Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteinase-activated receptor-2 (PAR-2) is the second member of a putative larger class of proteolytically activated receptors that mediate cell activation events by receptor cleavage or synthetic peptidomimetics corresponding to the newly generated N-terminus. To further study the previously identified mitogenic effects of PAR-2, we used the interleukin-3 (IL-3)-dependent murine lymphoid cell line, BaF3, for generation of stable cell lines expressing PAR-2 (BaF3/PAR-2) or the noncleavable PAR-2 mutant PAR-2(Arg36 --> Ala36). Only BaF3 cells expressing either wild-type or mutated receptor exhibited mitogenic responses when grown in IL-3-deficient media supplemented with PAR-2 activating peptide (SLIGRL, PAR39-44). This effect was dose dependent with an EC50 of approximately 80 micromol/L, sustained at 24, 48, and 72 hours, and was also demonstrable using thrombin receptor peptide TR42-47. Because tryptase shares approximately 70% homology with trypsin (previously shown to activate PAR-2), we studied recombinantly expressed forms of alpha- and beta-tryptases as candidate protease agonists for PAR-2. Hydrolytic activity of the chromogenic substrate tosyl-glycyl-prolyl-argly-4-nitroanilide acetate was present as a sharp peak at Mr approximately 130, confirming the presence of secretable and functionally active homotetrameric alpha- and beta-tryptases in transfected COS-1 cells. Dose-dependent proliferative responses were evident using either secreted form of tryptase with maximal responses seen at approximately 3 pmol/L (0.1 U/L). Receptor proteolysis was necessary and sufficient for mitogenesis because active site-blocked tryptase failed to induce this response, and proliferative responses were abrogated in BaF3 cells expressing PAR-2(Arg36 --> Ala36). These results specifically identify both forms of mast cell tryptases as serine protease agonists for PAR-2 and have implications for elucidating molecular mechanisms regulating cellular activation events mediated by proteases generated during inflammatory, fibrinolytic, or hemostatic-regulated pathways.
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PMID:Mitogenic responses mediated through the proteinase-activated receptor-2 are induced by expressed forms of mast cell alpha- or beta-tryptases. 935 58

Recent evidence suggests a critical role for Syk in mast cell activation upon high affinity IgE receptor (FcepsilonRI) aggregation. A rat basophilic leukemia cell line, RBL-2H3, expresses similar levels of two Syk isoforms that differ with respect to the presence of a 23-amino acid insert within the "linker" region located between the second Src homology 2 and the catalytic domain. Although they exhibit comparable intrinsic enzymatic activity, functional differences between the two isoforms are unknown. Here we report that the deleted Syk isoform can mediate signal transduction in RBL-2H3 cells. Aggregation of chimeric kinase, consisting of either form of Syk fused to the transmembrane and extracellular domains of guinea pig type II IgG Fc receptor, on RBL transfectants resulted in degranulation, release of leukotrienes, and enhanced gene expression of tumor necrosis factor-alpha. The chimeras as well as phospholipase C-gamma1 and Vav became tyrosine-phosphorylated upon aggregation of chimeras. We also found that both Syk isoforms from transiently transfected COS-7 cells were capable of binding to phosphorylated FcepsilonRI, and their kinase activities were similarly up-regulated in the presence of tyrosine-phosphorylated synthetic peptides based on the sequence of the gamma subunit of FcepsilonRI. Thus, these results establish that both isoforms of Syk can mediate signal transduction in mast cells and suggest that the 23-amino acid insert in the linker region of Syk may not be obligatory for FcepsilonRI signaling.
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PMID:Evidence for involvement of two isoforms of Syk protein-tyrosine kinase in signal transduction through the high affinity IgE receptor on rat basophilic leukemia cells. 960 11

Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.
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PMID:Phospholipase D1 localises to secretory granules and lysosomes and is plasma-membrane translocated on cellular stimulation. 966 93

Paired Ig-like receptors (PIR) are polymorphic type I transmembrane proteins belonging to an Ig superfamily encoded by multiple isotypic genes. They are expressed on immune cells such as mast cells, macrophages, and B lymphocytes. Two subtypes of PIR have been classified according to the difference in the primary structure of the PIR transmembrane and cytoplasmic regions. These subtypes are designated as PIR-A and PIR-B. In this study, the transmembrane and cytoplasmic regions of the PIR-A subtype were shown to mediate activation signal events such as cytoplasmic calcium mobilization, protein tyrosine phosphorylations, and degranulation in rat mast cell line RBL-2H3. The association of the Fc receptor gamma and beta subunits with PIR-A was shown to be responsible for PIR-A function but not required for membrane expression of PIR-A on COS-7 cells. We further revealed the role of two charged amino acid residues in the transmembrane region, namely arginine and glutamic acid, in PIR-A function and its association with the above subunits. In contrast to the inhibitory nature of the PIR-B subtype, present findings reveal that PIR-A potentially acts as a stimulatory receptor in mast cells, suggesting a mechanism for regulation of mast cell functions by the PIR family.
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PMID:Stimulatory function of paired immunoglobulin-like receptor-A in mast cell line by associating with subunits common to Fc receptors. 1051 23

