TRANSMEMBRANE SIGNALING BY LY-49

描述（改编自申请人的摘要）：本发明的目标是： 应用是定义Ly-49跨膜信号传导的机制 小鼠自然杀伤（NK）细胞上的受体。 小鼠Ly-49 A（mLy 49-A）结合 靶细胞上的H-2Dd，并阻止mLY-49 A + NK细胞裂解靶标。 为了检查mLY-49 A阻止自然杀伤的途径， 申请人已经开发了一种模型，其中mLY-49 A被功能性表达 在大鼠NK细胞系RNK-16中。 他们还定义了12-mer肽， 对应于H-Dd的α 1螺旋的残基73-84， 通过mLy-49 A的跨膜信号传导。 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区， mLy-49 A的胞质结构域，并使用该模型来证明 mLy-49 A的胞质结构域在跨膜信号中的作用， 抑制NK细胞毒性。 他们现在的目标是定义 功能信号和mLy-49 A的性质，允许它们的 activation. 为此，他们有六个具体目标：1）创建截断 和在RNK-16中表达的mLY-49 A的位点特异性突变，以确定 对细胞毒性的功能影响。 2)比较完整或 突变的mLy-49 A产生响应α 1螺旋的跨膜信号 肽并比较信号传导与功能（在具体目标1中）。 3)作为 另外的和选择性的探针，表达mLy-49 A/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)免疫沉淀mLy-49 A， 未刺激的RNK-16.mLy-49 A细胞和相关分子的探针。 第五章） 利用酵母双杂交系统克隆、测序和表达， 来自小鼠IL-2激活的NK细胞的蛋白质，其与细胞质结合 mLY-49 A的结构域。 6)比较Ly-49 A的跨膜信号传导与 Ly-49基因家族其他成员的信号传导。

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.