Peptidomimetics of Arg-Phe-NH2 as small molecule agonists of Mas-related gene C (MrgC) receptors

A series of Arg-Phe-NH₂ peptidomimetics containing an Arg mimetic were synthesized and tested as agonists of human MrgX1, rat MrgC, and mouse MrgC11 receptors. As predicted from the previously established species specificity, these peptidomimetics were found to be devoid of MrgX1 agonist activity. In contrast, these compounds acted as agonists of MrgC and/or MrgC11 with varying degrees of potency. These new peptidomimetics should complement the existing small molecule human MrgX1 agonists and enhance our ability to assess the therapeutic utility of targeting Mrg receptors in rodent models.     

Ceramide kinase is a mediator of calcium-dependent degranulation in mast cells

Ceramide kinase (CERK) catalyzes the conversion of ceramide to ceramide 1-phosphate (C1P) and is known to be activated by calcium. Although several groups have examined the functions of CERK and its product C1P, the functions of C1P and CERK are not understood. We studied the RBL-2H3 cell line, a widely used model for mast cells, and found that CERK and C1P are required for activation of the degranulation process in mast cells. We found that C1P formation was enhanced during activation induced by IgE/antigen or by Ca(2+) ionophore A23187. The formation of C1P required the intracellular elevation of Ca(2+). We generated RBL-2H3 cells that stably express CERK, and when these cells were treated with A23187, a concomitant C1P formation was observed and degranulation increased 4-fold, compared with mock transfectants. The cell-permeable N-acetylsphingosine (C(2)-ceramide), a poor substrate of CERK, inhibited both the formation of C1P and degranulation, indicating that C1P formation was necessary for degranulation. Exogenous introduction of CERK into permeabilized RBL-2H3 cells caused degranulation. We identified a cytosolic localization of CERK that provides exposure to cytosolic Ca(2+). Taken together, these results indicate that C1P formation is a necessary step in the degranulation pathway in RBL-2H3 cells.     

Ceramide induces serotonin release from RBL-2H3 mast cells through calcium mediated activation of phospholipase A2

Ceramide has been suggested to function as a mediator of exocytosis in response to the addition of a calcium ionophore from PC12 cells. Here, we show that although cell-permeable C(6)-ceramide or a calcium ionophore alone did not increase either the degranulation of serotonin or the release of arachidonic acid (AA) from RBL-2H3 cells, their combined effect significantly stimulated these processes in a time- and dose-dependent manner. This effect was inhibited by the presence of an exogenous calcium chelator and significantly suppressed by the CERK inhibitor (K1) and phospholipase A(2) (PLA(2)) inhibitors. Moreover, cytosolic PLA(2) GIVA (cPLA(2) GIVA) siRNA-transfected RBL-2H3 cells showed a lower level of serotonin release than scramble siRNA-transfected cells. Little is known about the regulation of degranulation proximal to the activation of cytosolic phospholipase A(2) GIVA, the initial rate-limiting step in RBL-2H3 cells. In this study, we suggest that CERK, ceramide-1-phosphate, and PLA(2) are involved in degranulation in a calcium-dependent manner. Inhibition of p44/p42 mitogen-activated protein kinase partially decreased the AA release, but did not affect degranulation. Furthermore, treatment of the cells with AA (ω-6, C20:4), not linoleic acid (ω-6, C18:2) or α-linolenic acid (ω-6, C18:3), induced degranulation. Taken together, these results suggest that ceramide is involved in mast cell degranulation via the calcium-mediated activation of PLA(2).     

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

Role of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion

It has been recently claimed that the human B1 receptors for kinins bind angiotensin-converting enzyme (ACE) inhibitors via a potential zinc-binding domain and are pharmacologically stimulated by these drugs. We verified whether ACE inhibitors stimulate B1 receptors in vitro. The isolated rabbit aorta or mouse stomach responded by negligible contractions to the application of captopril, enalaprilat, or zofenoprilat. The human isolated umbilical vein also failed to respond to enalaprilat. All of these preparations were responsive to the B1 receptor agonists des-Arg9-bradykinin (BK) or Lys-des-Arg9-BK. Furthermore, enalaprilat applied continuously had no significant interaction with the effects of Lys-des-Arg9-BK on the rabbit aorta. Enalaprilat failed to stimulate [3H]arachidonate release, translocate the receptors (confocal microscopy), or stimulate ERK1/2 phosphorylation (immunoblot) in HEK-293 cells stably expressing the rabbit B1 receptor conjugated to yellow fluorescent protein. The phospho-ERK1/2 content of arterial smooth muscle cells of human or rabbit origin was increased by treatment with Lys-des-Arg9-BK but not with enalaprilat. ACE inhibitors do not act as bona fide agonists of the kinin B1 receptors.     

