Cloning, sequencing, and cell surface expression pattern of bovine immunoreceptor NKG2D and adaptor molecules DAP10 and DAP12

Abstract

NKG2D is an activating lectin-like receptor that initiates natural killer (NK) cell responses against transformed tumor cells expressing its ligands, i.e., molecules related to major histocompatibility complex (MHC) class I molecules. NKG2D lacks signaling elements in its cytoplasmic domain and can deliver stimulatory signals only in association with transmembrane adaptor proteins DAP10 or DAP12. The complementary DNAs (cDNAs) encoding the bovine homologues of NKG2D and the adaptor proteins DAP10 and DAP12 were cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from resting bovine peripheral blood mononuclear cells (PBMC) and sequenced. Comparison with human, pig, and mouse sequences showed that bovine NKG2D is most similar to pig NKG2D and short mouse NKG2D (NKG2D-S). Similar to its human, mouse, and pig homologues, the cDNA for bovine DAP10 codes for a phosphatidyl-inositol-3 (PI-3) kinase-binding site (YxxM) in its cytoplasmic region. Finally, similar to its human, mouse, and pig homologues, the cDNA encoding bovine DAP12 demonstrates one tyrosine-based activated motif (ITAM) in its cytoplasmic domain. Bovine NKG2D cell surface expression was analyzed by flow cytometry on HEK 293 cells transiently transfected with cDNA expression vectors encoding COOH-terminal polyhistidine-tagged NKG2D and NH(2)-terminal Flag-tagged DAP10 and DAP12. Confirming previous findings for short mouse NKG2D-S, bovine NKG2D immunoreceptor could associate with either DAP10 or DAP12 adaptor protein for its cell surface expression.

Cell mediated innate responses of cattle and swine are diverse during foot-and-mouth disease virus (FMDV) infection: A unique landscape of innate immunity

“…Similar observations have been made by others regarding activation of NK cells by other microorganisms [18,19]. Bovine NK cells also express a short form of natural-killer group 2, member D protein (NKG2D, an activating receptor found on NK and CD8 + T cells) which associates with signaling adaptors such as DNAX activation proteins of 10 kDa (DAP10) and 12 kDa, DAP12 [20]. Furthermore, NKG2D expressed on CD335 + cells in peripheral blood, mesenteric lymph nodes and intraepithelial cells binds to bovine MIC1 (bovine leukocyte antigen-like molecule 1) and MIC4.…”

Definition of the Cattle Killer Cell Ig–like Receptor Gene Family: Comparison with Aurochs and Human Counterparts

“…5D ). The Fcγ and DAP12 genes are both present in the cattle genome and appear functional, which in the case of DAP12 has been demonstrated ( 46 ).…”

DAP10‐ and DAP12‐associated receptors in innate immunity

“…Orthologs of the gene encoding DAP12, designated TYROBP , have been identified in mammals [e.g. humans (3), chimps (GenBank ), Rhesus monkey (GenBank ), pigs (6), cows (7), water buffalo (GenBank ), sheep (GenBank ), pigs (GenBank ), dogs (GenBank ), mice (3), and rats (GenBank )), amphibians (frogs, GenBank ), and fish, i.e. zebrafish (8) and Fugu (9)] and DAP12 has remained remarkably conserved (), demonstrating that it provides essential signaling functions in most vertebrates.…”

“…Remarkably, the gene encoding DAP10 ( HCST ) is adjacent to DAP12 in the genome (the DAP12‐and DAP10‐encoding genes are separated only by 130 nucleotides in the human genome) but in the opposite transcriptional orientation. DAP10‐encoding genes have been identified in mammals (7, 10, 11), amphibians (9), and fish (8, 9) (). The DAP10‐ and DAP12‐encoding genes in zebrafish are adjacent in the genome, indicating that DAP10 and DAP12 have been preserved as a pair for millions of years and that they probably arose by duplication from a common more ancient gene.…”