The membrane-proximal immunoreceptor tyrosine-based inhibitory motif is critical for the inhibitory signaling mediated by Siglecs-7 and -9, CD33-related Siglecs expressed on human monocytes and NK cells.
Siglec-7 and Siglec-9 are two members of the recently characterized CD33-related Siglec family of sialic acid binding proteins and are both expressed on human monocytes and NK cells. In addition to their ability to recognize sialic acid residues, these Siglecs display two conserved tyrosine-based motifs in their cytoplasmic region similar to those found in inhibitory receptors of the immune system. In the present study, we use the rat basophilic leukemia (RBL) model to examine the potential of Siglecs-7 and -9 to function as inhibitory receptors and investigate the molecular basis for this. We first demonstrate that Siglecs-7 and -9 are able to inhibit the FcepsilonRI-mediated serotonin release from RBL cells following co-crosslinking. In addition, we show that under these conditions or after pervanadate treatment, Siglecs-7 and -9 associate with the Src homology region 2 domain-containing phosphatases (SHP), SHP-1 and SHP-2, both in immunoprecipitation and in fluorescence microscopy experiments using GFP fusion proteins. We then show by site-directed mutagenesis that the membrane-proximal tyrosine motif is essential for the inhibitory function of both Siglec-7 and -9, and is also required for tyrosine phosphorylation and recruitment of SHP-1 and SHP-2 phosphatases. Finally, mutation of the membrane-proximal motif increased the sialic acid binding activity of Siglecs-7 and -9, raising the possibility that "inside-out" signaling may occur to regulate ligand binding.
Avril
Tony
T
Division of Cell Biology and Immunology, The Wellcome Trust Biocentre, University of Dundee, Dundee, United Kingdom.
Floyd
Helen
H
Lopez
Frederic
F
Vivier
Eric
E
Crocker
Paul R
PR
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics / Q000276:immunology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism*; D037102:Lectins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000037:antagonists & inhibitors / Q000502:physiology; D011971:Receptors, Immunologic / Q000235:genetics / Q000502:physiology*; D012701:Serotonin / Q000378:metabolism; D012702:Serotonin Antagonists / Q000494:pharmacology; D063268:Sialic Acid Binding Ig-like Lectin 3; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012794:Sialic Acids / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D014443:Tyrosine / Q000378:metabolism
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C571163:CD33 protein, human; D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins; D017455:Receptors, IgE; D011971:Receptors, Immunologic; C402206:SIGLEC7 protein, human; C412227:SIGLEC9 protein, human; D012702:Serotonin Antagonists; D063268:Sialic Acid Binding Ig-like Lectin 3; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; D012794:Sialic Acids; D012701:Serotonin; D014443:Tyrosine; C516724:PTPN11 protein, human; C509249:PTPN6 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C516727:Ptpn11 protein, rat; C509250:Ptpn6 protein, rat