ABSTRACT
Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.
Subject(s)
Killer Cells, Natural/metabolism , Mast Cells/metabolism , Monocytes/metabolism , Neutrophils/metabolism , Receptors, Immunologic/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Intracellular Signaling Peptides and Proteins , Killer Cells, Natural/immunology , Macrophages/immunology , Macrophages/metabolism , Mast Cells/immunology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Molecular Sequence Data , Monocytes/immunology , Neutrophils/immunology , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/metabolism , Rats , Receptors, Immunologic/biosynthesis , Transcription, GeneticABSTRACT
NKp46 has been identified in the human, rat, mouse, monkey, and cattle. We have generated a monoclonal antibody, WEN23, against rat NKp46. By flow cytometry, NKp46 is expressed by all natural killer (NK) cells but not by T cells, B cells, granulocytes, monocytes, dendritic cells, or macrophages. Thus, NKp46/WEN23 is the first NK cell-specific marker in the rat. In a redirected lysis assay, preincubation of the effector cells with WEN23 augmented lysis of the Fc receptor (FcR)+ murine tumor target cells, indicating that NKp46 is an activating NK cell receptor. Moreover, preincubation of the effector cells with WEN23 F(ab')2 fragments reduced killing of target cells, confirming the activating function of NKp46 and indicating that the mouse tumor target cells express a ligand for rat NKp46. Lysis of FcR- mouse and human tumor target cells was reduced after incubation of effector cells with WEN23, suggesting that rat NKp46 recognizes a ligand that is conserved between primates and rodents. By Western blot and immunoprecipitation using WEN23, NKp46 is expressed as a monomer of approximately 47 kDa in interleukin-2-activated NK cells. The immunoreceptor tyrosine-based activation motif bearing adaptor proteins CD3zeta and the gamma chain of FcRI for IgE (FcepsilonRIgamma) with NKp46 from lysates of NK cells, indicating that rat NKp46 activates NK cell cytotoxicity by similar pathways as CD16.
