Engagement of the Fcɛ receptor I (FcɛRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non–T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow–derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcɛRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C γ1 and phospholipase C γ2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain–containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid-and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca 2؉ . However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcRI does not exhibit an increased association with lipid rafts, suggest that FcRI phosphorylation and early activation events can be initiated outside of lipid rafts.The high-affinity immunoglobulin E (IgE) receptor (FcεRI)-mediated activation of mast cells and basophils triggers a cascade of intracellular biochemical events that ultimately lead to the secretion of preformed pharmacological agents and the transcription of cytokine genes. This process is initiated by aggregation of the receptor by means of multivalent antigen (Ag)-IgE complexes, followed by tyrosine phosphorylation of the receptor subunits by Src family protein tyrosine kinases (14, 31).FcεRI has a tetrameric structure comprised of an IgE-binding ␣ subunit, a ␤ subunit, and a disulfide-bonded ␥ dimer (32). The ␤ and ␥ subunits possess immunoreceptor tyrosine-based activation motifs (ITAMs), which are rapidly phosphorylated by protein tyrosine kinase Lyn. Tyrosine-phosphorylated ITAMs of the the ␥ subunits serve as novel binding sites for Src homology 2 (SH2) domains of Syk kinase (6, 22, 28), leading to phosphorylation and activation of Syk. Thereafter, a number of other signaling and adaptor molecules become phosphorylated and recruited into the regions of activated FcεRI/Syk complexes. These include PLC␥1 (26), the proto-oncogene product Vav (41), PKC-␦ (18), and the linker for activation of T cells (LAT) (37, 52).Detailed molecular mechanisms of the initial engagement of the Lyn kinase and FcεRI are not completely understood, but two different models have been proposed. One model, based on protein-protein interactions, postulates that a small fraction of Lyn is constitutively associated with the ␤ subunit of the FcεRI prior...

Aggregation of high-affinity receptors for immunoglobulin E (Fc⑀RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc⑀RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc⑀RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc⑀RI aggregation. Fc⑀RI-and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit-and Fc⑀RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation. IntroductionMast cell activation leading to degranulation, arachidonic acid metabolism, and cytokine production is initiated following antigendependent aggregation of high-affinity receptors for immunoglobulin E (IgE) (Fc⑀RI) on the cell surface. 1 Stem cell factor (SCF), although primarily required for the growth, differentiation, and survival of mast cells by binding to Kit,2,3 potentiates secretory responses elicited via the Fc⑀RI. 4 Both Fc⑀RI and Kit responses follow tyrosine kinase activation and subsequent protein tyrosine phosphorylation. 5,6 Fc⑀RI possess no inherent tyrosine kinase activity thus require the recruitment of the Src family tyrosine kinase, Lyn, and the zeta-associated protein 70 (ZAP 70)-related tyrosine kinase, spleen tyrosine kinase (Syk), into the signaling complex, where, following receptor aggregation, they become sequentially activated. 7,8 Subsequent tyrosine phosphorylation of the ␤ and ␥ chains of the Fc⑀RI, and of the transmembrane adaptor molecule linker for activated T cells (LAT), provides multiple docking sites for downstream Src homology 2 (SH2) domaincontaining signaling molecules. 9 These initial tyrosine-phosphorylation events appear to be crucial for subsequent Fc⑀RI-mediated degranulation to proceed.Unlike the Fc⑀RI, Kit does possess inherent tyrosine kinase activity. 10,11 Binding of SCF induces autophosphorylation of Kit with consequential binding of SH2 domain-containing signaling molecules to the Kit cytosolic domain. 12 In contrast to Fc⑀RI signaling, to date there is little evidence that LAT or similar transmembrane adaptor molecules become phosphorylated following Kit activation. Thus, despite, Kit's ability to potentiate Fc⑀RI-mediated ...

Migration of mast cells is essential for their recruitment within target tissues where they play an important role in innate and adaptive immune responses. These processes rely on the ability of mast cells to recognize appropriate chemotactic stimuli and react to them by a chemotactic response. Another level of intercellular communication is attained by production of chemoattractants by activated mast cells, which results in accumulation of mast cells and other hematopoietic cells at the sites of inflammation. Mast cells express numerous surface receptors for various ligands with properties of potent chemoattractants. They include the stem cell factor (SCF) recognized by c-Kit, antigen, which binds to immunoglobulin E (IgE) anchored to the high affinity IgE receptor (FcεRI), highly cytokinergic (HC) IgE recognized by FcεRI, lipid mediator sphingosine-1-phosphate (S1P), which binds to G protein-coupled receptors (GPCRs). Other large groups of chemoattractants are eicosanoids [prostaglandin E2 and D2, leukotriene (LT) B4, LTD4, and LTC4, and others] and chemokines (CC, CXC, C, and CX3C), which also bind to various GPCRs. Further noteworthy chemoattractants are isoforms of transforming growth factor (TGF) β1–3, which are sensitively recognized by TGF-β serine/threonine type I and II β receptors, adenosine, C1q, C3a, and C5a components of the complement, 5-hydroxytryptamine, neuroendocrine peptide catestatin, tumor necrosis factor-α, and others. Here we discuss the major types of chemoattractants recognized by mast cells, their target receptors, as well as signaling pathways they utilize. We also briefly deal with methods used for studies of mast cell chemotaxis and with ways of how these studies profited from the results obtained in other cellular systems.

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Mast cells are powerful immune modulators of the tissue microenvironment. Within seconds of activation, these cells release a variety of preformed biologically active products, followed by a wave of mediator synthesis and secretion. Increasing evidence suggests that an intricate network of inhibitory and activating receptors, specific signaling pathways, and adaptor proteins governs mast cell responsiveness to stimuli. Here, we discuss the biological and clinical relevance of negative and positive signaling modalities that control mast cell activation, with an emphasis on novel FcεRI regulators, immunoglobulin E (IgE)-independent pathways [e.g., Mas-related G protein-coupled receptor X2 (MRGPRX2)], tetraspanins, and the CD300 family of inhibitory and activating receptors.

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcɛ-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells.

Thy-1 is a surface glycoprotein that is attached to the plasma membrane by a glycosyl-phosphatidylinositol anchor. Crosslinking of Thy-1 in rat mast cells and basophilic leukemia cefls (RBL-2H3) induces cell activation including histamine release and tyrosine phosphorylation of several proteins. Here we show that glycosyl-phosphatidylinositol-linked Thy-1 forms noncovalent complexes with srcrelated protein-tyrosine kinase p53/p56'Yn and other proteintyrosine kinases and/or their substrates. These complexes are resistant to solubilization by a nonionic detergent, sedimentable at 200,000 x g, and very large (>10 MDa) as determined by gel chromatography. Activation of RBL-2H3 cells by crosslinking of the high-affinity IgE receptors resulted in decreased recovery of the complexes. The combined data indicate the existence of large detergent-resistant domains in the surface membrane of mast cells that may play an important role in their activation.