P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.     

Orexin/hypocretin receptor chimaeras reveal structural features important for orexin peptide distinction

We wanted to analyze the basis for the distinction between OX(1) and OX(2) orexin receptors by the known agonists, orexin-A, orexin-B and Ala(11), D-Leu(15)-orexin-B, of which the latter two show some selectivity for OX(2). For this, chimaeric OX(1)/OX(2) and OX(2)/OX(1) orexin receptors were generated. The receptors were transiently expressed in HEK-293 cells, and potencies of the agonists to elicit cytosolic Ca(2+) elevation were measured. The results show that the N-terminal regions of the receptor are most important, and the exchange of the area from the C-terminal part of the transmembrane helix 2 to the transmembrane helix 4 is enough to lead to an almost total change of the receptor's ligand profile.     

Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcepsilonRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H(2)O(2)) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and beta-hexosaminidase, and the secretion of newly synthesized leukotriene C(4), whereas selective scavenging H(2)O(2) by ebselen impaired leukotriene C(4) secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H(2)O(2) caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cgamma and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.     

Enhancement of intracellular sphingosine-1-phosphate production by inositol 1,4,5-trisphosphate-evoked calcium mobilisation in HEK-293 cells: endogenous sphingosine-1-phosphate as a modulator of the calcium response

Sphingosine-1-phosphate (S1P) regulates many cellular functions, such as migration, differentiation and growth. The effects of S1P are thought to be primarily mediated by G-protein coupled receptors, but an intracellular function as a calcium releasing second messenger has also been proposed. Here we show that in HEK-293 cells, exogenous S1P mobilises sequestered calcium by a mechanism primarily dependent on the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3) pathway, and secondarily on the subsequent synthesis of intracellular S1P. Stimulating HEK-293 cells exogenously with S1P increased the production of both inositol phosphates and intracellular S1P. The calcium response was inhibited in cells treated with 2-APB, caffeine or U73122, showing that the PLC/IP3 pathway for calcium release is activated in response to exogenous S1P. The calcium response was partially inhibited in cells treated with the sphingosine kinase inhibitor DMS and in cells expressing a catalytically inactive sphingosine kinase, showing that endogenously produced S1P is also involved. Importantly, 2-APB and U73122 inhibited the S1P-evoked production of intracellular S1P. S1P is therefore not likely a major calcium releasing second messenger in HEK-293 cells, but rather a secondary regulator of calcium mobilisation.     

Synergistic activation of phospholipases Cgamma and Cbeta: a novel mechanism for PI3K-independent enhancement of FcepsilonRI-induced mast cell mediator release

Antigen/IgE-mediated mast cell activation via FcvarepsilonRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcvarepsilonRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxin-sensitive synergistic translocation to the membrane of phospholipase (PL)Cgamma and PLCbeta and to an enhancement of PLCgamma phosphorylation. This "trans-synergistic" activation of PLCbeta and gamma, in turn, enhanced production of inositol 1,4,5-trisphosphate, store-operated calcium entry, and activation of protein kinase C (PKC) (alpha and beta). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCgamma and PLCbeta that permits reinforcement of signals for degranulation in mast cells.     

