Downstream signaling molecules bind to different phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) peptides of the high affinity IgE receptor

Abstract

The cytoplasmic tails of both the β and γ subunits of the high affinity IgE receptor (FcεRI) contain a consensus sequence termed the immunoreceptor tyrosine-based activation motif (ITAM). This motif plays a critical role in receptor-mediated signal transduction. Synthetic peptides based on the ITAM sequences of the β and y subunits of FcεRI were used to investigate which proteins associate with these motifs. Tyrosine- phosphorylated β and γ ITAM peptides immobilized on beads precipitated Syk, Lyn, Shc, Grb2, and phospholipase C-γ1 from lysates of rat basophilic leukemia RBL-2H3 cells. Syk was precipitated predominantly by the tyrosine- diphosphorylated γ ITAM peptide, but much less by the diphosphorylated β ITAM peptide or by the monophosphorylated peptides. Phospholipase C-γ1, Shc, and Grb2 were precipitated only by the diphosphorylated β ITAM peptide. Non- phosphorylated ITAM peptides did not precipitate these proteins. In membrane binding assays, fusion proteins containing the Src homology 2 domains of phospholipase C-γ1, Shc, Syk, and Lyn directly bound the tyrosine- phosphorylated ITAM peptides. Although the ITAM sequences of the β and γ subunits of FcεRI are similar, once they are tyrosine-phosphorylated they preferentially bind different downstream signaling molecules. Tyrosine phosphorylation of the ITAM of the y subunit recruits and activates Syk, whereas the β subunit may be important for the Ras signaling pathway.