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: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human kidney 293 TSA cells were transfected by a calcium phosphate method with human formylpeptide and C5a receptor cDNAs with high efficiency. Formylpeptide receptor positive transfectants expressed a total of 968,000 +/- 34,000 receptors per cell with two affinity states (Kds of ca. 0.43 nM and 39 nM), which in the presence of 100 microM GTP gamma S decreased by ca. 4-fold the number of high-affinity sites. The ligand binding pharmacology of cloned and expressed formylpeptide receptors were indistinguishable from endogenous receptors on human neutrophils. Expressed formylpeptide and C5a receptors were functionally active in mobilizing intracellular calcium via a pertussis toxin sensitive mechanism with an ED50 for formylpeptide of ca. 0.5-1.0 nM. This expression system, in which receptor expression can be monitored by flow cytometric methods and in which intracellular calcium responses are measurable, unlike in the more popular COS-7 cell expression system, will provide a useful basis for the analysis of chemoattractant receptor structure-function relationships.
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PMID:Functional high efficiency expression of cloned leukocyte chemoattractant receptor cDNAs. 154 8

We have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a Kd of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the alpha and beta subunits of Gi, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm our earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.
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PMID:Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor-Gi complex. 189 85

C5a elicits a variety of responses from the polymorphonuclear leukocyte all of which utilize G proteins as transducing elements. In the present study, we report the consequences of the interaction between the C5a receptor and the G proteins and describe a system which may allow identification of the transducing proteins. C5a binding to polymorphonuclear leukocyte membranes is inhibited by pertussis, but not cholera, toxin and by a variety of guanine nucleotides. In the absence of nucleotide, we observed a single class of sites with a Kd of 17 pM. The presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) did not alter this affinity but did result in a concentration-dependent decrease in the number of binding sites. Surprisingly, we did not observe the concomitant appearance of a low affinity state implying that, if such a state exists, its affinity is below our limit of detection (5 nM). The receptor and G protein retained their functional interaction following solubilization of the membrane in digitonin. In the absence of nucleotide, we observed a single class of sites with a Kd of 28 pM. Addition of GTP gamma S suppressed binding, and, as was found in membranes, this inhibition is due almost entirely to a decrease in the number of sites. Again we failed to detect the appearance of a lower affinity state. Gel filtration studies of the detergent-solubilized receptor and receptor-C5a complexes indicate that the receptor is precoupled to G protein in the absence of ligand (C5a).
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PMID:Interaction between the C5a receptor and Gi in both the membrane-bound and detergent-solubilized states. 212 89

Platelet activating factor (PAF) and the active cleavage product of the fifth component of complement, C5a, are potent anaphylotoxins and mediators of inflammation. Both substances engage distinct guanine nucleotide binding regulatory protein-coupled receptors on a variety of cell types, thereby activating a signaling cascade that results in the mobilization of intracellular calcium stores, and in functional responses such as neutrophil chemotaxis and smooth muscle contraction. Little is known about the structure of PAF and C5a receptors or about the intracellular signaling pathways used by them. We have used the Xenopus oocyte expression system to demonstrate acquired C5a and PAF receptor activity in oocytes injected with mRNA from the promyelocytic leukemia cell line HL60 differentiated with dibutyryl cAMP. Activity was determined by measuring acquired ligand-dependent efflux of intracellular 45Ca2+ and by measuring ligand-activated transmembrane currents in voltage clamped oocytes. C5a receptor activity was confined to a single sharp peak in sucrose gradient fractionated RNA that corresponded to a transcript size of 2 kb. In contrast, PAF receptor activity was broadly distributed in size fractionated RNA from 3.5 to 6 kb. This suggests that multiple transcripts of different sizes may encode a functional PAF receptor. Both ligands activated their respective receptor in a concentration-dependent and a structure-dependent manner. The acquired C5a-dependent calcium efflux activity was inhibited by pertussis toxin whereas the PAF receptor activity was not, suggesting that the two receptors couple to different G-proteins. These data establish the Xenopus oocyte as a model system for studying the molecular and functional properties of the C5a receptor and the PAF receptor(s) of phagocytic cells.
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PMID:Characterization of human phagocytic cell receptors for C5A and platelet activating factor expressed in Xenopus oocytes. 216 18