Regulation of cytosolic phospholipase A2 phosphorylation by proteolytic cleavage of annexin A1 in activated mast cells

Annexin A1 (ANXA1) is cleaved at the N terminal in some activated cells, such as macrophages, neutrophils, and epithelial cells. We previously observed that ANXA1 was proteolytically cleaved in lung extracts prepared from a murine OVA-induced asthma model. However, the cleavage and regulatory mechanisms of ANXA1 in the allergic response remain unclear. In this study, we found that ANXA1 was cleaved in both Ag-induced activated rat basophilic leukemia 2H3 (RBL-2H3) cells and bone marrow-derived mast cells. This cleavage event was inhibited when intracellular Ca(2+) signaling was blocked. ANXA1-knockdown RBL-2H3 cells produced a greater amount of eicosanoids with simultaneous upregulation of cytosolic phospholipase A(2) (cPLA(2)) activity. However, there were no changes in degranulation activity or cytokine production in the knockdown cells. We also found that cPLA(2) interacted with either full-length or cleaved ANXA1 in activated mast cells. cPLA(2) mainly interacted with full-length ANXA1 in the cytosol and cleaved ANXA1 in the membrane fraction. In addition, introduction of a cleavage-resistant ANXA1 mutant had inhibitory effects on both the phosphorylation of cPLA(2) and release of eicosanoids during the activation of RBL-2H3 cells and bone marrow-derived mast cells. These data suggest that cleavage of ANXA1 causes proinflammatory reactions by increasing the phosphorylation of cPLA(2) and production of eicosanoids during mast-cell activation.     

Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells

Although cysteinyl leukotrienes (cysLTs) are known to be principal inflammatory lipid mediators released from IgE-stimulated mast cells, the signaling mechanisms involved in the synthesis of cysLTs remain largely unknown. In the present study, therefore, we investigated the signaling pathway by which IgE induces cysLTs synthesis after binding to its high affinity receptor (FcepsilonRI) in RBL-2H3 mast cells. We found that IgE-induced cysLT synthesis is completely abolished in RBL-2H3(Rac-N17) cells, a stable cell line expressing Rac(N17), a dominant negative Rac1 mutant; conversely, synthesis was enhanced in cells expressing Rac(V12), a constitutively active Rac1 mutant, suggesting that Rac1 is a key mediator of IgE signaling to cysLT synthesis. Further analysis aimed at identifying mediators downstream of Rac1 revealed that pretreating cells with a protein kinase C-delta (PKC-delta) inhibitor or infection with an adenoviral vector harboring a dominant negative PKC-delta mutant significantly attenuates IgE-induced ERKs phosphorylation, cytosolic phospholipase A(2) phosphorylation/translocation, and cysLT synthesis. In addition, the expression of Rac(N17) blocked PKC-delta translocation and impaired the phosphorylation of ERKs and cytosolic phospholipase A(2) otherwise elicited by IgE stimulation. Taken together these results suggest that PKC-delta also plays a critical mediatory role in the IgE signaling pathway leading to cysLT synthesis, acting downstream of Rac1. Finally, the physiological significance of PKC-delta in the IgE signaling pathway was demonstrated in an Ag (OVA)-challenged in vivo mouse model, in which induced levels of cysLTs and airway responsiveness in lung airways were significantly diminished by prior i.p. injection of a PKC-delta inhibitor.     

The role of calcium and protein kinase C in the IgE-dependent activation of phosphatidylcholine-specific phospholipase D in a rat mast (RBL 2H3) cell line.

Our previous studies have suggested that phosphatidylcholine-specific phospholipase D (PtdCho-PLD) plays a role in IgE-dependent diacylglycerol production, protein kinase C activation and mediator release in the RBL 2H3 mast cell line. We have extended these studies to examine the mechanisms by which PtdCho-PLD may be regulated in these cells. RBL 2H3 cellular lipids were labeled with [14C]arachidonic acid or [3H]myristic acid, then PtdCho-PLD activity was monitored by the formation of radiolabeled phosphatidylethanol when ethanol was included in the incubation medium. Trinitrophenol-ovalbumin conjugate (10 ng/ml), when added to cells previously sensitized with anti-(trinitrophenelated mouse IgE) (0.5 microgram/ml), ionomycin (1 microM) and thapsigargin (0.1 microM), stimulated PtdCho-PLD activation and mediator release in cells incubated in buffer containing 1.8 mM calcium, but not in cells incubated in calcium-free, buffer. Phorbol 12-myristate 13-acetate (0.1 microM) activated PtdCho-PLD in both buffers, but on its own did not trigger mediator release. When intracellular calcium was chelated with 5,5'-dimethyl-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, trinitrophenol-ovalbumin conjugate failed to activate PtdCho-PLD and histamine release. Similarly, down-regulation of protein kinase C activity by long-term exposure to the phorbol ester (0.1 microM) and preincubation of the cells with protein kinase inhibitors resulted in the loss of the trinitrophenol-ovalbumin response on PtdCho-PLD activity and histamine release. Taken together, the above results suggest that IgE-dependent PtdCho-PLD activation is dependent on both activation of protein kinase C and a rise in the intracellular free calcium concentration.     