Receptor-mediated signal transduction pathways of cells involved in allergy and inflammations are extremely significant. Lyn is a member of the Src family of non-receptor protein tyrosine kinases and is associated with a number of cell surface receptors, including the B-cell antigen receptor and immunoglobulin E receptor (FcepsilonRI). Lyn is necessary for FcepsilonRI-mediated mast cell activation. To investigate how the level of Lyn is maintained in mast cell activation, it was studied whether Lyn binds to ubiquitin and is ubiquitinated for proteasomal degradation in cells. In the yeast two hybrid system, Lyn specifically interacted with ubiquitin in vivo. Furthermore, Lyn bound to ubiquitin-conjugated Sepharose beads in vitro and was efficiently competed by soluble ubiquitin. Pulse-chase experiments indicated intracellular degradation of Lyn was associated with the generation of a high molecular weight complex in the presence of proteasome-specific inhibitor, lactacystin. This high molecular weight complex cross-reacted with anti-Lyn and anti-ubiquitin demonstrating the ubiquitination Lyn. Overexpression of Lyn and ubiquitin in COS 7.2 cells also resulted in the ubiquitination of Lyn in the presence of lactacystin, supporting the ubiquitination of Lyn by a proteasome specific pathway.
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PMID:Ubiquitination of Lyn-kinase in rat basophilic leukemia RBL-2H3 cells. 1113 37

Inoculation of KCMH-1 cells, a keratinocyte-derived cell line established from a chemically induced skin tumor, into the skin of mice results in accumulation of mast cells around the resulting tumors. The conditioned medium of KCMH-1 cells enhances the growth of mast cells in vitro when they are cultured in the presence of NIH/3T3 fibroblasts, suggesting an important role for keratinocytes in mast cell hyperplasia in the skin. The aim of this study was to identify this mast cell growth-enhancing factor (MCGEF) by screening a KCMH-1 cDNA library. We first established a polyclonal antibody raised against the partially purified factor obtained from KCMH-1-conditioned medium which neutralized the MCGEF activity in KCMH-1-conditioned medium. Expression cloning of 1 x 10(6) cDNAs from the KCMH-1 cDNA library led to 16 cDNAs. One of these cloned cDNAs was found to be leukemia inhibitory factor (LIF). Both LIF produced by COS cells and the recombinant protein obtained commercially showed MCGEF activity when added to mast cell/fibroblast cocultures. MCGEF activity in KCMH-1-conditioned medium was completely neutralized by an anti-LIF monoclonal antibody. These results suggest that MCGEF produced by KCMH-1 cells is identical to LIF.
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PMID:Identification of leukemia inhibitory factor as a potent mast cell growth-enhancing factor produced by mouse keratinocyte cell line, KCMH-1. 1128 77

Mutations of c-KIT causing spontaneous activation of the KIT receptor kinase are associated with sporadic adult human mastocytosis (SAHM) and with human gastrointestinal stromal tumors. We have classified KIT-activating mutations as either "enzymatic site" type (EST) mutations, affecting the structure of the catalytic portion of the kinase, or as "regulatory" type (RT) mutations, affecting regulation of an otherwise normal catalytic site. Using COS cells expressing wild-type or mutant KIT, 2 compounds, STI571 and SU9529, inhibited wild-type and RT mutant KIT at 0.1 to 1 microM but did not significantly inhibit the Asp816Val EST mutant associated with SAHM, even at 10 microM. Using 2 subclones of the HMC1 mast cell line, which both express KIT with an identical RT mutation but which differ in that one also expresses the Asp816Val EST mutation, both compounds inhibited the RT mutant KIT, thereby suppressing proliferation and producing apoptosis in the RT mutant-only cell line. Neither compound suppressed activation of Asp816Val EST mutant KIT, and neither produced apoptosis or significantly suppressed proliferation of the cell line expressing the Asp816Val mutation. These studies suggest that currently available KIT inhibitors may be useful in treating neoplastic cells expressing KIT activated by its natural ligand or by RT activating mutations such as gastrointestinal stromal tumors but that neither compound is likely to be effective against SAHM. Furthermore, these results help establish a general paradigm whereby classification of mutations affecting oncogenic enzymes as RT or EST may be useful in predicting tumor sensitivity or resistance to inhibitory drugs.
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PMID:The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. 1186 Dec 91

