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 simi larity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinki ng, reportedly occurs in a manner independent of the TCR/CD3 complex. To in vestigate the relationship between responses resulting from Thy-1 or TCR en gagement, a biochemically well defined system employing only affinity purif ied 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 ac tivated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss m utant cell line, did not respond to stimulation through CD despite expressi ng 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 enhanc ed mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These bioche mical properties are identical to those described for a recently cloned ada ptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyc lonal Abs raised against a LAT-peptide (amino acids 103-131) specifically r ecognized 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 exclu ded from these domains at least until stimulation takes place. Hence, we pr opose that Thy-1 promotes TCR:CD3 dependent signaling by facilitating LAT p hosphorylation on tyrosine and the subsequent recruitment of downstream eff ector molecules. (C) 1999 Elsevier Science Ltd. All rights reserved.