Cloning and analysis of the promoter region of 2B4, a murine NK cell receptor

We have previously identified, cloned and characterized a receptor, 2B4, expressed on murine NK cells and a subset of T cells. Functional studies indicate that ligation of 2B4 transduce activation signals and is associated with increased target cell lysis. 2B4 is a member of the immunoglobulin superfamily and shows homology to CD48, LFA-3 and SLAM. In order to further understand the transcriptional regulation of the 2B4 gene, the promoter region was subcloned and analyzed. Using nuclear protein extract from a T cell line, CTLL-2, the interaction of the promoter with nuclear proteins was studied by electrophoretic mobility shift assay (EMSA).The result showed that a 100bp 5′ fragment (-89 to +11) of the promoter region interacted with nuclear protein(s) expressed only in CTLL-2 cells, which express 2B4, whereas, such specific binding was not seen in Jurkat, Peer (both T cell line ) or Sp2/o (B cell line ) cells which do not express 2B4. Competition EMSA showed that the binding was sequence specific. Nested deletion of the 2B4 promoter region and analysis by the reporter gene chloramphenicol acetyl transferase yielded varying levels of CAT expression suggesting the presence of two cis-acting elements in the 2B4 promoter.