Pre-T cell receptor (TCR) expression plays a fundamental role in the generation of alpha beta T-cells by initiating expansion and differentiation of thymocytes that have successfully rearranged a TCR beta gene. We found that pre-TCR expression on the cell membrane is characterized by a partition into rafts and co-localization with the surface phosphatase CD45. This result suggests that lck activation by the pre-TCR may depend on co-localization with CD45 in the cell membrane. PreTCR containing rafts are not only found among thymocytes but also on the surface of a pre-TCR expressing T-cell line indicating that raft formation is a spontaneous event that does not need the ligation of the pre-TCR by ligands on thymic stromal cells. The contribution of actin polymerization to pT-alpha signaling is suggested by specific inhibition of beta selection in fetal thymus organ cultures (FTOC) conditioned by pharmacological agents that inhibit polymerization. Characterization of preTCR containing rafts by both morphological and biochemical analysis, as well as the pharmacological modification of the lipid composition of cell membrane microdomains should provide fundamental clues on the functional organization of the pre-TCR. Analysis of beta selection in FTOC treated with F-actin inhibitors will provide clues on the possible involvement of actin-scaffolded signaling pathways in the accomplishment of isotype and allelic exclusion which critically depend on pre-TCR function. Two independent approaches (RNA fingerprinting and screening of an ordered fetal thymus library with complex probes) will be undertaken to identify changes in gene expression that are caused by pre-TCR expression. The proposed experiments will elucidate the molecular mechanisms that mediate the function of the pre-TCR.
