Structural Requirements of SLP-76 in Signaling via the High-Affinity Immunoglobulin E Receptor (FcεRI) in Mast Cells

The high-affinity receptor for immunoglobulin E (IgE) (FcɛRI) is a multimolecular complex of the IgE-binding α subunit, two signal-transducing γ subunits, and a β subunit that promotes assembly of the receptor and amplifies signal transduction (3, 32). Both γ and β chains contain immunoreceptor tyrosine-based activation motifs (ITAMs) within their intracellular domains. Upon FcɛRI cross-linking, the ITAMs of the γ and β subunits become phosphorylated by the Src family tyrosine kinase lyn and recruit the protein tyrosine kinase Syk, which in turn phosphorylates intracellular proteins such as LAT, phospholipase C-γ (PLC-γ), Vav, and the adapter protein SLP-76 (9, 21, 28, 35). SLP-76 is predominantly expressed in hematopoietic cells and has three major protein-interacting domains (7, 25, 38, 46). Three tyrosine residues (Y113, Y128, and Y145) in the N-terminal domain become phosphorylated by Syk family protein tyrosine kinases following T-cell receptor (TCR) engagement and provide binding sites for the SH2 domains of Vav, Nck, and Itk. The binding of Vav and Nck to phosphotyrosine residues Y113 and Y128 may link SLP-76 to the JNK (Jun amino-terminal kinase) pathway and to the actin cytoskeleton (5, 10, 54-56). Y145 has been implicated in the binding of SLP-76 to Itk (6, 53). Direct interaction of PLC-γ with SLP-76 as well as formation of a complex involving LAT and Itk, which, respectively, bind and phosphorylate PLC-γ, may be required for PLC-γ activation (49, 57, 59). SLP-76 associates constitutively via its central proline-rich domain with the SH3 domain of Gads, which recruits it to LAT following TCR stimulation (1, 31, 33). This allows the translocation of SLP-76 to glycolipid-enriched microdomains (GEMs) (24) and may also link it via Sos to the Ras/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) pathway (29, 36). Proteins that directly interact with the SLP-76 SH2 domain include ADAP (formerly known as SLAP-130/FYB), the Ser/Thr kinase HPK1, and a 62-kDa phosphoprotein (11, 36, 37, 48). SLP-76−/− mice lack T cells, indicating that signals integrated by SLP-76 are critical for T-cell development (8, 43). SLP-76 also plays an important role in TCR signal transduction and T-cell activation. SLP-76-deficient Jurkat cells exhibit severely impaired signaling after stimulation through the TCR-CD3 complex. PLC-γ1 activation, calcium mobilization, ERK1/2 phosphorylation, and interleukin-2 (IL-2) production are all severely compromised (59). SLP-76-deficient mice have normal numbers of mast cells in their skin and bronchi, and their bone marrow cells differentiate normally in vitro into mast cells upon culture in IL-3-containing medium (44). However, SLP-76−/− bone marrow-derived mast cells (BMMC) fail to release the granular enzyme β-hexosaminidase and to secrete IL-6 after FcɛRI cross-linking. These findings indicate that SLP-76 plays an essential role in FcɛRI signaling. We took advantage of the availability of SLP-76−/− BMMC and transduced them retrovirally with SLP-76 mutants to address the role of SLP-76 domains and residues for its adapter function in signaling via FcɛRI.