TRANSMEMBRANE SIGNALING BY LY-49

描述(改编自申请人的摘要)：本报告的目标 应用Ly-49确定跨膜信号转导机制 小鼠自然杀伤(NK)细胞上的受体。小鼠Ly-49A(mLy49-A)结合 与靶细胞H-2DD结合，阻止mLY-49A+NK细胞杀伤靶细胞。 为了研究mly-49A阻止自然死亡的途径， 申请人已经开发了一种模型，其中mLY-49A是功能表达的 在大鼠NK细胞系RNK-16中。他们还定义了12聚体多肽， 对应于H-DD的A1螺旋上的73-84个残基，它们起始于 MLy-49A的跨膜信号转导。他们创造了一个嵌合体 受体，将NK1.1的胞外结构域连接到跨膜和 MLy-49A的细胞质结构域，并用这个模型证明了 MLy-49A胞质结构域在跨膜信号转导中的作用 抑制NK细胞杀伤作用。他们现在的目标是定义 功能信号和mly-49A的性质，允许他们的 激活。为此，它们有六个具体目标：1)创建截断 以及在RNK-16中表达的mLY-49A的定点突变 对细胞毒性的功能影响。2)比较完好无损或 突变的mLy-49A产生跨膜信号以响应A1螺旋 多肽和比较信号与功能(在特定的目标1)。3)作为 额外的选择性探针，表达mLy-49A/NK1.1嵌合受体 RNK-16细胞。将跨膜信号的产生与效应相关联 对NK细胞活性的影响。4)免疫共沉淀mLy-49A 未受刺激的RNK-16.mLy-49A细胞及其相关分子的探针。5) 利用酵母双杂交系统克隆、测序和表达细胞质 小鼠IL-2激活的NK细胞与胞浆结合的蛋白质 MLY-49A的结构域。6)比较Ly-49A与Ly-49A的跨膜信号转导 Ly-49基因家族其他成员发出的信号。

DESCRIPTION (Adapted from the Applicant's Abstract): The goal of this application is to define mechanisms of transmembrane signaling by Ly-49 receptors on mouse natural killer (NK) cells. Mouse Ly-49A (mLy49-A) binds to H-2Dd on target cells and prevents mLY-49A+ NK cells from lysing targets. To examine the pathways by which mLY-49A prevents natural killing, the applicants have developed a model in which mLY-49A is functionally expressed in the rat NK cell line, RNK-16. They have also defined 12-mer peptides, corresponding to residues 73-84 from the a1 helix of H-Dd, which initiate transmembrane signaling through mLy-49A. They have created a chimeric receptor, linking the extracellular domain of NK1.1 to the transmembrane and cytoplasmic domains of mLy-49A, and have used this model to demonstrate a role for the cytoplasmic domain of mLy-49A in transmembrane signals that inhibit NK cell cytotoxicity. Their objective now is to define the functional signals and the properties of mLy-49A that permit their activation. To this end, they have six specific aims: 1) Create truncation and site-specific mutations of mLY-49A expressed in RNK-16 to determine functional effects on cytotoxicity. 2) Compare the capacity of intact or mutated mLy-49A to generate transmembrane signals in response to a1 helix peptides and compare signaling to function (in Specific Aim 1). 3) As an additional and selective probe, express mLy-49A/NK1.1 chimeric receptors on RNK-16 cells. Correlate the generation of transmembrane signals with effect on NK cell activity. 4) Immunoprecipitate mLy-49A from stimulated or unstimulated RNK-16.mLy-49A cells and probe for associated molecules. 5) Use the yeast two hybrid system to clone, sequence, and express, cytoplasmic proteins from mouse IL-2-activated NK cells that bind to the cytoplasmic domain of mLY-49A. 6) Compare transmembrane signaling by Ly-49A with signaling by other members of the Ly-49 gene family.