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An Activating Immunoreceptor Complex Formed by NKG2D and DAP10

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Science  30 Jul 1999:
Vol. 285, Issue 5428, pp. 730-732
DOI: 10.1126/science.285.5428.730

Abstract

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 histocompatibility 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 mediated by receptors for major histocompatibility complex (MHC) class I, including Ly49 and the killer cell immunoglobulin-like receptor (KIR) (1). These receptors have immunoreceptor tyrosine-based inhibition motifs in their cytoplasmic domains that recruit cytoplasmic tyrosine phosphatases, resulting in inactivation of NK cell function (2). However, certain receptors within the KIR and Ly49 families activate NK cells (3). These receptors lack signaling motifs but associate with DAP12, a CD3-like protein with an immunoreceptor tyrosine-based activation motif (4). Engagement of such receptor complexes triggers a signaling cascade similar to that initiated by the T cell receptor (4).

A sequence with ∼20% amino acid homology 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 subunits. 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 restriction enzyme digestion pattern predicted by the genomic sequence, consistent with a single DAP10 gene (6). The humanDAP10 and DAP12 genes are on human chromosome 19q13.1 in opposite transcriptional orientation, separated by only ∼130 base pairs (bp) (6). Abundant ∼500-bp DAP10 transcripts were detected in human peripheral blood leukocytes, spleen, thymus, NK cells, α/β− and γ/δ−T cell receptor+ T cells, and U937 (myeloid cell), but not substantially in other tissues or JY (B lymphoblastoid cell), 293T (epithelial cell), or primary fibroblasts (Fig. 2A) (6). Analysis by the reverse transcription polymerase chain reaction (RT-PCR) indicated the presence ofDAP10 mRNA in CD4+ and CD8+ T cell clones and in monocytes, granulocytes, and dendritic cells (6,7). Thus, DAP10 is predominantly expressed in hematopoietic cells.

Figure 1

Human DAP10. The extracellular cysteine residues, the transmembrane charged residue, and a cytoplasmic signaling motif are bold. Human DAP10 cDNA (AF122904) and a splice variant (AF072844) were identified, as were mouseDAP10 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.

Figure 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 125I, 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).

Protein immunoblot analysis of the NK cell line NKL, using an affinity-purified antibody to DAP10 (anti-DAP10), revealed multiple bands migrating slower than the predicted molecular mass of 10 kD for DAP10, primarily as a result of O-linked glycosylation (Fig. 2B). The multiple bands observed after treatment with O-glycanase may be due to incomplete saccharide removal, other posttranslational modifications, or alternative splicing. To examine whether DAP10 associates with other membrane receptors, we lysed a125I-labeled polyclonal NK line and NKL in 1% digitonin to preserve multisubunit receptor complexes. Although DAP10 did not label with 125I, anti-DAP10 coprecipitated a125I-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 125I-labeled DAP10-associated proteins were detected in several NK and T cell clones (7).

The precedent for interactions between proteins of multisubunit receptor complexes through oppositely charged amino acids in their transmembranes suggested that DAP10 may pair with another ligand-binding subunit. One candidate was NKG2D, a C-type lectin encoded by a gene in the “NK complex” on human chromosome 12p12-p13 (8). The mouse pre-B cell line Ba/F3 was transfected with an NH2-terminal Flag-tagged human DAP10 cDNA (Flag-DAP10), either alone or together with human NKG2D. Stable transfectants were stained using Flag monoclonal antibody (mAb) to visualize DAP10 and NKG2D mAb to detect NKG2D (9). Cotransfection of Flag-DAP10 and NKG2D resulted in surface expression of both proteins, whereas alone they were minimally expressed (Fig. 3A). Radioiodination and coimmunoprecipitation confirmed that DAP10 and NKG2D form a stable complex on the double transfectant (7). As with other receptor complexes, charged residues in the TM of NKG2D and DAP10 were critical for complex formation because mutation of these amino acids allowed surface expression and abolished the stable association between DAP10 and NKG2D (Fig. 3B). Small amounts of Flag-DAP10 were on the surface of Ba/F3 cells expressing the NKG2D TM mutant. However, we were unable to coimmunoprecipitate the NKG2D TM mutant with DAP10 (7). Residues in the TM other than Arg may affect the pairing of NKG2D with DAP10.

