nature immunology • volume 3 no 12 • december 2002 • www.nature.com/natureimmunology A RTICLES 1142 Andreas Diefenbach 1 , Elena Tomasello 2 , Mathias Lucas 2 , Amanda M. Jamieson 1 , Jennifer K. Hsia 1 , Eric Vivier 2 and David H. Raulet 1 Published online 11 November 2002; doi:10.1038/ni858 Optimal lymphocyte activation requires the simultaneous engagement of stimulatory and costimulatory receptors. Stimulatory immunoreceptors are usually composed of a ligand-binding transmembrane protein and noncovalently associated signal-transducing subunits. Here, we report that alternative splicing leads to two distinct NKG2D polypeptides that associate differentially with the DAP10 and KARAP (also known as DAP12) signaling subunits. We found that differential expression of these isoforms and of signaling proteins determined whether NKG2D functioned as a costimulatory receptor in the adaptive immune system (CD8 + T cells) or as both a primary recognition structure and a costimulatory receptor in the innate immune system (natural killer cells and macrophages). This strategy suggests a rationale for the multisubunit structure of stimulatory immunoreceptors. 1 Department of Molecular and Cell Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720-3200, USA. 2 Centre d’Immunologie de Marseille-Luminy, INSERM-CNRS-Univ. Méditerranée, 13288 Marseille cedex 09, France. Correspondence should be addressed to D. H. R. (raulet@uclink4.berkeley.edu) or E.V. (vivier@ciml.univ-mrs.fr). Selective associations with signaling proteins determine stimulatory versus costimulatory activity of NKG2D Antigen receptors and other stimulatory receptor complexes expressed by lymphoid and myeloid cells typically transduce intracellular signals by associating with immunoreceptor tyrosine-based activation motif (ITAM)–bearing signaling polypeptides, such as CD3ζ, immunoglobu- lin α (Igα, also known as CD79a) Igβ (CD79b) and FcRγ. Killer cell–activating receptor–associated polypeptide (KARAP 1 , also known as DAP12 2 ) is another ITAM-containing signaling adaptor molecule that is associated with various receptors expressed by natural killer (NK) and myeloid cells 3,4 . The immunoreceptors associate with the sig- naling adaptors via interactions between charged or polar amino acid residues in the transmembrane domains. Receptor engagement induces tyrosine phosphorylation of the ITAMs by Src family protein tyrosine kinases (PTKs), recruitment and activation of Syk–ZAP-70 family PTKs and downstream signaling events. Full activation often depends on engagement of a second (or costimulatory) receptor, such as CD28, inducible costimulator (ICOS) or CD19, which provides another degree of control over the immune response. Costimulatory receptors lack ITAMs, but display an intracytoplasmic YxxM motif, the phosphoryla- tion of which leads to recruitment and activation of phosphatidylinosi- tol-3 kinase (PI3K) family lipid kinases. NKG2D is an activating cell surface receptor that reportedly associates selectively with DAP10, an YxxM-bearing adaptor protein 5–7 . In the mouse, NKG2D is expressed by NK cells, activated macrophages, activated CD8 + T cells, γδ T cells and NK1.1 + T cells 8,9 . Cell surface ligands for NKG2D include the retinoic acid early transcript 1 (Rae-1) and H-60 protein families in mice 8,10 and the MIC 11 and ULBP families 12 in humans. Some of these ligands are up-regulated on transformed, infected or distressed cells 13,14 . Ligand expression by a tumor cell leads to potent activation of NK cells and CD8 + T cells and induction of tumor immunity 15,16 . Consistent with the signaling properties of DAP10, NKG2D engagement provides a costimulatory rather than primary stimulatory signal to activated CD8 + T cells 9,15,17 . Evidence suggests that NKG2D provides a direct stimula- tory signal rather than a costimulatory signal in NK cells and activated macrophages 9 . Because receptors that deliver stimulatory signals in some cells and costimulatory signals in others have not been demon- strated, we investigated here the basis of the differential signaling prop- erties of NKG2D in distinct cell types. Results Regulated expression of NKG2D splice variants A comparison of two murine NKG2D cDNA sequences 18,19 revealed differences in both the 5′ and 3′ ends (Fig. 1a). Alignment with genomic sequences indicated that the two cDNAs had been generated by alternative splicing from a single gene encoding NKG2D (D6H12S2489E). The two open-reading frames (ORFs) were identical except for the 5′ end: one sequence—NKG2D long (NKG2D-L)— contained an additional upstream AUG codon compared to the other sequence—NKG2D short (NKG2D-S). This resulted in a predicted 13–amino acid extension at the NH2 terminus of the protein (that is, in the cytoplasmic domain of these type II transmembrane proteins). Analysis of mRNA by semiquantitative polymerase chain reaction (PCR) demonstrated that the two isoforms were expressed differen- tially in distinct cell types and were regulated by cellular activation. Freshly isolated “naïve” NK cells contained large amounts of © 2002 Nature Publishing Group http://www.nature.com/natureimmunology