An Activating Immunoreceptor Complex Formed by NKG2D and DAP10 Jun Wu, 1 Yaoli Song, 1 Alexander B. H. Bakker, 1 Stefan Bauer, 2 Thomas Spies, 2 Lewis L. Lanier, 1 * Joseph H. Phillips 1 Many immune receptors are composed of separate ligand-binding and signal- transducing subunits. In natural killer (NK) and T cells, DAP10 was identified as a cell surface adaptor protein in an activating receptor complex with NKG2D, a receptor for the stress-inducible and tumor-associated major histocompat- ibility complex molecule MICA. Within the DAP10 cytoplasmic domain, an Src homology 2 (SH2) domain– binding site was capable of recruiting the p85 subunit of the phosphatidylinositol 3-kinase (PI 3-kinase), providing for NKG2D- dependent signal transduction. Thus, NKG2D-DAP10 receptor complexes may activate NK and T cell responses against MICA-bearing tumors. The ability of NK cells to kill tumors and virus-infected cells and to produce cytokines is regulated by a balance between activating and inhibitory receptors. Inhibition is medi- ated by receptors for major histocompatibility complex (MHC) class I, including Ly49 and the killer cell immunoglobulin-like receptor (KIR) (1). These receptors have immunore- ceptor tyrosine-based inhibition motifs in their cytoplasmic domains that recruit cyto- plasmic tyrosine phosphatases, resulting in inactivation of NK cell function (2). Howev- er, certain receptors within the KIR and Ly49 families activate NK cells (3). These recep- tors lack signaling motifs but associate with DAP12, a CD3-like protein with an immuno- receptor tyrosine-based activation motif (4 ). Engagement of such receptor complexes trig- gers a signaling cascade similar to that initi- ated by the T cell receptor (4 ). A sequence with 20% amino acid ho- mology to DAP12 was identified as a human expressed sequence tag. This cDNA encodes DAP10, a type I membrane protein of 93 amino acids (Fig. 1). Its transmembrane (TM) contains a negatively charged residue that is conserved in DAP12 and in the CD3 sub- units. The short cytoplasmic region of DAP10 has a Tyr-X-X-Met (YXXM) motif, a potential SH2 domain– binding site for the p85 subunit of the PI 3-kinase (5), suggesting a role for DAP10 as a signaling adaptor. Southern (DNA) blot analysis revealed a re- striction enzyme digestion pattern predicted by the genomic sequence, consistent with a single DAP10 gene (6 ). The human DAP10 and DAP12 genes are on human chromosome 19q13.1 in opposite transcriptional orienta- tion, separated by only 130 base pairs (bp) (6 ). Abundant 500-bp DAP10 transcripts were detected in human peripheral blood leu- kocytes, spleen, thymus, NK cells, /- and /-T cell receptor + T cells, and U937 (my- eloid cell), but not substantially in other tis- sues or JY (B lymphoblastoid cell), 293T (epithelial cell), or primary fibroblasts (Fig. 2A) (6 ). Analysis by the reverse transcription polymerase chain reaction (RT-PCR) indicat- ed the presence of DAP10 mRNA in CD4 + and CD8 + T cell clones and in monocytes, granulocytes, and dendritic cells (6, 7 ). Thus, DAP10 is predominantly expressed in hema- topoietic cells. Protein immunoblot analysis of the NK cell line NKL, using an affinity-purified an- tibody to DAP10 (anti-DAP10), revealed multiple bands migrating slower than the pre- dicted molecular mass of 10 kD for DAP10, primarily as a result of O-linked glycosyla- tion (Fig. 2B). The multiple bands observed after treatment with O-glycanase may be due to incomplete saccharide removal, other post- translational modifications, or alternative splicing. To examine whether DAP10 associ- ates with other membrane receptors, we lysed a 125 I-labeled polyclonal NK line and NKL in 1% digitonin to preserve multisubunit recep- tor complexes. Although DAP10 did not la- bel with 125 I, anti-DAP10 coprecipitated a 125 I-labeled glycoprotein migrating at 42 kD under reducing conditions and at 42 and 80 kD under nonreducing conditions (Fig. 2C). Removal of N-linked sugars revealed a 28-kD polypeptide (Fig. 2D). Similar 125 I- 1 DNAX Research Institute, 901 California Avenue, Palo Alto, CA 94304, USA. 2 Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA 98109, USA. *To whom correspondence should be addressed. E- mail: lanier@dnax.org Fig. 1. Human DAP10. The extracel- lular cysteine residues, the trans- membrane charged residue, and a cy- toplasmic signaling motif are bold. Human DAP10 cDNA (AF122904) and a splice variant (AF072844) were identified, as were mouse DAP10 cDNA (AF072846) and a splice variant (AF122905). The genomic organization of human DAP10 (AF072845) was deduced from a fragment of human chromosome 19q13.1 (AD0008333). All numbers in parentheses are GenBank accession numbers. Fig. 2. DAP10 RNA and protein. (A) Northern blot analysis of DAP10 in human tissues and a T leukemia cell ( Jurkat), a B lymphoblastoid cell ( JY), an NK leukemia cell (YT), an NK cell line (NKL), a myeloid cell (U937), and an epithelial cell (293T ) (14). Blots were stripped and rehybridized with an actin probe to confirm that all lanes were equally loaded (not shown). The small amounts of DAP10 detected in the nonhematopoietic organs may be contributed by tissue macrophages. (B) Lysates prepared from NKL were immunoprecipitated with a control immunoglobulin (cIg) or affinity-purified anti-DAP10. Immune complexes were either untreated or treated with neuraminidase and O-glycosidase (O-Gly/Neur). Samples were analyzed by protein immunoblot using affinity-purified anti-DAP10 (15). (C and D) NKL or a polyclonal NK cell line were labeled with 125 I, lysed in 1% digitonin, and immunoprecipitated with cIg or affinity-purified anti-DAP10. Samples were analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) under reducing or nonreducing conditions (C) or were treated with neuraminidase (Neur), O-glycosidase (O-gly), or N-glycosidase F (N-gly), separately and in combination, and analyzed under reducing conditions (D) (16). R EPORTS 30 JULY 1999 VOL 285 SCIENCE www.sciencemag.org 730