Phosphorylated ITIMs enable ubiquitylation of an inhibitory cell surface receptor

Immune responses are modulated by activating and inhibitory receptors that traffic to and from the cell surface. Ligands that bind to inhibitory receptors induce phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails, followed by recruitment of inhibitory signaling molecules. Mechanisms that control the surface levels of inhibitory receptors are largely unexplored. Here, we show, using CD33/sialic acid-binding immunoglobulin-like lectin (Siglec)-3 as a paradigm, that ITIMs can bind to the ubiquitin ligase Cbl and that ITIMs are ubiquitylated following Src family kinase-mediated tyrosine phosphorylation. Ubiquitylation is a known signal for endocytosis. Accordingly, cells expressing CD33 mutants that cannot become ubiquitylated show significantly increased cell surface expression of CD33 and have impaired CD33 internalization, whereas in-frame fusion of ubiquitin to CD33 reverses this phenotype. Our results identify a novel function of ITIMs and demonstrate that phosphorylation-dependent ubiquitylation regulates cell surface expression and internalization, and thus possibly function, of CD33/Siglec-3, suggesting an important role of ubiquitin in endocytosis of ITIM-bearing inhibitory immunoreceptors.