Defective signaling to Fyn by a T cell antigen receptor lacking the α-chain connecting peptide motif

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