Figure 3

An NKG2D-DAP10 receptor complex. (Aand B) Ba/F3 cells stably expressing the indicated receptors were stained with cIg, Flag mAb M2, or NKG2D mAb 5C6 [or 1D11 mAb (7)] and analyzed by flow cytometry. Flag-DAP10 TM and NKG2D TM contain point mutants in the transmembranes in which the charged residues were substituted with Ala and Leu, respectively (17). (C) 125I-labeled NKL cells were lysed in 1% digitonin and immunoprecipitated with cIg, affinity-purified anti-NKG2D, affinity-purified anti-DAP10, or NKG2D mAb 5C6. DAP10-associated proteins were eluted with 50 mM diethylamine (pH 12) and reimmunoprecipitated with either cIg or affinity-purified anti-NKG2D. Samples were analyzed by SDS-PAGE (reducing condition).

To establish that NKG2D is the physiological partner for DAP10, we immunoprecipitated 125I-labeled NKL and a polyclonal NK line with an affinity-purified rabbit antibody specific for the cytoplasmic domain of human NKG2D or with a human NKG2D mAb. The size of the DAP10-associated glycoprotein was identical to that of NKG2D (Fig. 3C). Moreover, NKG2D was reimmunoprecipitated from the eluate of the dissociated DAP10 complex (Fig. 3C). DAP10 could not pair with KIR2DS2 or CD94/NKG2C, two receptors associated with DAP12 (4), and DAP12 did not associate with NKG2D; these results confirmed the specificity of the interaction (7). The extracellular domains of DAP10 and DAP12 contain cysteines that form disulfide-bonded homodimers; however, heterodimers were not observed (7). NKG2D, despite its name, has limited homology with NKG2A, -C, and -E (8) and does not form heterodimers with CD94 (7).

NKG2D is an activating receptor (6) that initiates NK and T cell–mediated cytotoxicity against transfectants and tumors expressing its ligands, MICA and MICB (9). However, NKG2D lacks signaling elements in its cytoplasmic domain. A potential activation motif in the cytoplasmic domain of DAP10 is the YXXM sequence, a predicted binding site for the SH2 domain of the p85 subunit of PI 3-kinase (5). A tyrosine-phosphorylated peptide corresponding to the DAP10 cytoplasmic domain specifically bound to p85 (Fig. 4A). Treatment of NKL and NKG2D-Flag-DAP10+ Ba/F3 with the phosphatase inhibitor pervanadate resulted in DAP10 tyrosine phosphorylation and enhanced association of p85 with the NKG2D-DAP10 complex (Fig. 4B). This suggested that DAP10 functions as a signal transducer leading to PI 3-kinase activation.

Figure 4

DAP10 recruits PI 3-kinase. (A) NKL lysates were incubated with a biotinylated unphosphorylated (DAP10) or phosphorylated DAP10 peptide (biotin-PAQE- DGKVY* INMPGRG; Y* indicates the phosphotyrosine residue) (P-DAP10) and precipitated with avidin-agarose. Samples were analyzed by protein immunoblot using anti-p85 (18). (B) NKG2DDAP10+ Ba/F3 cells or NKL were stimulated with pervanadate, lysed, and immunoprecipitated with cIg, Flag mAb M2, or NKG2D mAb 5C6. Samples were analyzed by protein immunoblot using HRP-conjugated phosphotyrosine mAb 4G10 or anti-p85 (19). Blots were stripped and rehybridized with anti-Shc (specificity control).

CD28 is a well-characterized receptor with a cytoplasmic YXXM motif that activates PI 3-kinase (10). On T cells CD28 is a costimulatory molecule, whereas on NK cells CD28 initiates cytotoxicity against target cells expressing CD28 ligands (CD80 or CD86) (11). Unlike CD28, DAP10 has a small extracellular domain and is unlikely to mediate ligand binding, whereas NKG2D directly binds MICA (7, 9). Having separate components of a multisubunit receptor responsible for ligand binding and signaling may permit DAP10 to function as an adaptor molecule for other receptors (for example, in myeloid cells expressing DAP10 but lacking NKG2D). The finding that the NKG2D-DAP10 complex is a receptor for the nonclassical MHC class I molecule MICA (9) and evidence that these molecules are stress-inducible and broadly expressed in epithelial tumors (12) suggest that the activating NKG2D-DAP10 complex may be involved in innate immune surveillance.

  • * To whom correspondence should be addressed. E-mail: lanier{at}dnax.org

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