Anti-NTAL Antibody [NAP-07] (A85921)

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.

Immunolabeling of isolated plasma membrane (PM) sheets combined with high-resolution electron microscopy is a powerful technique for understanding the topography of PM-bound signaling molecules. However, this technique has been mostly confined to analysis of membrane sheets from adherent cells. Here we present a rapid, simple and versatile method for isolation of PM sheets from non-adherent cells, and show its use for examination of the topography of Fcepsilon receptor I (FcepsilonRI) and transmembrane adaptors, LAT (linker for activation of T cells) and NTAL (non-T cell activation linker), in murine bone marrow-derived mast cells (BMMC). The data were compared with those obtained from widely used but tumor-derived rat basophilic leukemia (RBL) cells. In non-activated cells, FcepsilonRI was distributed either individually or in small clusters of comparable size in both cell types. In multivalent antigen-activated BMMC as well as RBL cells, FcepsilonRI was internalized to a similar extent, but, strikingly, internalization in BMMC was not preceded by formation of large (~200 nm) aggregates of FcepsilonRI, described previously in activated RBL cells. On the other hand, downstream adaptor proteins, LAT and NTAL, were localized in independent domains in both BMMC and RBL cells before and after FcepsilonRI triggering. The combined data demonstrate unexpected properties of FcepsilonRI signaling assemblies in BMMC and emphasize the importance of studies of PM sheets isolated from non-tumor cells.

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Linker for activation of B cell (LAB)/non-T cell activation linker (NTAL) and phosphoprotein associated with glycophospholipid-enriched membrane microdomain (PAG)/Csk-binding protein (Cbp) are raft-associated transmembrane adaptor proteins with distinct functions in immediate/early phases of receptor signaling pathways. Heterogeneous rafts are thought to compartmentalize membrane-associated signaling events. In order to investigate the subcellular localization of LAB/NTAL and PAG/Cbp, they were expressed as fluorescent chimeric fusion proteins in a human B cell line and their distribution was examined, along with the corresponding endogenous proteins, before and after B cell receptor (BCR) stimulation. Both adaptors were distributed predominantly at the plasma membrane in resting cells and co-clustered with other raft-associated proteins; however, they distributed differently in buoyant membranes isolated by either detergent resistance or non-detergent methods, indicating that they might localize to distinct rafts. After activation, LAB/NTAL was internalized and co-localized with the BCR while PAG/Cbp remained on the cell surface. BCR internalization was reduced in LAB/NTAL-deficient murine B cells, suggesting a regulatory role for LAB/NTAL in activation-induced internalization of the BCR. The cytoplasmic domain of LAB/NTAL, and not the transmembrane/juxtamembrane region, was found to be essential for its internalization.

Thy-1 (CD90) is a glycoprotein bound to the plasma membrane by a GPI anchor. Aggregation of Thy-1 in mast cells and basophils induces activation events independent of the expression of Fcepsilon receptor I (FcepsilonRI). Although we and others have previously suggested that plasma membrane microdomains called lipid rafts are implicated in both Thy-1 and FcepsilonRI signaling, properties of these microdomains are still poorly understood. In this study we used rat basophilic leukemia cells and their transfectants expressing both endogenous Thy-1.1 and exogenous Thy-1.2 genes and analyzed topography of the Thy-1 isoforms and Thy-1-induced signaling events. Light microscopy showed that both Thy-1 isoforms were in the plasma membrane distributed randomly and independently. Electron microscopy on isolated membrane sheets and fluorescence resonance energy transfer analysis indicated cross-talk between Thy-1 isoforms and between Thy-1 and FcepsilonRI. This cross-talk was dependent on actin filaments. Thy-1 aggregates colocalized with two transmembrane adaptor proteins, non-T cell activation linker (NTAL) and linker for activation of T cells (LAT), which had been shown to inhabit different membrane microdomains. Thy-1 aggregation led to tyrosine phosphorylation of these two adaptors. The combined data indicate that aggregated GPI-anchored proteins can attract different membrane proteins in different clusters and thus can trigger different signaling pathways.

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon 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 epsilon 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 epsilon 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 epsilon RI aggregation. Fc epsilon 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 epsilon 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.