2E, top row), despite lower levels of expression overall (Fig. an intracellular protein. (28) Results NKG2E is translated and associates with CD94 and DAP12, but does not translocate to the cell surface To first assess whether or not NKG2E could form a complex with CD94 and DAP12, we transfected the human renal epithelial 293T cell line and the murine Ba/F3 B cell line previously used to study CD94 with CD94, DAP12, and either NKG2E or NKG2C expressing plasmids, with both NKG2 members tagged with C-terminal MYC(14). We then stained for surface MYC, and found that while NKG2C was present on the cell surface as expected, NKG2E-transfected cells expressed minimal NKG2E extracellularly (Fig. 1A, left panels). The presence of both proteins after transfection and lack of NKG2 expression in untransfected controls was confirmed by immunoblotting (Fig. 1A, right panel). To verify that the lack of NKG2E surface expression was not caused by an unanticipated side effect of tagging with MYC, we transfected Ba/F3 cells with N-terminal FLAG tagged DAP12, CD94, and NKG2E or C cloned into bicistronic IRES-EGFP. EGFP positive cells were then surface stained using anti-CD94 and anti-FLAG antibodies. We found that NKG2C transfection led to Rabbit Polyclonal to OPRD1 major upregulation of CD94 and DAP12 on the surface compared to cells transfected with CD94 and DAP12 alone, whereas the presence of NKG2E failed to induce surface expression (Fig. 1B). Overall, these data demonstrate that NKG2E transcripts are translated within cells, but NKG2E proteins fail to be expressed on cell surfaces in the presence of CD94 and DAP12. NKG2E forms an intracellular complex with CD94 and DAP12 Given the finding that NKG2E is not present on cell surfaces, we were intrigued by the possibility that NKG2E might form intracellular complexes with CD94 and DAP12. We first sought to determine whether NKG2E is capable of retaining CD94 within the intracellular compartment. To study a potential effect of NKG2E on surface CD94 expression, we transfected 293T cells with CD94 alone, CD94 with DAP12, or CD94, DAP12, and NKG2E or C. Cells were then stained in two steps-first with anti-CD94 conjugated to APC for surface detection, then with anti-CD94-PE for intracellular detection after fixation and permeabilization. We found that 5,6-Dihydrouridine there was a basal level of surface CD94 expression that was unaffected by the presence of DAP12, and that co-expression of NKG2C caused a significant increase in CD94 extracellularly (Fig. 2A). In contrast, transfection with NKG2E resulted in substantial downregulation of surface CD94 compared to cells transfected with CD94 alone or CD94 in conjunction with DAP12, suggesting that NKG2E not only fails to be expressed on cell surfaces, but that it additionally is capable of preventing surface CD94 expression. Open in a separate window Figure 2 NKG2E forms an intracellular complex with DAP12 and CD94, and is trafficked to the ER but not the plasma membrane. (A) 293T cells 5,6-Dihydrouridine were transfected with CD94 alone (first panel), CD94 and DAP12 (second panel), CD94/DAP12/NKG2C (third panel), or CD94/DAP12/NKG2E (fourth panel). Cells were stained 5,6-Dihydrouridine for surface expression of CD94 and were permeabilized and stained for intracellular CD94. Data are representative of at least three independent experiments. (B) 293T 5,6-Dihydrouridine cells were transfected with CD94, DAP12, and either NKG2C (left panels) or NKG2E (right panels) and assessed by confocal microscopy. CD94 is shown in green while differential interference contrast (DIC) is shown in gray for all samples. Pan-cadherin is shown in red in the top two panels, while protein disulfide-isomerase (PDI) is shown in red on the bottom two panels. Images are representative of at least three independent experiments. (C) NKG2C and NKG2E have identical transmembrane domains, suggesting that NKG2E can associate with CD94. (D) DAP12 associates.