terminal region downstream of the Y catalytic domain and a region rich in acidic amino acids between the two separated X and Y catalytic domains. To determine the sites on PLC-beta 2 that participate in the interaction of the enzyme with alpha q and beta gamma subunits, we introduced specific truncations and substitutions in the PLC-beta 2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be activated by alpha q and beta gamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta 2 affected neither alpha q- nor beta gamma-dependent activation significantly. Carboxyl-terminal truncation to residue Gly-934 or to residue Ala-867 resulted in enzymes that were activated by beta gamma but not by alpha q. This result suggests that the carboxyl-terminal region of PLC-beta 2 is required for activation by alpha q, and that beta gamma subunits interact with a different region of the enzyme. Thus, alpha q and beta gamma subunits may independently modulate a single PLC-beta 2 molecule concurrently.
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PMID:Activation of phospholipase C-beta 2 mutants by G protein alpha q and beta gamma subunits. 824 28

The presence of high concentrations of membrane-bound carboxypeptidase M in human, baboon, dog, and rat lung was established by employing a variety of techniques. The activity of the enzyme in the membrane-enriched fractions of human, baboon, dog, and rat lung, measured with fluorescent dansyl substrate (DNS-Ala-Arg), was 198, 261, 484, and 153 nmol/h/mg protein, respectively. This activity in the lung was much higher than that found in the heart, liver, or kidney. The enzyme, optimally active around neutral pH, was completely inhibited by 10 microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and was activated by 1 mM CoCl2 to 170%. Antibody to human carboxypeptidase M immunoprecipitated the solubilized carboxypeptidase from human (98%), baboon (81%), and dog (88%) lung membrane fractions. Carboxypeptidase M is attached to lung membranes by a phosphatidylinositol glycan anchor; thus, it is released with bacterial phospholipase C. Membrane fractions from cultured human pulmonary arterial endothelial cells also contained high carboxypeptidase M activity (254 nmol/h/mg protein). A Northern blot of poly(A)+ RNA from various human tissues showed the presence of a high level of carboxypeptidase M mRNA in human lung and placenta. Finally, immunohistochemistry, employing purified antibody to the enzyme, revealed in fluorescent light microscopy that carboxypeptidase M is present in alveolar type I pneumocytes and in macrophages in apparently lower concentration. In contrast, type II alveolar epithelial cells gave negative results. Because carboxypeptidase M cleaves a variety of active peptides (e.g., bradykinin, anaphylatoxins), it may protect the alveolar surface from the effects of these peptides. In addition, carboxypeptidase M could be a marker enzyme for type I cells.
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PMID:High concentration of carboxypeptidase M in lungs: presence of the enzyme in alveolar type I cells. 833 89


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