Differential Effects of the Toll-Like Receptor 2 Agonists,PGN and Pam3CSK4 on Anti-IgE Induced Human Mast Cell Activation

Mast cells are pivotal in the pathogenesis of allergy and inflammation. In addition to the classical IgE-dependent mechanism involving crosslinking of the high-affinity receptor for IgE (FcεRI), mast cells are also activated by Toll-like receptors (TLRs) which are at the center of innate immunity. In this study, we demonstrated that the response of LAD2 cells (a human mast cell line) to anti-IgE was altered in the presence of the TLR2 agonists peptidoglycan (PGN) and tripalmitoyl-S-glycero-Cys-(Lys)4 (Pam3CSK4). Pretreatment of PGN and Pam3CSK4 inhibited anti-IgE induced calcium mobilization and degranulation without down-regulation of FcεRI expression. Pam3CSK4 but not PGN acted in synergy with anti-IgE for IL-8 release when the TLR2 agonist was added simultaneously with anti-IgE. Studies with inhibitors of key enzymes implicated in mast cell signaling revealed that the synergistic release of IL-8 induced by Pam3CSK4 and anti-IgE involved ERK and calcineurin signaling cascades. The differential modulations of anti-IgE induced mast cell activation by PGN and Pam3CSK4 suggest that dimerization of TLR2 with TLR1 or TLR6 produced different modulating actions on FcεRI mediated human mast cell activation.     

Complex formation with the Type B gamma-aminobutyric acid receptor affects the expression and signal transduction of the extracellular calcium-sensing receptor. Studies with HEK-293 cells and neurons

We co-immunoprecipitated the Ca(2+)-sensing receptor (CaR) and type B gamma-aminobutyric acid receptor (GABA-B-R) from human embryonic kidney (HEK)-293 cells expressing these receptors and from brain lysates where both receptors are present. CaRs extensively co-localized with the two subunits of the GABA-B-R (R1 and R2) in HEK-293 cell membranes and intracellular organelles. Coexpressing CaRs and GABA-B-R1s in HEK-293 cells suppressed the total cellular and cell surface expression of CaRs and inhibited phospholipase C activation in response to high extracellular [Ca(2+)] ([Ca(2+)](e)). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca(2+)](e). The latter effects of the GABA-B-R2 on the CaR were blunted by coexpressing the GABA-B-R1. Coexpressing the CaR with GABA-B-R1 or R2 enhanced the total cellular and cell surface expression of the GABA-B-R1 or R2, respectively. Studies with truncated CaRs indicated that the N-terminal extracellular domain of the CaR participated in the interaction of the CaR with the GABA-B-R1 and R2. In cultured mouse hippocampal neurons, CaRs co-localized with the GABA-B-R1 and R2. CaRs and GABA-B-R1s also co-immunoprecipitated from brain lysates. The expression of the CaR was increased in lysates from GABA-B-R1 knock-out mouse brains and in cultured hippocampal neurons with their GABA-B-R1 genes deleted in vitro. Thus, CaRs and GABA-B-R subunits can form heteromeric complexes in cells, and their interactions affect cell surface expression and signaling of CaR, which may contribute to extracellular Ca(2+)-dependent receptor activation in target tissues.     

