Reconstitution of CD3 zeta coupling to calcium mobilization via genetic complementation.
The integrity of the T cell receptor complex (CD3-TCR) transduction machinery is central to T cell development and to T cell effector function. Molecular dissection of the multimeric CD3-TCR complex revealed that at least two associated polypeptides, CD3 zeta and CD3 epsilon, autonomously couple antigenic recognition event to early and late events of the intracytoplasmic activation cascade. A 18-amino acid motif based on a tandem YXXL stretch, the activation receptor homology sequence 1 (ARH-1) motif, is necessary and sufficient to the transducing properties of both CD3 zeta and CD3 epsilon. Stimulation of chimeric molecules made of ecto- and transmembrane domains of various cell surface proteins and intracytoplasmic domains of CD3 epsilon or CD3 zeta leads to an increase in the intracellular Ca2+ concentration ([Ca2+]i) in Jurkat cells. We describe here that a similar CD25/zeta chimeric molecule was unable to induce a detectable [Ca2+]i rise upon CD25 cross-linking once expressed in the murine thymoma BW-. A Ca2+ influx could, however, be triggered in BW- cells by thapsigargin, i.e. following depletion of Ca2+ stores. Somatic cell hybrids made from BW- and either thymocytes or mature lymph node T cells reconstituted the coupling of CD3 zeta to the Ca2+ signal via an ARH-1 motif-dependent pathway. However, pervanadate-induced Ca2+ mobilization, a phenomenon attributed to tyrosine phosphorylation, was impaired in BW-cells and reconstituted in hybridomas. In contrast to the Ca2+ response, IL-2 production was induced in both BW- and hybrids cells, which questions the functional relevance of [Ca2+]i augmentation in T cell activation. In conclusion, the properties of the BW- thymoma, which define a novel group of CD3 zeta transduction cell mutants, as well as its complementation by somatic cell fusion demonstrate that this cell line represents a useful model to dissect the signaling pathway that couples CD3 zeta to Ca2+ mobilization by genetic reconstitution.
Donnadieu
E
E
Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Marseille, France.
Trautmann
A
A
Malissen
M
M
Trucy
J
J
Malissen
B
B
Vivier
E
E
D000818:Animals; D017252:CD3 Complex / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D007376:Interleukin-2 / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D051966:Phospholipase C gamma; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
D017252:CD3 Complex; D007376:Interleukin-2; D007527:Isoenzymes; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium