Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT.
Clustering of the glycosyl-phosphatidylinositol (GPI)-anchored protein Thy-1 on the cell surface leads to T cell activation. However, despite the similarity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinking, reportedly occurs in a manner independent of the TCR/CD3 complex. To investigate the relationship between responses resulting from Thy-1 or TCR engagement, a biochemically well defined system employing only affinity purified antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium influx and IL-2 production. By these criteria, anti-CD3 mAbs moderately activated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss mutant cell line, did not respond to stimulation through CD3 despite expressing all essential signaling molecules. Together these results emphasized the existence of a poorly appreciated mutual interdependence between Thy-1 and CD3 for efficient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These biochemical properties are identical to those described for a recently cloned adaptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyclonal Abs raised against a LAT-peptide (amino acids 103-131) specifically recognized the 40 kDa protein. LAT is present in microdomains of the plasma membrane enriched in sphingolipids, cholesterol, GPI-anchored proteins and a variety of signaling molecules. By contrast, the TCR/CD3 complex is excluded from these domains at least until stimulation takes place. Hence, we propose that Thy-1 promotes TCR/CD3 dependent signaling by facilitating LAT phosphorylation on tyrosine and the subsequent recruitment of downstream effector molecules.
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Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland. lleyton@eliot.unil.ch
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D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017252:CD3 Complex / Q000378:metabolism*; D020013:Calcium Signaling; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018800:Thy-1 Antigens / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D007376:Interleukin-2; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D018800:Thy-1 Antigens; D014443:Tyrosine