Cloning, sequencing, and cell surface expression pattern of bovine immunoreceptor NKG2D and adaptor molecules DAP10 and DAP12.
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.
Fikri
Youssef
Y
ISP-Institut Pasteur de Bruxelles, Rue Engeland 642, 1180 Bruxelles, Belgium.
Nyabenda
Jean
J
Content
Jean
J
Huygen
Kris
K
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D055607:Receptors, Natural Killer Cell; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D018076:DNA, Complementary; C527085:KLRK1 protein, human; C527086:Klrk1 protein, mouse; D008565:Membrane Proteins; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011971:Receptors, Immunologic; D055607:Receptors, Natural Killer Cell; D011994:Recombinant Proteins