Molecular cloning and characterization of the promoter region of murine natural killer cell receptor 2B4

Abstract Natural killer (NK) cells are bone marrow-derived lymphocytes that have the ability to kill certain tumor cells and virally infected cells. The activation of NK cells is mediated by a balance of negative and positive signals from cell–cell interactions and from responses to cytokines. However, the molecular basis of NK cell activation and recognition of target cells is poorly understood. We have previously identified, cloned and characterized a receptor, 2B4, expressed on murine NK cells. 2B4 is not only expressed on all NK cells, but also on a subset of T-cells which have NK-like killing properties. Structural analysis indicated that 2B4 belongs to the CD2 subset of immunoglobulin superfamily. We have also shown 2B4 to interact with CD48 with nine times more affinity than that of CD2–CD48 interaction. In order to understand the transcriptional regulation as well as the mechanisms controlling the restricted expression of the 2B4 gene, we obtained a genomic 2B4 clone including the sequence of the 5′-flanking region. To define the start site of transcription, we performed primer extension and 5′-RACE assays and found that the 2B4 gene may be initiated at multiple start sites and driven by a TATA-less promoter. Transient transfections of nested 5′-fragments of the 2B4 promoter to drive CAT expression revealed tissue specific expression in CTLL-2 cells, a mouse T-cell line. A promoter fragment of 348 bases upstream from the first base of the mouse 2B4 cDNA clone p2B4.8 produced maximal CAT activity in CTLL-2 cells. The presence of the region −653 to −540 on the other hand, drastically reduced transcription. Sequence analysis of this promoter region has identified potential recognition motifs for a number of lymphocyte-restricted in addition to ubiquitous transcription factors, which may play a role in the transcriptional regulation of the mouse 2B4 gene.