Binding of multivalent ligands to surface receptors induces receptor aggregation and subsequent biochemical pathways leading to cell activation. Our laboratory has been traditionally dealing with analysis of early molecular events in activated mast cells, which are involved in innate immunity, inflammatory diseases and allergy. These cells, can be triggered by aggregation of the high affinity receptor for IgE (FceRI). Binding of multivalent antigen to IgE anchored to FceRI results in rapid tyrosine phosphorylation of the beta and gamma subunits of the receptor and other signal transduction molecules in seconds after triggering. This is followed by activation and translocation of numerous signaling molecules leading to secretion of preformed mediators of allergy reactions (e. g. histamine) in minutes after cell triggering. More than a decade ago we have discovered that mast cells can also be activated by aggregation of glycosyl-phosphatidyl-inositol (GPI)-anchored protein Thy-1. Further studies indicated that Thy-1 and other GPI-anchored proteins are located in glycosphingolipid- and cholesterol-enriched plasma membrane microdomains, called lipid rafts. These microdomains have been suggested to play an important role in signaling not only via GPI-anchored proteins but also other surface receptors, including FceRI. We are interested in understanding composition and functional properties of these membrane microdomains in the course of mast cell activation.

     In order to better understand the complexity of lipid raft components, we prepared monoclonal antibodies against lipid rafts isolated from model mast cell line (RBL-2H3). One of the antibodies was found to recognize a novel GPI-anchored plasma membrane glycoprotein of 250 aminoacids, designated TEC-21, containing a cysteine-rich domain homologous to urokinase-plasminogen activator receptor/Ly-6/snake neurotoxin family. TEC-21 was found to be abundant on the surface of RBL-2H3 cells , but was absent in numerous rat tissues except for testes. Aggregation of TEC-21 induced a rapid increase in tyrosine phosphorylation of several substrates including Syk kinase and LAT adaptor, calcium flux, and release of secretory components. However, aggregation of TEC-21 did not induce changes in density of IgE-FceRI complexes, tyrosine phosphorylation of FceRI b and g subunits and co-aggregation of Lyn kinase. The combined data indicate that TEC-21 is a novel lipid raft component of RBL-2H3 cells whose aggregation induces cell activation independently of FceRI.

     In colaboration with Laboratory of Leukocyte Antigens we studies properties of a novel lipid raft transmembrane adapter protein NTAL (non-T cell activation linker). We have found that NTAL, which is located in lipid rafts, is rapidly tyrosine-phosphorylated upon FceRI aggregation in both RBL and bone marrow-derived mast cells.

     We have also analyzed molecular mechanisms of the inhibitory effect of gangliosides on FceRI-mediated mast cell activation. We have found that pretreatment of RBL-2H3 cells with isolated brain gangliosides inhibited degranulation from cells activated via FceRI but not Thy-1 glycoprotein. Exogenously administered gangliosides also inhibited formation of filamentous actin and production of phosphoinositides. Gangliosides had no or only marginal effect on the association of aggregated FceRI with lipid rafts, on tyrosine phosphorylation of FceRI and LAT adaptor. Though pretreatment with gangliosides did not inhibit the association of LAT with phospholipase C (PLC)g1 and PLCg2, tyrosine phosphorylation of these enzymes, as well as their enzymatic activities and association with detergent-insoluble signaling assemblies were reduced. This resulted in a decreased production of inositol 1,4,5-trisphosphate and an inhibition of Ca2+ mobilization. These data support the concept that exogenously administered gangliosides interfere with those properties of GEM that are important for the formation of plasma membrane-associated signaling assemblies containing PLCg but not for initial tyrosine phosphorylation of FceRI subunits.