Regulation of muscarinic acetylcholine receptor sequestration and function by beta-arrestin

After activation, agonist-occupied G protein-coupled receptors are phosphorylated by G protein-coupled receptor kinases and bind cytosolic beta-arrestins, which uncouple the receptors from their cognate G proteins. Recent studies on the beta2-adrenergic receptor have demonstrated that beta-arrestin also targets the receptors to clathrin-coated pits for subsequent internalization and activation of mitogen-activated protein kinases. We and others have previously shown that muscarinic acetylcholine receptors (mAChRs) of the m1, m3, and m4 subtype require functional dynamin to sequester into HEK-293 tsA201 cells, whereas m2 mAChRs sequester in a dynamin-independent manner. To investigate the role of beta-arrestin in mAChR sequestration, we determined the effect of overexpressing beta-arrestin-1 and the dominant-negative inhibitor of beta-arrestin-mediated receptor sequestration, beta-arrestin-1 V53D, on mAChR sequestration and function. Sequestration of m1, m3, and m4 mAChRs was suppressed by 60-75% in cells overexpressing beta-arrestin-1 V53D, whereas m2 mAChR sequestration was affected by less than 10%. In addition, overexpression of beta-arrestin-1 V53D as well as dynamin K44A significantly suppressed m1 mAChR-mediated activation of mitogen-activated protein kinases. Finally, we investigated whether mAChRs sequester into clathrin-coated vesicles by overexpressing Hub, a dominant-negative clathrin mutant. Although sequestration of m1, m3, and m4 mAChRs was inhibited by 50-70%, m2 mAChR sequestration was suppressed by less than 10%. We conclude that m1, m3, and m4 mAChRs expressed in HEK-293 tsA201 cells sequester into clathrin-coated vesicles in a beta-arrestin- and dynamin-dependent manner, whereas sequestration of m2 mAChRs in these cells is largely independent of these proteins.     

Spiro(indoline-3,4'-piperidine) growth hormone secretagogues as ghrelin mimetics.

A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4'-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.     

Rat basophilic leukemia cells (RBL-2H3) generate prostaglandin D2 (PGD2) after regulated upon activation, normal T-cell expressed and secreted (RANTES) activation

Increasing evidence indicates that local neurogenic inflammation, possibly in response to different stimuli, may be involved in sensory nerve sensitization, migraine generation and some other precipitating events leading to neuronal dysfunction in the brain. In addition, mast cells generate eicosanoids that are linked to asthma and other inflammatory diseases. Regulated upon activation, normal T-cell expressed and secreted (RANTES) is a small protein and a prototype member of the CC chemokine-beta subfamily with chemoattractant and inflammatory properties. In this study we used the RBL-2H3 cell line to determine whether or not these cells generate prostaglandin D2 (PGD2) after treatment with RANTES. After 4 hours of incubation, RBL-2H3 cells cultured with RANTES at 20 ng/mL released large amounts of PGD2 in a dose-response manner compared to control. Moreover, RBL-treated RANTES generated a large quantity of histamine. Our study confirms once again the proinflammatory action of RANTES, in this case acting on the stimulation of the arachidonic acid cascade product PGD2.     

Positive regulation of c-Jun N-terminal kinase and TNF-alpha production but not histamine release by SHP-1 in RBL-2H3 mast cells

The SH2-containing protein tyrosine phosphatase1 (SHP-1) is important for signaling from immune receptors. To investigate the role of SHP-1 in mast cells we overexpressed the wild-type and the phosphatase-inactive forms of SHP-1 in rat basophilic leukemia 2H3 (RBL-2H3) mast cell line. The phosphatase-inactive SHP-1 (C453S or D419A) retains its ability to bind tyrosine phosphorylated substrates and thereby competes with the endogenous wild-type enzyme. Overexpression of wild-type SHP-1 decreased the FcepsilonRI aggregation-induced tyrosine phosphorylation of the beta and gamma subunits of the receptor whereas the dominant negative SHP-1 enhanced phosphorylation. There were also similar changes in the tyrosine phosphorylation of Syk. However, receptor-induced histamine release in the cells expressing either wild-type or dominant negative SHP-1 was similar to that in the parental control cells. In contrast, compared with the parental RBL-2H3 cells, FcepsilonRI-induced c-Jun N-terminal kinase phosphorylation and the level of TNF-alpha mRNA was increased in the cells overexpressing wild-type SHP-1 whereas the dominant negative SHP-1 had the opposite effect. The substrate-trapping mutant SHP1/D419A identified pp25 and pp30 as two major potential substrates of SHP-1 in RBL-2H3 cells. Therefore, SHP-1 may play a role in allergy and inflammation by regulating mast cell cytokine production.