Regulation of <i>N</i>-Formyl Peptide-Mediated Degranulation by Receptor Phosphorylation

Vines, Charlotte M.; Xue, Mei; Maestas, Diane C.; Cimino, Daniel F.; Prossnitz, Eric R.

doi:10.4049/jimmunol.169.12.6760

Cited by 18 publications

(24 citation statements)

References 22 publications

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“…5E. We attribute this variation to reported differences in measurements of TNF-␣ levels possibly due to differences in mast cell cultures, which has also been noted by others (26)(27)(28). These data were similar for huMC (n ϭ 3 donors) (data not shown).…”

Section: Complete Abrogation Of Ros In Wt and 5-lo-deficient Mbmmc Dosupporting

confidence: 77%

FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

Swindle

Coleman

DeLeo

et al. 2007

The Journal of Immunology

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We investigated the enzymes responsible for FcεRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcεRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcεRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91phox- or p47phox-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcεRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcεRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcεRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcεRI-dependent degranulation and cytokine production.

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Section: Complete Abrogation Of Ros In Wt and 5-lo-deficient Mbmmc Dosupporting

confidence: 77%

FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

Swindle

Coleman

DeLeo

et al. 2007

The Journal of Immunology

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“…ALEXA 546-6-pep (18) and ALEXA 488-GM1 (19) were synthesized as described previously. 6-pep-FITC was from Molecular Probes.…”

Section: Methodsmentioning

confidence: 99%

N-Formyl Peptide Receptors Cluster in an Active Raft-associated State Prior to Phosphorylation

Xue¹,

Vines²,

Buranda

et al. 2004

Journal of Biological Chemistry

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In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G proteincoupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, ⌬ST-FPR, which lacks all phosphorylation sites in the carboxylterminal domain. We found that activation of the signaling-competent ⌬ST-FPR resulted in rapid receptor clustering on the plasma membrane independent of G i protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.A central question in understanding the process of cellular activation is defining the events that localize and restrict signaling activity at the membrane. This is of particular importance in chemotactic cells where cell polarization dictates the direction of movement. Activation of cell surface receptors initiates cellular signaling pathways that ultimately control cell polarity and migration. Polarization correlates with the redistribution of distinct lipid raft domains and their associated signaling molecules to the leading and trailing edges of the cell (1). However, at what point and even whether chemotactic receptors translocate into raft domains remain controversial (1, 2).Chemoattractant G protein-coupled receptors (GPCRs) 1 are seven transmembrane receptors that activate numerous cellular functions in part through the stimulation of heterotrimeric GTP-binding (G) proteins. Agonist binding to the GPCR triggers a conformational change within the receptor that facilitates G protein binding and activation. This binding leads to exchange of GDP for GTP on the G␣ subunit of the G protein followed by the dissociation of the ␣ subunit from the dimeric ␤/␥ subunit. These G protein subunits then regulate the activity of multiple cellular effectors such as adenylyl cyclase, phosphatidylinosit...

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“…Internalization-deficient mutants of PAFR were used in addition to transient transfections with dominant negative forms for arrestins 2 and 3 and dynamin to demonstrate this phenomenon. Moreover, Vines et al (38) demonstrated that the termination of fMLP-mediated degranulation in transfected RBL-2H3 cells was dependent on receptor phosphorylation and arrestin binding but independent of receptor internalization. We demonstrate here that BLT1 does internalize, and by different strategies we show the importance of BLT1 endocytosis for PLB-BLT cell degranulation.…”

Section: Discussionmentioning

confidence: 99%

Involvement of BLT1 Endocytosis and Yes Kinase Activation in Leukotriene B4-Induced Neutrophil Degranulation

Gaudreault

Thompson

Staňková

et al. 2005

The Journal of Immunology

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One of the important biological activities of human neutrophils is degranulation, which can be induced by leukotriene B4 (LTB4). Here we investigated the intracellular signaling events involved in neutrophil degranulation mediated by the high affinity LTB4 receptor, BLT1. Peripheral blood neutrophils as well as the promyeloid PLB-985 cell line, stably transfected with BLT1 cDNA and differentiated into a neutrophil-like cell phenotype, were used throughout this study. LTB4-induced enzyme release was inhibited by 50–80% when cells were pretreated with the pharmacological inhibitors of endocytosis sucrose, Con A and NH4Cl. In addition, transient transfection with a dominant negative form of dynamin (K44A) resulted in ∼70% inhibition of ligand-induced degranulation. Pretreating neutrophils or BLT1-expressing PLB-985 cells with the Src family kinase inhibitor PP1 resulted in a 30–60% inhibition in BLT1-mediated degranulation. Yes kinase, but not c-Src, Fgr, Hck, or Lyn, was found to exhibit up-regulated kinase activity after LTB4 stimulation. Moreover, BLT1 endocytosis was found to be necessary for Yes kinase activation in neutrophils. LTB4-induced degranulation was also sensitive to inhibition of PI3K. In contrast, it was not affected by inhibition of the mitogen-activated protein kinase MEK kinase, the Janus kinases, or the receptor tyrosine kinase epidermal growth factor receptor or platelet-derived growth factor receptor. Taken together, our results suggest an essential role for BLT1 endocytosis and Yes kinase activation in LTB4-mediated degranulation of human neutrophils.

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Regulation of N-Formyl Peptide-Mediated Degranulation by Receptor Phosphorylation

Cited by 18 publications

References 22 publications

FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

N-Formyl Peptide Receptors Cluster in an Active Raft-associated State Prior to Phosphorylation

Involvement of BLT1 Endocytosis and Yes Kinase Activation in Leukotriene B4-Induced Neutrophil Degranulation

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