Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Receptors for the anaphylactic portion of complement, C5a, are not initially expressed in the monoblastic U937 cell line, but appear as the cell is induced to differentiate by the synergistic actions of 1,25(OH)2D and cyclic adenosine monophosphate (cAMP). Phorbol myristate acetate (PMA), which activates the protein kinase C pathway (PKC), does not cause C5a receptor (C5aR) expression when used as a single agent. The induction of C5aR by the synergistic actions of 1,25(OH)2D and cAMP, however, can be augmented as much as 180% by the addition of PMA. C5aR arising in cells exposed to 1,25(OH)2D and 8,4-chlorophenylthio-cAMP have an affinity constant of about 0.4 nM as assessed by cold competition analysis. We show here that when phorbol augmentation of receptor number occurs, the affinity constant is increased by 3.6-fold. In an effort to ascertain whether the change in C5aR Kd involved a PKC-dependent event we examined whether 5-60 min exposure of C5aR-positive cells to PMA would change C5aR Kd. Acutely, PMA caused a downregulation of receptor binding with decreases in apparent receptor number out of proportion to changes in Kd. One hundred nanomolar PMA, which effects nearly complete translocation of PKC to the membrane, consistently caused a 70-90% decrease in C5a surface binding. This downregulation was proportional to PMA dose and exposure time. Micromolar concentrations of the microtubule depolymerizing agents colchicine and vinblastine caused a less drastic downregulation, about 50% of the maximal phorbol effect. Our data suggest that activation of the PKC system might acutely limit the macrophage's ability to respond to C5a; chronically, phorbols upregulate receptor expression, most likely through positive effects on C5aR gene expression.
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PMID:Regulation of complement 5a receptor expression in U937 cells by phorbol ester. 166 Sep 14

U937 cells can be induced to express receptor for complement 5a (C5aR) by sequential 2 day treatments of cells with dihydroxyvitamin D-3 (1,25(OH)2D3) followed by prostaglandin E2. We asked whether the action of prostaglandin E2 to cause maximal C5aR expression required only activation of the cAMP-dependent protein kinase (PKA). Prostaglandin E2 dose dependently activated PKA in control and 1,25(OH)2D3 treated cells; by 4 h the PKA did not respond to further prostaglandin E2 challenge. We hypothesized that prostaglandin E2 actions transduced via PKA should be complete by 4 h; i.e., C5aR induction should be equivalent in cells treated with prostaglandin E2 for 4 h and for 2 days. All cells were treated for the first 2 days with 1,25(OH)2D3 and the second 2 days with prostaglandin E2 or cAMP analogs. C5aR number was measured after 4 days total culture. 4 h pulse treatments with agents were given at the end of the 1,25(OH)2D3 treatment. Cells exposed to a 4 h pulse of prostaglandin E2 had only 68.2 +/- 4.4% the amount of C5aR seen in cells continuously exposed to prostaglandin E2. Continuous culture with a cAMP analog pair (50 microM each of 8-thiomethyl-cAMP + N6-benzoyl-cAMP), which caused a 41.7% +/- 10.8% increase PKA activation above basal, resulted in only 51% +/- 16% of the C5aR numbers seen in cells cultured for 2 days with prostaglandin E2, where PKA remained at basal activity. We therefore concluded that C5aR expression caused by prostaglandin E2 could not be ascribed entirely to duration or degree of activation of cAMP-dependent signalling pathways. We investigated the possibility that the calcium sensitive protein kinase C was involved. Cytoplasmic protein kinase C was increased 154% +/- 14% above control in cells treated with sequential 2 days treatments of 1,25(OH)2D3 and prostaglandin E2. A 147% +/- 2% increase in membrane associated protein kinase C was also seen 10 min after phorbol myristate acetate stimulation in the above treatment group. Finally, phorbol myristate acetate augmented the C5aR induction caused by cAMP analog. We propose that the mechanism of prostaglandin E2 synergism with 1,25(OH)2D3 in causing C5aR induction in U937 cells includes signal transduction not only by the cAMP cascade, but also via protein kinase C modulated pathways.
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PMID:Prostaglandin E2 induction of monoblastic differentiation utilizes both cAMP and non-cAMP dependent signalling systems. 184 2

Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization. Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 12-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser314, Ser317, Ser327, Ser332, Ser334, and Ser338. Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser332 to Ser338 is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.
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PMID:Identification of the major phosphorylation sites in human C5a anaphylatoxin receptor in vivo. 764 84

Human neutrophils respond to chemoattractants, resulting in their accumulation at an inflammatory site. Chemoattractants such as the C5a peptide, derived from the C5 complement factor, bind to inhibitory guanine nucleotide binding protein (Gi)-coupled seven membrane-spanning receptors expressed in neutrophils. C5a receptor activation results in the Gi-dependent activation of the mitogen-activated protein (MAP) kinase pathway in human neutrophils. C5a receptor ligation activates both B-Raf and Raf-1, with B-Raf activation overlapping but temporally distinct from that of Raf-1. B-Raf and Raf-1 both efficiently phosphorylate MAP kinase kinase (MEK-1). C5a also stimulates guanine nucleotide exchange and activation of Ras. Ras and Raf activation in response to C5a involves protein kinase C-dependent and -independent pathways. Activation of both Raf-1 and B-Raf was inhibited by protein kinase A stimulation, consistent with the inhibitory effects of increased cAMP levels on neutrophil function. The findings define a functional signal transduction pathway linking the neutrophil C5a chemoattractant receptor to the regulation of Ras, B-Raf, Raf-1, and MAP kinase.
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PMID:Mapping of the C5a receptor signal transduction network in human neutrophils. 809 Jul 90

To define the regulation of chemoattractant receptors, epitope-tagged human formyl peptide and C5a receptor cDNAs (ET-FR and ET-C5aR) were stably expressed in rat basophilic leukemia, RBL-2H3 cells. An antibody (12CA5) specific to "ET" was used to immunoprecipitate ET-FR and ET-C5aR. fMLP and C5a caused time- and dose-dependent phosphorylation of their respective receptors. Phosphorylated ET-FR migrated as a single broad band between 50 and 70 kDa on SDS-polyacrylamide gel electrophoresis, whereas ET-C5aR exhibited both fast (39-45 kDa) and broadly (39-52 kDa) migrating forms. Fast form phosphorylation alone was observed at low concentrations of C5a (0.001-0.01 microM), or at early times (5-30 s) with a higher concentration of C5a (0.1 microM). Phorbol 12-myristate 13-acetate, thrombin, or antigen caused no phosphorylation of ET-FR but stimulated exclusively fast form phosphorylation of ET-C5aR. The protein kinase C inhibitor staurosporine did not inhibit phosphorylation of ET-FR but blocked the fast migrating component of phosphorylated ET-C5aR. Homologous desensitization correlated with ligand-induced phosphorylation of both receptors. Of note, ET-C5aR but not ET-FR underwent heterologous desensitization by antigen, phorbol 12-myristate 13-acetate, and thrombin. The data suggest that protein kinase C mediates heterologous phosphorylation and desensitization of C5aR but not FR, yet, both receptors are homologously desensitized by a staurosporine-resistant kinase.
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PMID:Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization. 822 71

Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.
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PMID:Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells. 846 87

The hemolytically inactive complement component complex C5b67, designated iC5b67, can signal human polymorphonuclear leukocytes (PMN) both as a pertussis toxin-inhibitable agonist for chemotaxis and as an antagonist for C5a- and FMLP-stimulated chemotaxis and superoxide production. The signaling pathways utilized by iC5b67 have been further investigated. In contrast to mastoparan, iC5b67 failed to directly activate G proteins to stimulate inositol phosphate formation in COS cells that had been transfected with G alpha 16. In COS cells co-transfected with both G alpha 16 and the C5a receptor, iC5b67 could neither activate phospholipase C nor inhibit C5a receptor-mediated activation of phospholipase C. iC5b67 stimulated GTPase activity in a membrane-enriched fraction from PMN. These data support the hypothesis that iC5b67 signals through a unique receptor, likely G protein linked, but distinct from the C5a receptor. iC5b67 was able to mobilize intracellular stores to elicit increases in intracellular Ca2+. Based on the effects of herbimycin A, wortmannin, and chelerythrine on iC5b67-induced PMN chemotaxis, iC5b67 signaling involved activation of tyrosine and phosphatidylinositol 3-kinases, but not protein kinase C. Relevant to the capacity of iC5b67 to antagonize PMN superoxide production, iC5b67 induced rapid and sustained increases in intracellular cAMP, which others have shown can inhibit superoxide formation. Although iC5b67 antagonizes C5a and FMLP receptor-mediated superoxide generation, iC5b67 had no effect on PMA-induced superoxide formation. The distinct agonist and antagonist signaling pathways activated by iC5b67 in the PMN diverge soon after initial iC5b67 receptor-mediated transduction steps.
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PMID:Signaling by hemolytically inactive C5b67, an agonist of polymorphonuclear leukocytes. 854 34

