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

自然杀伤（NK）细胞能够特异性识别和溶解 某些肿瘤或病毒感染的细胞没有预先致敏， 目标的 细胞表面分子负责的特异性， NK-靶细胞相互作用未被定义。 最近的结构和 功能证据表明，细胞表面糖蛋白NKR-P1在 大鼠NK细胞可能作为一种重要的受体， 靶细胞鉴定靶细胞上结合 NKR-P1将有助于确定NK细胞应答的机制。 NKR-P1是一种II型整合膜蛋白，具有细胞外C型 凝集素结构域。 凝集素区域在结构上类似于已知的 受体分子我们的实验室已经产生了一个突变的大鼠NK细胞系 （来源于RNK- 16）缺乏NKR-P1的表达，其选择性地被 不能裂解来自C57 BL/6（H-2b）小鼠的某些靶细胞。 其它靶细胞系被突变型和野生型RNK-16同等地杀死 细胞 这些结果支持NKR-P1可能是必需的假设。 用于识别和/或裂解一些但不是全部靶细胞。 我提出的研究将集中在确定NKR-P1的配体， 靶细胞我制备了一种重组可溶性嵌合蛋白 由NKR-P1的细胞外结构域融合到NKR-P1的Fc部分组成。 人IgG 1（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，通过细胞与 固定嵌合蛋白，并通过抗体依赖性细胞介导 细胞毒 为了证明靶细胞与可溶性 嵌合rNKR-P1/Fc类似于细胞表面蛋白相互作用， 转染以表达高水平细胞表面NKR-P1的CHO细胞将 用于检查特定的细胞-细胞粘附到目标。用于以下的配体 NKR-P1将从适当的靶细胞中亲和纯化，使用 固定化rNKR-P1/Fc。推定配体的特征为： 考虑到大小、对蛋白水解的敏感性、碳水化合物含量，以及 与已知的细胞表面分子的关系。 这些研究将鉴定NKR-P1的配体，NKR-P1是一种被提出的受体， NK细胞。参与特异性的分子的鉴定 NK靶细胞识别将为了解NK细胞的作用提供重要见解 NK细胞在免疫调节中的作用以及它们如何区分 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.