RESUMO
The pre-T cell receptor (TCR) signals constitutively in the absence of putative ligands on thymic stroma and signal transduction correlates with translocation of the pre-TCR into glycolipid-enriched microdomains (rafts) in the plasma membrane. Here, we show that the pre-TCR is constitutively routed to lysosomes after reaching the cell surface. The cell-autonomous down-regulation of the pre-TCR requires activation of the src-like kinase p56(lck), actin polymerization, and dynamin. Constitutive signaling and degradation represents a feature of the pre-TCR because the gammadeltaTCR expressed in the same cell line does not exhibit these features. This is also evident by the observation that the protein adaptor/ubiquitin ligase c-Cbl is phosphorylated and selectively translocated into rafts in pre-TCR- but not gammadeltaTCR-expressing cells. A role of c-Cbl-mediated ubiquitination in pre-TCR degradation is supported by the reduction of degradation through pharmacological inhibition of the proteasome and through a dominant-negative c-Cbl ubiquitin ligase as well as by increased pre-TCR surface expression on immature thymocytes in c-Cbl-deficient mice. The pre-TCR internalization contributes significantly to the low surface level of the receptor on developing T cells, and may in fact be a requirement for optimal pre-TCR function.
Assuntos
Endocitose , Receptores de Antígenos de Linfócitos T/metabolismo , Actinas/metabolismo , Animais , Biopolímeros/metabolismo , Membrana Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Dinaminas , GTP Fosfo-Hidrolases/metabolismo , Hidrólise , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos SCID , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Transdução de SinaisRESUMO
A transgenic reporter mouse strain, which expressed the human CD25 (hCD25) surface marker as a reporter under the control of the pre-T cell receptor alpha(pTalpha) promoter, was used to identify lymphoid precursors that expressed pTalpha intracellularly. The hCD25 reporter marked intra- and extrathymic precursors of lymphocytes but not myeloid cells. The earliest intrathymic precursors were CD4(lo)CD8(-)CD25(-)CD44(+)c-Kit(+) cells that expressed elevated levels of Notch-1 mRNA. Clonogenic assays showed that the extrathymic precursors were common lymphoid progenitors (CLPs) that included CD19(-), B220(+), Thy1(+) and CD4(+) cells. Thus, the pTalpha reporter can be used to trace lymphopoiesis between CLPs and alphabeta T cells. The slower extinction of the hCD25 reporter compared to pTalpha enabled us to define points at which pTalpha(-) lineages branched off.