The biological effects of the potent inflammatory mediator C5a, a complement split product, on human neutrophils and monocytes are limited by the rapid internalization of its specific receptor (C5aR, CD88). The C terminus of the C5aR is phosphorylated after stimulation with C5a of phorbol ester, and this phosphorylation might lead to receptor internalization. In this context, we have studied the effects on C5aR internalization of C5a, phorbol 12-myristate 13-acetate (PMA), the protein kinase inhibitor staurosporine, and pertussis toxin on rat basophilic RBL.2H3 cells stably transfected with the human wild-type or mutant C5aR. C5aR mutants lacked either part of the cytosolic C terminus, including suggested major phosphorylation sites, or a putative phosphorylation motif for protein kinase C in the third cytosolic loop. Additionally, agonist-induced internalization was analyzed on HEK293 cells co-transfected with C5aR and the pertussis toxin-resistant G protein alpha subunit, G alpha 16. Staurosporine-sensitive agonist-dependent C5aR internalization could be detected, suggesting that C5aR phosphorylation, most likely of the C terminus, participates in this type of internalization. In contrast, PMA-induced C5aR internalization seems to be independent of putative phosphorylation sites in either the truncated section of the C terminus or the third cytosolic loop. The phorbol ester-induced C5aR internalization may, therefore, be caused by an indirect and less specific effect of protein kinase C on the internalization machinery. Manipulation of the pertussis toxin-sensitive or -resistant G protein-dependent signal transduction had no effect on ligand-induced internalization.
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PMID:The C terminus of the human C5a receptor (CD88) is required for normal ligand-dependent receptor internalization. 920 6

The C3a receptor (C3aR) is expressed on most human peripheral blood leukocytes with the exception of resting lymphocytes, implying a much higher pathophysiological relevance of the anaphylatoxin C3a as a proinflammatory mediator than previously thought. The response to this complement split product must be tightly regulated in situations with sustained complement activation to avoid deleterious effects caused by overactivated inflammatory cells. Receptor internalization, an important control mechanism described for G protein-coupled receptors, was investigated. Using rabbit polyclonal anti-serum directed against the C3aR second extracellular loop, a flow cytometry-based receptor internalization assay was developed. Within minutes of C3a addition to human granulocytes, C3aR almost completely disappeared from the cell surface. C3aR internalization could also be induced by PMA, an activator of protein kinase C. Similarly, monocytes, the human mast cell line HMC-1, and differentiated monocyte/macrophage-like U937-cells exhibited rapid agonist-dependent receptor internalization. Neither C5a nor FMLP stimulated any cross-internalization of the C3aR. On the contrary, costimulation of granulocytes with C5a, but not FMLP, drastically decreased C3aR internalization. This effect could be blocked by a C5aR-neutralizing mAb. HEK293-cells transfected with the C3aR, with or without Galpha16, a pertussis toxin-resistant G protein alpha subunit required for C3aR signal transduction in these cells, did not exhibit agonist-dependent C3aR internalization. Additionally, preincubation with pertussis toxin had no effect on C3a-induced internalization on PMNs. C3aR internalization is a rapid negative control mechanism and is influenced by the C5aR pathway.
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PMID:Modulation of C3a activity: internalization of the human C3a receptor and its inhibition by C5a. 1035 94

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


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