Defective signaling to Fyn by a TCR lacking the a-chain connecting peptide motif

A key role in the communication between the TCR and the CD3/ complex is played by a specific motif within the connecting peptide domain of the TCR chain (-CPM). T cell hybridomas expressing an -CPM-mutated TCR show a dramatic impairment in antigen-driven interleukin-2 pro- duction. This defect can be complemented by a calcium ionophore, indicating that activation of the calcium path- way is impaired. Several lines of evidence implicate Fyn in the regulation of calcium mobilization, at least in part through the activation of phospholipase C. Here we have investigated the potential involvement of Fyn in the TCR -CPM signaling defect. Using T cell hybridomas express- ing either a wild-type TCR or an -CPM mutant, we show that Fyn fails to be activated by the mutant receptor follow- ing SEB binding and fails to generate tyrosine-phospho- rylated Pyk2, a member of the focal adhesion kinase family. This defect correlated with an impairment in phospho- lipase C phosphorylation. Production of interlukin-2 and activation of the transcription factor NF-AT in response to triggering of the TCR -CPM mutant with SEB were fully restored in the presence of constitutively active Fyn. Hence the signaling defect generated by the TCR -CPM mutation results at least in part from an impaired coupling of the TCRCD3 complex to Fyn activation.