On human mature monocytes the immunomodulator IFN-gamma has been shown to down-regulate the receptor for the anaphylatoxic peptide C5a (CD88, C5aR). In this study, we show that in immature myelo-/monoblastic U937, HL60, and MonoMac6 cells, IFN-gamma induces C5aR-ligand binding activity. In U937 cells, this induction cannot be blocked by the protein kinase C inhibitor staurosporine. An increase in free cytosolic Ca2+ upon ligand binding indicates functional coupling of this receptor in U937 and HL-60 cells. G-Proteins involved in this C5a responsiveness after IFN-gamma induction are completely pertussis toxin sensitive. Our data suggest that an additional pertussis toxin-resistant pathway exists in U937 cells after induction by dibutyryl cAMP. However, this is not due to changes in the mRNA level of the pertussis toxin-insensitive G-protein subunit G alpha 16. Induction by dibutyryl cAMP, but not that by IFN-gamma, resulted in C5a-dependent release of N-acetyl-beta-D-glucosaminidase, further highlighting functional differences in the effects of the inducers. Our data show an IFN-gamma-dependent increase in C5aR expression and suggest a maturation-related change in signaling of the C5aR, presumably at the level of receptor coupling.
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PMID:IFN-gamma up-regulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines. 759 3

The anaphylatoxin C5a receptor activates the Ras/Raf/mitogen-activated protein (MAP) kinase pathway in human neutrophils. The signal pathways involved in Ras/Raf/MAP kinase activation in response to C5a and other chemoattractant receptors is poorly understood. Stimulation of the C5a receptor expressed in HEK293 cells results in modest MAP kinase activation, which is inhibited by pertussis toxin-catalyzed ADP-ribosylation of G(i). Coexpression of the C5a receptor and the G16 alpha subunit (alpha 16) results in the G16-mediated activation of phospholipase C beta and a robust MAP kinase activation. Pertussis toxin treatment of C5a receptor/alpha 16-cotransfected cells inhibits C5a stimulation of MAP kinase activity approximately 60% relative to the control response. Similarly, the protein kinase C inhibitor, GF109203X inhibits activation of MAP kinase activation in C5a receptor/alpha 16-cotransfected cells by 60%; the protein kinase C inhibitor does not affect the modest C5a receptor response in the absence of alpha 16 expression. These results demonstrate that two independent signals are required for the maximal activation of MAP kinase by G protein-coupled receptors.
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PMID:Mitogen-activated protein kinase activation requires two signal inputs from the human anaphylatoxin C5a receptor. 764 93

Binding of activation peptide from the fifth component of C (C5a) to its receptor triggers events leading to both stimulation of cellular responses and receptor desensitization in myeloid cells. However, although transmission of a signal to pertussis toxin-sensitive G proteins is a prerequisite to neutrophil activation, we show that the process of receptor phosphorylation is mainly independent from activation of this pathway. Treatment of cells with pertussis toxin did not modify the incorporation of phosphate mediated by a saturating concentration of C5a, indicating that agonist-occupied C5aR can be fully phosphorylated, presumably by a specific G protein-coupled receptor kinase, in the absence of activation of the Gi protein. Receptor phosphorylation was transient, with a half-life of 30 to 40 min, which suggested a role for protein phosphatases in the regulation of the state of phosphorylation of C5aR in dHL60 cells. Pretreatment of cells with okadaic acid, an inhibitor of protein phosphatases 1 and 2A, increased the basal phosphorylation of unoccupied receptor and extended the phosphorylation mediated by C5a binding. Okadaic acid delayed, but did not suppress, the dephosphorylation process, which suggests either the involvement of additional phosphatase(s) or the degradation of nondephosphorylated receptors in the endocytic pathway. The data strongly suggest that internalized C5aR are recycled to the plasma membrane with a time course consistent with the kinetics of dephosphorylation. Dephosphorylation of C5aR might be essential to receptor recycling and resensitization during chemotaxis.
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PMID:Phosphorylation, dephosphorylation, and recycling of the C5a receptor in differentiated HL60 cells. 770 44

The key event in receptor-catalyzed activation of heterotrimer G proteins is binding of GTP, which leads to subunit dissociation generating GTP-bound alpha subunits and free beta gamma complexes. We have previously identified a mutation that abolished GTP binding in G alpha o (S47C) and demonstrated that the mutant retained the ability to bind beta gamma and could act in a dominant negative fashion when expressed in Xenopus oocytes (Slepak, V.Z., Quick, M.W., Aragay, A.M., Davidson, N., Lester, H.A., and Simon, M.I. (1993) J. Biol. Chem. 268, 21889-21894). In the current work, we investigated the effects of the homologous mutant of G alpha i2 (S48C) upon signaling pathways reconstituted in transiently transfected COS-7 cells. We found that expression of the G alpha i2 S48C mutant prevented stimulation of phospholipase C (PLC) beta 2 by free beta gamma subunit complexes. This effect of G alpha i S48C was not readily reversible in contrast to the inhibitory effect of wild-type G alpha i2, which could be reversed upon activation of the cotransfected muscarinic M2 receptor, presumably by release of beta gamma from the G protein heterotrimer. Coexpression of G alpha i S48C or the wild-type G alpha i2 also dramatically decreased G16-mediated stimulation of PLC by C5a in the cells transfected with cDNAs encoding C5a receptor and G alpha 16. Activation of PLC via endogenous Gq or G11 in the presence of alpha 1C adrenergic receptors was similarly attenuated by coexpression of G alpha i or G alpha i S48C. Pertussis toxin treatment of the transfected cells enhanced the inhibition of the receptor-stimulated PLC by wild-type G alpha i subunits but did not influence the effects of the dominant negative mutant. The enhancement of the wild-type G alpha i inhibitory effect by pertussis toxin can be explained by stabilization of G alpha i binding to beta gamma as a result of ADP-ribosylation, while G alpha i S48C mutant binds beta gamma irreversibly even without pertussis toxin treatment. Therefore, a feasible mechanism to rationalize the attenuation of the G alpha 16 and Gq/11-mediated activation of PLC by cotransfected G alpha i is the competition between G alpha i and G alpha 16 or Gq/11 for the beta gamma complexes, which are necessary for the G protein coupling with receptors. These experiments provide new evidence for the role of beta gamma in the integration of signals controlling phosphoinositide release through different G alpha families.
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PMID:Functional analysis of a dominant negative mutant of G alpha i2. 787 52

The human C5a receptor is known to signal through Gi proteins. The ability of the cloned C5a receptor to inhibit adenylyl cyclase or to stimulate phospholipase C through Gi proteins was examined in transfected cells. Activation of recombinant C5a receptors resulted in the stimulation of phospholipase C in Ltk- cells and inhibition of adenylyl cyclase in 293 cells. Pertussis toxin potently abolished both responses indicating the involvement of Gi proteins. Previous studies have shown that Gi-mediated inhibition of adenylyl cyclase can be similarly regulated by the pertussis toxin-insensitive GZ. In 293 cells co-transfected with the alpha subunit of GZ, the C5a-mediated inhibition of cAMP accumulation became pertussis toxin-resistant, signifying functional coupling between the C5a receptor and GZ. However, GZ cannot substitute for Gi in the C5a-induced stimulation of phospholipase C or inhibition of adenylyl cyclase in Ltk- cells.
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PMID:The human chemoattractant complement C5a receptor inhibits cyclic AMP accumulation through Gi and Gz proteins. 788 33

The signaling properties of the receptor for the chemoattractant C5a (C5aR) were investigated in differentiated U-937 cells and in NIH/3T3 cells transfected with the C5aR. In both U-937 cells and transfected cells (2A3 cells), C5a induced the mobilization of intracellular calcium, phosphoinositide breakdown, and activation of mitogen-activated protein kinase. In addition, in 2A3 cells C5a induced the inhibition of forskolin-stimulated cAMP generation. Pretreatment with pertussis toxin suppressed all C5a-mediated signal transduction in both cell lines. In the presence of cholera toxin, C5a induced the ribosylation of a 39-40-kDa protein in membranes of both U-937 cells and 2A3 cells. Similar phenomena have been described in other systems, whereby Gi alpha subunits are substrates for cholera toxin-induced ribosylation in the presence of receptor agonists. Moreover, the C5a-induced ribosylation was eliminated in membranes of cells that had been pretreated with pertussis toxin. The G protein alpha subunit G alpha 16, which is insensitive to pertussis toxin, has been reported to couple selectively to C5aR in cells co-transfected with C5aR and G alpha 16 cDNAs. G alpha 16 expression was not detected in U-937 cells or in 2A3 cells, either by reverse transcription-polymerase chain reaction or by immunoblotting. Because pertussis toxin modifies only G alpha subunits of the Gi/o family and all signaling by C5aR was abolished by pertussis toxin pretreatment, the results strongly suggest that, in U-937 and 2A3 cells, C5a-mediated responses can be accounted for entirely through coupling with G proteins of the Gi subtype.
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PMID:Coupling of the C5a receptor to Gi in U-937 cells and in cells transfected with C5a receptor cDNA. 796 69


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