TCRbeta transmembrane tyrosines are required for pre-TCR function

The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.