GAMMA INTERFERON RECEPTOR ON TUMORILYTIC MACROPHAGES

未来五年的目标是了解如何表达 小鼠IFN γ受体（MuIFN-γ R）在 激活巨噬细胞杀死肿瘤细胞，并获得线索， 它如何传递信号。 提出这项工作的理由是， 这种受体是激活的起始物， 重要的宿主防御和体内平衡途径。 更好地了解 受体的表达是如何调节的，以及它是如何启动功能的， 应该能让我们更好地理解它所服务的途径。 那里 将有四个具体目标。 首先，单克隆抗体（MoAbs）将被 用于绘制受体的外部和内部结构域，我们是 现在的特点，将有助于在这些研究的第一。 细胞内结构域在信号转导中的作用的分析将是 通过向巨噬细胞（J774细胞系）中引入结合 空间上不同的表位 阻塞/增强 将寻求IFN γ诱导的功能。 阳离子脂质体或 电穿孔将是引入MoAb的手段。 第二、 受体mRNA的表达，新的蛋白质的数量， 从受体特异性信息合成，以及受体的表达 细胞表面上的蛋白质将在时间过程研究中定量， 激活巨噬细胞杀死肿瘤细胞的条件。 初步 研究表明，激活剂下调受体的表达， 具体信息。 这些研究中的关键试剂将是核糖探针， 我们已经从受体cDNA的3'片段和我们的单克隆抗体合成， 与受体的外部结构域结合 MoAb也将用于 用免疫沉淀法研究受体特异性蛋白质合成 生物合成放射性标记的（35 S-甲硫氨酸）受体蛋白。 三是 将从小鼠基因组粘粒文库中克隆IFN-γ R基因， 测序其外显子、内含子边界和5'侧翼区。 的 这样做的理由是，测序将提供信息， 将需要确定基因的转录调控。 第四， 我们将从功能上描述调节 编码MuIFN-γ R的基因的转录。 我们将 首先集中于定义发起人的组织， 将测试DNA片段瞬时转染到小鼠细胞中 巨噬细胞系，J774。 荧光素酶基因将被用作报告基因 这些建筑中的元素。 将寻找增强子/阻遏子序列 如果激活剂对受体mRNA表达的负调节， 不同细胞中受体蛋白表达水平的差异 类型被证明具有转录基础。 拟议的研究是 重要的是，因为负责调节表达的机制 巨噬细胞中IFN-γ R可能是调节 激活肿瘤细胞杀伤和其他炎症/免疫 这些功能可归因于IFN-γ。 关于受体如何 也可以获得转换信号。 结果应具有一般性 有趣，因为几乎所有其他有核细胞类型表达和使用 受体在自我平衡和/或防御反应。

The objectives of the next five years are to understand how expression of the receptor for mouse IFNgamma (MuIFN-gammaR) is controlled during activation of macrophages for tumor cell killing, and to obtain clues as to how it transduces signals. The rationale for proposing the work is that this receptor is the initiator of activation and a variety of other important host defensive and homeostatic pathways. Better understanding of how expression of the receptor is regulated, and how it initiates functions should lead to better understanding of the pathways that it serves. There will be four specific aims. First, monoclonal antibodies (MoAbs) will be used to map the external and internal domains of the receptor, which we are now characterizing, will be instrumental in the first of these studies. Analysis of the intracellular domain's role in signal transduction will be examined by introducing into macrophages (J774 cell line) MoAbs that bind to defined, spatially distinct epitopes. Blockage/augmentation of IFNgamma-inducible functions will be sought. Either cationic liposomes or electroporation will be the means of introducing the MoAbs. Second, expression of the receptor's mRNA, the amount of new protein that is synthesized from receptor-specific message, and expression of the receptor protein on the cell surface will be quantified in time course studies under conditions that activate macrophages for tumor cell killing. Preliminary studies suggest that activators down-regulate expression of receptor- specific message. Key reagents in these studies will be the riboprobe that we have synthesized from a 3' fragment of the receptor's cDNA and our MoAbs that bind to the receptor's external domain. The MoAbs will also be used to study receptor-specific protein synthesis by immunoprecipitating newly biosynthetically radiolabeled (35S-methionine) receptor protein. Third, we will clone the IFN-gammaR's gene from a mouse genomic cosmid library and sequence its exons, intronic boundaries and 5' flanking region. The rationale for doing so is that sequencing will provide the information that will be needed to identify the gene's transcriptional regulators. Fourth, we will functionally characterize the cis-acting sequences that regulate transcription of the gene that encodes the MuIFN-gammaR. We will concentrate initially on defining the organization of the promoter by transiently transfecting test DNA fragments into cells of the mouse macrophage line, J774. The luciferase gene will be used as a reporter element in these constructs. Enhancer/repressor sequences will be sought if either negative regulation of receptor mRNA expression by activators or differences in levels of receptor protein expression in different cell types is shown to have a transcriptional basis. The proposed research is important because the mechanisms responsible for regulating expression of the IFN-gammaR in macrophages could be important means of regulating activation for tumor cell killing and other inflammatory/immunologic functions that are attributable to IFN-gamma. Clues as to how the receptor transduces signals may also be obtained. The results should be of general interest, because virtually all other nucleated cell types express and use the receptor in homeostatic and/or defensive responses.