Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation. Dynamic light scattering and fluorescent microscale thermophoresis of the isolated SAM domain (residues 1-78) revealed evidence of dimers and tetramers. Consistently, deletion of the SAM region eliminated SLP-76 co-precipitation of itself, concurrent with a loss of microcluster formation, nuclear factor of activated T-cells (NFAT) transcription, and interleukin-2 production in Jurkat or primary T-cells. Furthermore, the H5 α helix within the SAM domain contributed to self-association. Retention of H5 in the absence of H1-4 sufficed to support SLP-76 self-association with smaller microclusters that nevertheless enhanced anti-CD3-driven AP1/NFAT transcription and IL-2 production. By contrast, deletion of the H5 α helix impaired self-association and anti-CD3 induced AP1/NFAT transcription. Our data identified for the first time a role for the SAM domain in mediating SLP-76 self-association for T-cell function.
Keywords: Adaptor Proteins; Biophysics; Immunology; Protein Complexes; SAM Domain; SLP-76; Signal Transduction; T-cell.