Siglec-5 (CD170) Can Mediate Inhibitory Signaling in the Absence of Immunoreceptor Tyrosine-based Inhibitory Motif Phosphorylation

Abstract

Siglec-5 (CD170) is a member of the recently described human CD33-related siglec subgroup of sialic acid binding Ig-like lectins and is expressed on myeloid cells of the hemopoietic system. Similar to other CD33-related siglecs, Siglec-5 contains two tyrosine-based motifs in its cytoplasmic tail implicated in signaling functions. To investigate the role of these motifs in Siglec-5-dependent signaling, we used transfected rat basophil leukemia cells as a model system. Tyrosine phosphorylation of Siglec-5 led to recruitment of the tyrosine phosphatases SHP-1 and SHP-2, as seen in both pull-down assays and microscopy. Siglec-5 could efficiently inhibit Fc⑀RI-mediated calcium fluxing and serotonin release after cocross-linking. Surprisingly, a double tyrosine to alanine mutant of Siglec-5 could still mediate strong inhibition of serotonin release in the absence of detectable tyrosine phosphorylation, whereas a double tyrosine to phenylalanine mutant lost all inhibitory activity. In comparison, suppression of Siglec-5-dependent adhesion to red blood cells was reversed by either tyrosine to alanine or tyrosine to phenylalanine mutations of the membrane proximal tyrosine-based motif. Using an in vitro phosphatase assay with synthetic and recombinant forms of the cytoplasmic tail, it was shown that a double alanine mutant of Siglec-5 had weak, but significant SHP-1 activating properties similar to those of wild type, non-phosphorylated cytoplasmic tail, whereas a double phenylalanine mutant was inactive. These findings establish that Siglec-5 can be classified as an inhibitory receptor with the potential to mediate SHP-1 and/or SHP-2-dependent signaling in the absence of tyrosine phosphorylation.Regulation of responses by cells in the hemopoietic and immune systems depends on a balance between activatory signaling and inhibitory signaling. Their relative strengths set an appropriate activation threshold that helps fine-tune the response. Inhibitory signals are typically initiated by receptors containing one or more cytoplasmic immunoreceptor tyrosinebased inhibitory motifs (ITIMs) 1 (1). The consensus sequence for ITIMs is (V/I/L)XYXX(L/V), where X is any amino acid. A recent bioinformatics study showed that the human proteome contains ϳ109 membrane proteins with cytoplasmic ITIMs, and many of these have been shown already to function as inhibitory receptors (2). The established dogma is that Src family tyrosine kinases phosphorylate the tyrosine residue in ITIMs during cellular activation and create high affinity docking sites for SH2 domain-containing phosphatases such as protein-tyrosine phosphatases SHP-1 and SHP-2 and the 5Ј inositol phosphatase, SHIP (1). The recruited and activated phosphatases can then dephosphorylate relevant substrates in the vicinity and regulate cellular activation (3). The human CD33-related siglecs are a recently described subgroup of the siglec family of sialic acid binding immunoglobulin (Ig)-like lectins (4). All eight human CD33-related siglecs are expressed by cells of the hemopoietic...

Alleviation of Neurotoxicity by Microglial Human Siglec-11

“…Several CD33-related Siglecs have been shown to inhibit activation pathways induced by immunostimulatory receptors (Avril et al, 2005;Crocker et al, 2007). For example, stimulation of Siglec-5 recruited SHP-1 and SHP-2 and efficiently inhibited FcRI-mediated calcium fluxing (Avril et al, 2005). The cytoplasmic tail of Siglec-11 is very closely related to that of Siglec-5 in respect to sequence similarity and conservation of the protein structure (Angata et al, 2002Hayakawa et al, 2005).…”

“…We further demonstrated that Siglec-11-expressing microglial cells were less toxic to neurons leading to reduced neurite and neuronal cell body loss in microglia-neuron cocultures. Several CD33-related Siglecs have been shown to inhibit activation pathways induced by immunostimulatory receptors (Avril et al, 2005;Crocker et al, 2007). For example, stimulation of Siglec-5 recruited SHP-1 and SHP-2 and efficiently inhibited FcRI-mediated calcium fluxing (Avril et al, 2005).…”

Probing the cis interactions of the inhibitory receptor Siglec-7 with α2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of α2,8-sialyltransferase gene expression

“…Tyrosine phosphorylation of the ITIM can lead to recruitment of Src homology-2-containing tyrosine phosphatase 1 (SHP-1) and SHP-2 tyrosine phosphatases and initiation of inhibitory signaling [12]. Recent studies have raised the possibility that low-level, constitutive recruitment of tyrosine phosphatases by CD33-related siglecs could be important for maintaining a tonic suppression of leukocyte activation [13].…”

Enhanced lentiviral vector production in 293FT cells expressing Siglec-9

“…Siglecs are sialic acid-recognizing Ig-superfamily lectins prominently expressed on immune cells (Crocker et al 2012;Varki and Angata 2006). Members of CD33-related Siglecs have been shown to downregulate both innate and acquired immune responses, and this may occur via cytosolic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) residing in the cytoplasmic region of the protein (Avril et al 2005;Ikehara et al 2004;Lajaunias et al 2005;Nicoll et al 2003;Paul et al 2000;Ulyanova et al 1999;von Gunten et al 2005;Zhang et al 2007). We previously suggested that CD33-related Siglecs may antagonize Toll-like receptor (TLR)-mediated inflammatory responses, leading to a reduction in proinflammatory cytokines and increase in the production of an antiinflammatory cytokine (Ando et al 2008).…”