Mutational Analysis of Immunoreceptor Tyrosine-Based Inhibition Motifs of the Ig-Like Transcript 2 (CD85j) Leukocyte Receptor

• Published in: The Journal of immunology (1950) 2002-04, Vol.168 (7), p.3351-3359
• Main Authors: Bellon, Teresa, Kitzig, Friederike, Sayos, Joan, Lopez-Botet, Miguel
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs - genetics; Amino Acid Motifs - immunology; Animals; Antigens, CD - genetics; Antigens, CD - metabolism; Antigens, CD - physiology; CD85j antigen; Cell Line, Transformed; Cells, Cultured; Coculture Techniques; COS Cells; Cytoplasm - enzymology; Cytoplasm - genetics; Cytoplasm - metabolism; DNA Mutational Analysis; Enzyme Precursors - metabolism; Humans; ILT2 protein; immunoreceptor tyrosine-based inhibition motif; Intracellular Signaling Peptides and Proteins; Leukocyte Immunoglobulin-like Receptor B1; Ligands; Phosphorylation; Protein Binding - genetics; Protein-Tyrosine Kinases - metabolism; Rats; Receptors, IgE - antagonists & inhibitors; Receptors, IgE - physiology; Receptors, Immunologic - genetics; Receptors, Immunologic - metabolism; Receptors, Immunologic - physiology; Serotonin Antagonists - metabolism; Serotonin Antagonists - pharmacology; SHP-1 protein; src Homology Domains - genetics; Syk Kinase; Tumor Cells, Cultured; Tyrosine - genetics; Tyrosine - metabolism
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.168.7.3351

The inhibitory receptor Ig-like transcript (ILT)2 (leukocyte Ig-like receptor or CD85j) is a type I transmembrane protein expressed by different leukocyte lineages. The extracellular region of ILT2 binds HLA class I molecules, and its cytoplasmic domain displays four immunoreceptor tyrosine-based inhibition motifs. Upon tyrosine phosphorylation ILT2 recruits the Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) that is involved in negative signaling. To address the structural basis of ILT2-mediated inhibitory signaling, deletion and single tyrosine mutants were generated and transfected in the COS-7 and rat basophilic leukemia cell lines; their abilities to bind SHP-1 and to inhibit FcepsilonR-induced serotonin release in rat basophilic leukemia cells were studied. Both biochemical and functional analyses revealed tyrosines 644 (SIYATL) and 614 (VTYAQL) as the SHP-1 docking sites required for ILT2 inhibitory function. Substitution of tyrosine 562 (VTYAEV) did not alter receptor function. By contrast, mutation of tyrosine 533 (NLYAAV) interfered with ILT2 tyrosine phosphorylation and the subsequent SHP-1 recruitment, thus supporting a regulatory role for this motif.