Membrane rafts and signaling molecules associated with them are thought to play important roles in immunoreceptor signaling. Rafts differ in their lipid and protein compositions from the rest of the membrane and are relatively resistant to solubilization by Triton X-100 or similar detergents, producing buoyant, detergent-resistant membranes (DRMs) that can be isolated by density gradient ultracentrifugation. One of the key signaling molecules present in T cell DRMs is the transmembrane adaptor protein LAT (linker for activation of T cells). In contrast to previous results, a recent study demonstrated that a LAT construct not present in the buoyant DRMs is fully able to support TCR signaling and development of T cells in vivo. This finding caused doubts about the real physiological role of rafts in TCR signaling. In this study, we demonstrate that these results can be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization "heavy DRMs" containing a number of membrane molecules. At a moderate level of expression, LAT supported TCR signaling more efficiently than constructs targeted to the microdomains producing heavy DRMs or to nonraft membrane. We suggest that different types of membrane microdomains provide environments regulating the functional efficiencies of signaling molecules present therein.
- MeSH
- adaptorové proteiny signální transdukční imunologie metabolismus MeSH
- aktivace lymfocytů imunologie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové mikrodomény chemie imunologie MeSH
- membránové proteiny chemie imunologie izolace a purifikace metabolismus MeSH
- receptory antigenů T-buněk imunologie metabolismus MeSH
- signální transdukce imunologie MeSH
- T-lymfocyty chemie imunologie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.
- MeSH
- aktivace enzymů účinky léků genetika imunologie MeSH
- antigeny imunologie metabolismus farmakologie MeSH
- fosforylace účinky léků genetika imunologie MeSH
- inhibitory enzymů farmakologie MeSH
- krysa rodu rattus MeSH
- mastocyty imunologie metabolismus MeSH
- membránové mikrodomény genetika imunologie metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- oxidace-redukce účinky léků MeSH
- oxidancia farmakologie MeSH
- peroxid vodíku farmakologie MeSH
- receptory IgE genetika imunologie metabolismus MeSH
- skupina kinas odvozených od src-genu genetika imunologie metabolismus MeSH
- transport proteinů účinky léků genetika imunologie MeSH
- tyrosinfosfatasy antagonisté a inhibitory genetika imunologie metabolismus MeSH
- vanadáty farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- membránové mikrodomény imunologie MeSH
- receptory imunologické fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- techniky in vitro MeSH
