Molecular cloning and characterization of pig immunoreceptor DAP10 and NKG2D

Pig immunoreceptor DAP10 cDNA was cloned from a peripheral blood lymphocyte (PBL) cDNA library using human DAP10 cDNA as a probe. The length of the pi g DAP10 cDNA is 465 bp and it contains an open reading frame of 237 bp. The predicted polypeptide sequence is 79 amino acids, consisting of an 18-amin o acid leader, a 16-amino acid extracellular domain, a 24-amino acid transm embrane segment, and a 21-amino acid cytoplasmic domain. The amino acid seq uence of pig DAP10 has 68% and 78% sequence identity with human DAP10 and m ouse DAP10, respectively, Pig DAP10 has a conserved aspartic acid in the tr ansmembrane domain, two cysteines in the extracellular domain, and a phopha tidylinositol-3 kinase-binding site (YxxM) in the cytoplasmic region. Genom ic organization reveals that pig DAP10 comprises four exons and three intro ns. Pig DAP10 and DAP12 are genetically linked on Chromosome (Chr) 6 at 6q2 1 in opposite transcriptional orientation, separated by 152 bp, In Northern blot analysis, DAP10 transcripts were detected predominantly in lymphohema topoietic tissues. Pig NKG2D cDNA has an open reading frame of 642 bp. Its expected polypeptide sequence is 214 amino acids. Pig NKG2D has 66% sequenc e identity with human NKG2D and 56% identity with mouse NKG2D, The NKG2D ge ne maps to pig Chr 5q25. RT-PCR analysis reveals that pig NKG2D transcripts are expressed in PBLs, NK cells, macrophages, and monocytes, When transien tly transfected into COS-7 cells, pig NKG2D requires DAP10 for cell surface expression.