We have examined the specificity of oleate as an activator of phospholipase D2 (PLD2) and whether it can be used to study PLD2 localization and its involvement in cell function. Oleate stimulates PLD activity in intact RBL-2H3 mast cells. Comparing PLD1- with PLD2-overexpressing cells, oleate enhanced PLD activity only in PLD2-overexpressing cells. Membranes were also sensitive to oleate and when membranes prepared from PLD1- and PLD2-overexpressing cells were examined, oleate further increased PLD activity only in membranes from PLD2-overexpressing cells. Overexpressed green fluorescent protein (GFP)-PLD2 fusion protein was localized at the plasma membrane and GFP-PLD1 was found in an intracellular vesicular compartment. Oleate was used to examine whether overexpressed PLD2 co-localized with endogenous PLD2. RBL-2H3 mast cell homogenates were fractionated on a linear sucrose gradient and analysed for both oleate-stimulated activity and ADP ribosylation factor 1-stimulated PLD1 activity. The oleate-stimulated activity co-localized with markers of the plasma membrane including the beta-subunit of the FcepsilonRI and linker for activation of T cells. Fractionation of homogenates from PLD2-overexpressing cells demonstrated that the overexpressed PLD2 fractionated in an identical location to the endogenous oleate-stimulated activity and this activity was greatly enhanced in comparison with control membranes. Examination of membranes prepared from COS-7, Jurkat and HL60 cells indicated a relationship between oleate-stimulated PLD2 activity and PLD2 immunoreactivity. We examined whether oleate could be used to activate secretion and membrane ruffling in adherent RBL-2H3 mast cells. Oleate did not stimulate secretion but did stimulate membrane ruffling, which was short-lived. We conclude that oleic acid is a selective activator of PLD2 and can be used for localization studies, but its use as an activator of PLD2 in intact cells to study function is limited due to toxicity.
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PMID:Endogenous phospholipase D2 localizes to the plasma membrane of RBL-2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid. 1237 67

Adaptor protein 3BP2, a c-Abl-Src homology 3 (SH3) domain-binding protein, is known to play a regulatory role in T-cell receptor-mediated transcriptional activation of nuclear factor of activated T cell and activator protein 1 by interacting with Syk/ZAP-70 protein-tyrosine kinase. We have previously demonstrated that aggregation of high affinity IgE receptor (FcepsilonRI) induces tyrosine phosphorylation of 3BP2, and overexpression of the 3BP2-SH2 domain suppresses antigen-induced degranulation in rat basophilic leukemia RBL-2H3 mast cell line. In this report, we attempt to analyze the biological relevance of 3BP2 tyrosine phosphorylation. By using the transient expression system in COS-7 cells, we have demonstrated that 3BP2 was predominantly phosphorylated on Tyr174, Tyr183, and Tyr446 when it was coexpressed with Syk. An in vitro binding study revealed that phosphorylation of Tyr446 by Syk was likely to create a binding site for the Lyn-SH2 domain in RBL-2H3 cells. In addition, proline-rich region of 3BP2 bound to the Lyn-SH3 domain. Conformational microscopic analysis showed that Lyn and 3BP2 are constitutively colocalized in RBL-2H3 cells. Overexpression of 3BP2 in RBL-2H3 cells resulted in an enhancement of Lyn autophosphorylation. These results suggest that the adaptor protein 3BP2 is a potential regulator of Lyn protein-tyrosine kinase as a ligand of its SH3/SH2 domains in FcepsilonRI-mediated signaling in mast cells.
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PMID:Adaptor protein 3BP2 is a potential ligand of Src homology 2 and 3 domains of Lyn protein-tyrosine kinase. 1270 37

Four prostaglandin E (EP) receptor subtypes have been identified and cloned, designated as EP1, EP2, EP3 and EP4. These EP receptors are members of the G-protein coupled receptor family. EP3 receptor signals are primarily involved in inhibition of adenylyl cyclase via Gi activation, while EP2 and EP4 receptor signals cause a stimulation of adenylyl cyclase via Gs activation. Immune cells, such as mast cells, express multiple EP subtypes on their cell membranes, but few studies have been conducted to understand exactly what signals the main flow for the multiple subtypes expressing immune cells. We previously demonstrated that activation of Gi-coupled EP3 receptor exhibited a cooperative effect on cAMP synthesis induced by Gs-coupled EP2 receptor in COS-7 cells. Here we report that a selective EP4 agonist-induced adenylyl cyclase activity was augmented by simultaneous addition of a selective EP3 agonist in mastocytoma P-815 cells, which express mRNAs for both EP3 and EP4 subtypes. The augmentation in cAMP synthesis was found to be pertussis toxin-sensitive. P-815 cells are demonstrated to bind to Pronectin-F, a proteolytic fragment of fibronectin, in adhesion protein of the extracellular matrix, by addition of PGE2, which is mediated by PKA. The binding of P-815 cells to Pronectin-F mediated by EP4 receptor was augmented by the EP3 receptor. These findings indicate that two subtypes of PGE2 receptors, EP3 and EP4, cooperatively activate the cAMP-mediated adhesion event through induction of fibronectin ligand elicited by PGE2 in P-815 cells. Furthermore, the PGE2-induced adhesion response may contribute to the mast cell recruitment function on extracellular matrix during inflammation.
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PMID:[Cooperation of two subtypes of PGE2 receptor, Gi coupled EP3 and Gs coupled EP2 or EP4 subtype]. 1457 29


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