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.

Engagement of the FcεRI 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 FcεRI-induced tyrosine phosphorylation of the FcεRI 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 FcεRI-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 Ca2+ 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 FcεRI-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.

Although aggregation of the Fc 4 receptor I (Fc 4 RI) is necessary for Ag-mediated mast cell triggering, the relationship between the extent of the Fc 4 RI aggregation and subsequent biochemical and topographical events is incompletely understood. In this study, we analyzed the activation events induced by Fc 4 RI dimers, elicited by binding of anti-Fc 4 RI mAb to rat basophilic leukemia cells. We found that, in contrast to extensively aggregated Fc 4 RI, receptor dimers (1) induced a less extensive association of Fc 4 RI with detergent-resistant membranes, (2) delayed the tyrosine phosphorylation and membrane recruitment of several signaling molecules, (3) triggered a slower but more sustained increase in concentration of free cytoplasmic calcium, (4) induced degranulation which was not inhibited at higher concentrations of the cross-linking mAb, and (5) failed to produce clusters of Fc 4 RI, Syk kinase and Grb2 adapter in osmiophilic membranes, as detected by immunogold electron microscopy on membrane sheets. Despite striking differences in the topography of Fc 4 RI dimers and multimers, biochemical differences were less pronounced. The combined data suggest that Fc 4 RI-activated mast cells propagate signals from small signaling domains formed around dimerized/oligomerized Fc 4 RI; formation of large Fc 4 RI aggregates in osmiophilic membranes seems to promote both strong receptor triggering and rapid termination of the signaling responses.

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