Georgetown University Medical Center, Research Building, Room E404, Georgetown University Medical Center, 3970 Reservoir Rd., N.W., Washington, DC 20057, USA.
J Leukoc Biol. 2013 Aug;94(2):301-13. doi: 10.1189/jlb.0213093. Epub 2013 May 28.
KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules.
KIR 有助于调节 NK 细胞的活性。在这项研究中，研究了 KIR2DS 与其衔接子 DAP12 之间的相互作用的影响，超出了先前定义的信号转导功能。流式细胞术分析显示，当 NKL 细胞共转染 DAP12 时，KIR2DS 的表面表达增强。相反，当用 DAP12 特异性 siRNA 处理原代细胞时，KIR2DS4 的表面表达减少。用 CHX 或 BFA 处理 KIR2DS 和 DAP12 转染的细胞会抑制 KIR2DS 的表面表达，表明 DAP12 在将新合成的 KIR 转运到细胞表面方面发挥作用。DAP12 的免疫沉淀显示 DAP12 与 KIR2DS 的不成熟同工型相互作用，表明这种相互作用可能在 ER 内起始。内化测定表明 DAP12 对 KIR2DS 表面稳定性有重大影响。共聚焦显微镜显示，内化的 KIR2DS 分子被招募到溶酶体区室中，而与 DAP12 的表达无关。我们的研究结果表明，体内条件会不利地影响 DAP12 的表达，从而间接降低 KIR2DS 的表面表达和稳定性。这些影响可能会显著影响通过 KIR2DS 分子的配体识别和信号转导强度。
