SLP -76: Complex regulation of FcϵRI signaling in mast cells

The adaptor protein SRC homology 2 (SH2)-domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) mediates immunoreceptor signaling in multiple lineages including mast cells, but the mechanism by which it mediates FcϵRI signaling in mast cells is not well understood. I have developed a confocal real time imaging approach that allows direct observation of the sub-cellular localization pattern of proteins involved in proximal FcϵRI signaling in RBL cells and primary bone marrow-derived mast cells. SLP-76 is found in the cytosol of unstimulated cells and, upon FcϵRI crosslinking, it translocates to the cell membrane forming clusters that co-localize with the FcϵRI, the tyrosine kinase Syk, the adaptor LAT, and phosphotyrosine. Disruption of the SLP-76/Gads interaction through mutation of SLP-76 or expression of the Gads-binding region of SLP-76 inhibits translocation and clustering of SLP-76. I further demonstrate that expression of the Gads-binding region of SLP-76 in bone marrow-derived mast cells inhibits FcϵRI-induced calcium flux, degranulation, and cytokine secretion. These studies reveal, for the first time, that SLP-76 forms signaling clusters following FcϵRI stimulation and demonstrate that the Gads-binding region of SLP-76 regulates clustering of SLP-76 and FcϵRI-induced mast cell responses. These data suggest a model where SLP-76 translocates from the cytosol to the cell membrane adaptor LAT where SLP-76 mediates the formation of multi-molecular signaling complexes that regulate FcϵRI induced mast cell responses. While SLP-76 regulates FcϵRI signaling, I present evidence for SLP-76 independent FcϵRI signaling in vitro and in vivo, and test whether SLP-76 independent signaling is mediated by an alternative pathway regulated by the kinase Fyn. Initial characterization of mice deficient in both SLP-76 and Fyn indicates that Fyn does not mediate SLP-76 independent signaling, suggesting a model where FcϵRI signaling is partially independently of both SLP-76 and Fyn