Natural killer cell and T cell subsets express at their cell surface a repertoire of receptors for MHC class I molecules, the natural killer cell receptors (NKRs). NKRs are characterized by the existence of inhibitory and activating isoforms, which are encoded by highly homologous but separate genes present in the same locus. Inhibitory isoforms express an intracytoplasmic immunoreceptor tyrosine-based inhibition motif, whereas activating isoforms lack any immunoreceptor tyrosinebased inhibition motif but harbor a charged amino acid residue in their transmembrane domain. We previously characterized KARAP (killer cell activating receptorassociated protein), a novel disulfide-linked tyrosinephosphorylated dimer that selectively associates with the activating NKR isoforms. We report here the identification of the mouse KARAP gene, its localization on chromosome 7 and its genomic organization in five exons. Point mutation and transfection studies revealed that KARAP is a novel signaling transmembrane subunit whose transduction function depends on the integrity of an intracytoplasmic immunoreceptor tyrosinebased activation motif. In contrast to previous members of the immunoreceptor tyrosine-based activation motif polypeptide family, KARAP is ubiquitously expressed on hematopoietic and nonhematopoietic cells, suggesting its association with a broad range of activating receptors in a variety of tissues.

Subsets of T and natural killer (NK) lymphocytes express the CD94-NKG2A heterodimer, a receptor for major histocompatibility complex class I molecules. We show here that engagement of the CD94-NKG2A heterodimer inhibits both antigen-driven tumor necrosis factor (TNF) release and cytotoxicity on melanoma-specific human T cell clones. Similarly, CD16-mediated NK cell cytotoxicity is extinguished by cross-linking of the CD94-NKG2A heterodimer. Combining in vivo and in vitro analysis, we report that both I/VxYxxL immunoreceptor tyrosine-based inhibition motifs (ITIM) present in the NKG2A intracytoplasmic domain associate upon tyrosine phosphorylation with the protein tyrosine phosphatases SHP-1 and SHP-2, but not with the polyinositol phosphatase SHIP Determination of the dissociation constant, using surface plasmon resonance analysis, indicates that NKG2A phospho-ITIM interact directly with the SH2 domains of SHP-1 and SHP-2 with a high affinity. Engagement of the CD94-NKG2A heterodimer therefore appears as a protein-tyrosine phosphatase-based strategy that negatively regulates both antigen-induced T cell response and antibody-induced NK cell cytotoxicity. Our results suggest that this inhibitory pathway sets the threshold of T and NK cell activation.