LIGANDS FOR NKR P1--A RECEPTOR ON NATURAL KILLER CELLS

自然杀伤(NK)细胞能够特异性地识别和溶解 某些肿瘤或病毒感染的细胞，而不事先致敏 目标。细胞表面分子负责的特异性 NK细胞与靶细胞的相互作用没有明确的定义。最近的结构和 功能证据表明，细胞表面糖蛋白NKR-P1 大鼠NK细胞可能是一种重要的受体 目标单元格。靶细胞上与之结合的配体的鉴定 NKR-P1将有助于确定NK细胞反应的潜在机制。 NKR-P1是一种具有胞外C型结构的II型完整膜蛋白 凝集素结构域。凝集素区域在结构上与已知的相似 受体分子。我们实验室培育了一株突变的大鼠NK细胞系 (来源于RNK-16)缺乏NKR-P1的表达，选择性地 不能溶解来自C57BL/6(H-2b)小鼠的某些靶细胞。 突变型和野生型RNK-16对其他靶细胞的杀伤作用相同 细胞。这些结果支持了NKR-P1可能需要的假设 用于识别和/或裂解一些但不是全部靶细胞。 我建议的研究将集中在确定NKR-P1的配体(S)上 目标单元格。我已经准备了一种重组的可溶性嵌合蛋白 由NKR-P1的胞外区与Fc部分融合而成 人IgG1(rNKR-P1/Fc)。利用这种嵌合蛋白作为探针 配体，靶细胞将通过FACS筛选，通过细胞结合到 固定化嵌合蛋白及抗体依赖细胞介导法 细胞毒性。为了证明靶细胞与可溶性的相互作用 嵌合rNKR-P1/Fc类似于细胞表面蛋白相互作用， CHO细胞，转染后高水平表达细胞表面NKR-P1，将 用于检测特定细胞与靶细胞的粘附性。配体(S) NKR-P1将从适当的靶细胞中亲和纯化，使用 固定化rNKR-P1/Fc。假定的配体(S)将被表征为 关于大小，对蛋白质分解的敏感性，碳水化合物含量，以及 与已知的细胞表面分子的关系。 这些研究将确定NKR-P1的配体(S)，NKR-P1是一种建议的 NK细胞。特异性相关分子的鉴定 NK-靶细胞识别将为深入了解 NK细胞在免疫调节中的作用及其可能的区别 肿瘤和非肿瘤细胞。

Natural killer (NK) cells are able to specifically recognize and lyse certain tumor or virally-infected cells without prior sensitization to targets. The cell surface molecules responsible for the specificity of the NK-target cell interaction are not defined. Recent structural and functional evidence suggests that the cell surface glycoprotein NKR-P1 on rat NK cells may serve as an important receptor in the recognition of target cells. The identification of ligands on target cells which bind to NKR-P1 will help to define the mechanisms underlying the NK cell response. NKR-P1 is a type II integral membrane protein with an extracellular C-type lectin domain. The lectin region is structurally similar to known receptor molecules. Our laboratory has generated a mutant rat NK cell line (derived from RNK- 16) lacking expression of NKR-P1, which is selectively unable to lyse certain target cells derived from C57BL/6 (H-2b) mice. Other target cell lines are killed equally by mutant and wild type RNK-16 cells. These results support the hypothesis that NKR-P1 may be required for recognition and/or lysis of some but not all target cells. My proposed studies will focus on defining the ligand(s) for NKR-P1 on target cells. I have prepared a recombinant soluble chimeric protein composed of the extracellular domain of NKR-P1 fused to the Fc portion of human IgG1 (rNKR-P1/Fc). Utilizing this chimeric protein as a probe for ligand, target cells will be screened by FACS, by cell binding to immobilized chimeric protein, and by antibody-dependent cell mediated cytotoxicity. To demonstrate that target cell interactions with soluble chimeric rNKR-P1/Fc are analogous to cell surface protein interactions, CHO cells, transfected to express high levels of cell surface NKR-P1, will be used to examine specific cell-cell adhesion to targets. Ligand(s) for NKR-P1 will be affinity-purified from appropriate target cells using immobilized rNKR-P1/Fc. Putative ligand(s) will be characterized with regard to size, susceptibility to proteolysis, carbohydrate content, and relation to known cell-surface molecules. These studies will identify ligand(s) for NKR-P1, a proposed receptor on NK cells. Identification of the molecules involved in the specificity of NK-target cell recognition will provide important insight into the role of NK cells in immune regulation and how they might discriminate between tumors and non-neoplastic cells.