ABSTRACT
Proteins that bear immunoreceptor tyrosine based inhibitory motifs (ITIM) are believed to participate in the repression of cell activation via phosphatases such as SHP-1, SHP-2 and/or SHIP-1. CLECSF6, also called DCIR, is a transmembrane protein expressed on leukocytes and predominantly on neutrophils that bears one ITIM pattern. This feature confers to CLECSF6 a role in the repression of cell activation. In order to better understand its role in neutrophil signalling, we analysed the binding of phosphatases to the ITIM of CLECSF6. We showed that a peptide bearing the ITIM of CLECSF6 in its phosphorylated form associates with both SHP-1 and SHP-2. Phosphorylated SHP-1 binds the ITIM whereas phosphorylated SHP-2 does not. In addition, granulocyte macrophage-colony stimulating factor (GM-CSF) reduces the binding of SHP-2 to the ITIM of CLECSF6 while enhancing the phosphorylation level of SHP-2. GM-CSF is known to recruit SHP-2 to its receptor. These data suggest that the phosphorylation of SHP-2 by GM-CSF promotes the binding of SHP-2 to the GM-CSF receptor to the disadvantage of CLECSF6. Therefore, upon a treatment with GM-CSF, SHP-2 could move from a CLECSF6 associated signalosome with a repressor function to a GM-CSF receptor associated signalosome with an activator function.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Lectins, C-Type/metabolism , Membrane Glycoproteins/metabolism , Neutrophils/drug effects , Protein Tyrosine Phosphatases/metabolism , Receptors, Immunologic/metabolism , Amino Acid Motifs , Detergents/pharmacology , Humans , Lectins, C-Type/chemistry , Membrane Glycoproteins/chemistry , Neutrophils/metabolism , Peptides/chemistry , Peptides/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Receptors, Immunologic/chemistry , Tyrosine/metabolismABSTRACT
The recently discovered CLECSF6 protein displays the features of a receptor involved in the down-modulation of leukocyte activation. Although CLECSF6 has been the focus of the interest of many researchers lately, a Western blot characterization of the protein is still lacking. This highly reduces our ability to gain full knowledge of the biological relevance of this protein in cell responses. We produced two rabbit polyclonal antisera that detected a glycosylated protein at approximately 35kDa in neutrophils. Four different CLECSF6 mRNA species have been discovered to date. When deglycosylated, the protein displayed the molecular weight expected for the longest CLECSF6 form. Neutrophil membrane fractions were strongly enriched in the protein. We showed a down-modulation of the expression of this protein in neutrophils treated with granulocyte-macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor (TNF-alpha), lipopolysaccharide (LPS), and interleukin (IL)-4. This work supports the hypothesis that CLECSF6 is involved in the control of inflammation in neutrophils.
Subject(s)
Down-Regulation , Lectins, C-Type/biosynthesis , Membrane Glycoproteins , Neutrophils/metabolism , Receptors, Immunologic/biosynthesis , Animals , Baculoviridae/metabolism , Blotting, Western , Cell Line , Cell Membrane/metabolism , Cytokines/metabolism , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Genetic Vectors , Glycosylation , Humans , Immunoblotting , Insecta , Interleukin-4/metabolism , Leukocytes/metabolism , Lipopolysaccharides/metabolism , Peptides/chemistry , RNA, Messenger/metabolism , Rabbits , Recombinant Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In our study of the modulation of the expression of inflammation-related genes in neutrophils, we have found a gene called CLECSF6 (C-type lectin superfamily 6). CLECSF6 expresses two mRNA species at low levels in resting neutrophils. Here, we describe for the first time the sequence of the short mRNA version. It lacks amino acids that are likely to affect the functionality of its protein product. GM-CSF, IL-3, IL-4, and IL-13 caused an accumulation of the short CLECSF6 mRNA in neutrophils. The surface expression of the CLECSF6 protein was reduced by TNF-alpha, IL-1alpha, LPS, and Matrigel. CLECSF6 bears the immunoreceptor tyrosine-based inhibition motif (ITIM) involved in signal transduction resulting in the inhibition of leukocyte activation. We propose that some neutrophil activators modulate the expression of CLECSF6 at the mRNA (GM-CSF, IL-3, IL-4, and IL-13) or protein (TNF-alpha, IL-1alpha, LPS, and Matrigel) levels in ways that block ITIM-based transduction of anti-inflammatory signals and therefore promote inflammation.
