Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT L. Leyton*, A.F.G. Quest, C. Bron Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland Received 25 May 1999; accepted 19 July 1999 Abstract 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 de®ned system employing only anity puri®ed antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium in¯ux 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 ecient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identi®ed 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) speci®cally 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 eector molecules. # 1999 Elsevier Science Ltd. All rights reserved. 1. Introduction A number of functionally diverse cell surface pro- teins are anchored to the plasma membrane by a phosphatidylinositol-based glycolipid (GPI). This dis- tinctive mode of membrane association confers par- ticular properties to these proteins, such as increased membrane mobility and enhanced presence in glyco- lipid-enriched domains (Ishihara et al., 1987; Brown and Rose, 1992), but precludes direct association of GPI-anchored proteins with elements of the intra- cellular signal transduction machinery. Nonetheless, Ab-mediated crosslinking of many GPI-anchored proteins triggers transmembrane signaling events on lymphocytes, which leads to cellular activation or modulation of the TCR/CD3-induced response (Gunter et al., 1984; MacDonald et al., 1985; Stefanova et al., 1991). In particular, clustering of Thy-1, a mammalian GPI-anchored protein abun- dantly expressed on the surface of mouse T lympho- cytes, triggers lymphokine release and cell Molecular Immunology 36 (1999) 755±768 0161-5890/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0161-5890(99)00086-3 www.elsevier.com/locate/molimm * Tel.: +41-21-692-5745; fax: +41-21-692-5705. E-mail address: lleyton@eliot.unil.ch (L. Leyton) Abbreviations: [Ca 2+ ] i , intracellular calcium in¯ux; ECL, enhance chemiluminescence; EDTA, ethylenediaminetetraacetic acid; FACS, ¯uorescence-activated cell sorter; GPI, glycosyl-phosphatidylinositol; HEPES, N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid; HRP, horseradish peroxidase; LAT, Linker for Activation of T cells; NR- PTK, non-receptor protein tyrosine kinase; PLC, phospholipase C; PVDF, polyvinylidene ¯uoride; PY, phosphotyrosine; Tris, tris(hy- droxymethyl)aminomethane; TSA-1, thymic shared antigen-1.