GAMMA INTERFERON RECEPTOR ON TUMORILYTIC MACROPHAGES

未来五年的目标是了解如何表达 小鼠干扰素-γ受体(MuIFN-Gammar)在 激活巨噬细胞以杀死肿瘤细胞，并获得关于 它是如何传递信号的。提出这项工作的理由是 这种受体是激活的启动者和其他各种 重要的宿主防御和动态平衡途径。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 应该有助于更好地理解它所服务的路径。那里 将有四个具体目标。首先，单抗(MoAbbs)将是 用来映射受体的外部和内部结构域，我们 现在正在刻画，将在第一个这些研究中发挥作用。 分析细胞内结构域在信号转导中的作用将是 通过向巨噬细胞(J774细胞系)导入结合的MoAb来进行检测 到明确的、空间上不同的表位。阻塞/增强 将寻求IFNGamma诱导函数。阳离子脂质体或 电穿孔将是引入MoAbs的手段。第二, 受体的mRNA的表达，即新的蛋白质的量 由受体特异性信息合成，以及受体的表达 在时间进程研究中，细胞表面的蛋白质将被量化 激活巨噬细胞以杀死肿瘤细胞的条件。初步 研究表明，激活剂下调受体-2的表达 特定的消息。这些研究中的关键试剂将是核探针 我们已经用受体的3‘片段和我们的MoAbs合成了 与受体的外部结构域结合。MoAbs也将被使用 免疫沉淀法研究受体特异性蛋白质合成的新进展 生物合成放射性标记(35S-蛋氨酸)受体蛋白。第三，我们 将从小鼠基因组粘粒文库中克隆干扰素-伽玛基因，并 测序其外显子、内含子边界和5‘侧翼区。这个 这样做的理由是，测序将提供以下信息 将需要确定该基因的转录调控因子。第四, 我们将从功能上描述调控顺式作用序列 转录编码MuIFN-Gammar的基因。我们会 最初集中于通过以下方式定义发起人的组织 将测试DNA片段瞬时导入小鼠细胞 巨噬细胞系，J774。荧光素酶基因将被用作报告 元素在这些构造中。将寻找增强子/抑制子序列 如果激活剂或激活剂对受体mRNA表达的负调控 不同细胞中受体蛋白表达水平的差异 类型被证明有转录的基础。拟议的研究是 重要的是因为负责调节基因表达的机制 巨噬细胞中的干扰素-γ可能是调节的重要手段 激活肿瘤细胞杀伤和其他炎症/免疫 可归因于干扰素-伽马的功能。关于受体如何 也可以获得换能器信号。结果应该是一般性的 兴趣，因为几乎所有其他有核细胞类型都表达和使用 动态平衡和/或防御反应中的受体。

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.