NORMAL AND ABNORMAL INTERFERON ALPHA RECEPTORS

I型干扰素-R至少由三个不同的亚基组成：1) IFNaR3单抗识别的α亚基(110 KDa)，2) IFNaRbeta1单抗识别的β亚基(100 KDa) 抗体，以及3)克隆的受体亚基，分子量约为 75 kDa。克隆的受体亚单位与极低水平的IFNalpha8结合 仅在37摄氏度时，尽管Daudi细胞，为 克隆，在4℃下结合所有的IFNAlpha亚型。 Cos 7细胞中表达的亚基(约75 kDa)不同于主 相对分子质量为110和100 kDa(α和β)的IFNα结合蛋白 在亲和交联实验中观察到的。 因此，有许多问题尚未得到 该受体亚基的克隆和表达。例如，1)什么 受体的亚单位是与干扰素结合所必需的吗？2)什么是 克隆的亚基的作用？以及3)有多少受体亚基 获得IFNaplha绑定所必需的？对I型干扰素-受体的认识 蛋白质和DNA水平的结构将在 了解干扰素系统的功能。在过去三年中 多年来，我们已经证明了我们的假设，包括多链结构 研制抗α-干扰素的单抗研制I型干扰素-受体 和β亚基。这些抗体是克隆必不可少的工具。 这些I型干扰素-R亚基，并进一步了解 I型干扰素-受体在其他领域，如信号转导。 I型干扰素在病毒感染和治疗中发挥核心作用 某些类型的癌症。此外，干扰素系统的一些组件 如p68激酶和IRF-1可能是肿瘤抑制基因。因此， 干扰素受体结构和功能的研究进展 对于理解干扰素系统作为第一防御系统的作用是至关重要的 在抗病毒感染以及癌症的发展和治疗方面。 在本申请中，我们建议克隆I型的α亚基 干扰素-R我们的长期目标包括克隆其他组件 以确定IFNalphaR的作用，如β亚基 IFNpha信号转导中的每个受体成分。 在本申请中，我们建议克隆I型的α亚基 干扰素-R不同的阿尔法亚基克隆方法将 用于：1)利用亲和力纯化α亚基 小麦胚芽凝集素柱层析后亲和 特异性抗α亚基单抗的层析 IFNaR3、纯化蛋白的微测序及筛选 含IFNaR3的lambdagt11和3)真核表达文库 单抗。一旦我们克隆了阿尔法亚单位的cDNA，我们就 将在人类和小鼠细胞中单独表达，并与 已经克隆的受体亚基。这种方法将使我们能够 确定α和克隆的亚基在IFNpha结合中的作用。 最后，我们将描述编码该基因的结构 基因组水平上的α亚基。该项目的这一部分还将 确认人类21号染色体上的阿尔法亚单位的定位。

The Type I IFN-R is formed by at least three different subunits: 1) the alpha subunit (110kDa) recognized by the IFNaR3 monoclonal antibody, 2) the beta subunit (100 kDa) recognized by the IFNaRbeta1 monoclonal antibody, and 3) the cloned receptor subunit with an approximate MW of 75 kDa. The cloned receptor subunit binds very low levels of IFNalpha8 only at 37 degrees C, although Daudi cell, the source of mRNA for the cloning, bind all IFNalpha subtypes at 4 degrees C. The MW of the cloned subunit expressed in Cos 7 cells (approx. 75 kDa) differs from the main IFNalpha-binding proteins with MWs 110 and 100 kDa (alpha and beta subunits,respectively) observed in affinity crosslinking experiments. Thus, there are many questions that have not been addressed by the cloning and expression of this receptor subunit. For instance, 1) what subunits of the receptor are necessary for IFNalpha binding? 2) What is the role of the cloned subunit?, and 3) How many receptor subunits are necessary to obtain IFNaplha binding? The knowledge of the Type I IFN-R structure at the protein and DNA level will be important in the understanding of the function of the IFN system. Over the last three years we have proved our hypotheses involving a multichain structure for the Type I IFN-R by developing monoclonal antibodies against the alpha and beta subunits. These antibodies ar essential tools for the cloning of these Type I IFN-R subunits and to further our knowledge about the Type I IFN-R in other areas such as signal transduction. Type I IFNs play a central role in viral infections and in the treatment of some types of cancer. Furthermore, some components of the IFN system such as the p68 kinase and IRF-1 may be tumor suppressor genes. Thus, the unraveling of the structure and function of the interferon receptor is essential to understand the role of the IFN system as a first defense against viral infections and in the development and treatment of cancer. In this application we propose to clone the alpha subunit of the Type I IFN-R. Our long term objectives involve the cloning of other component of the IFNalphaR, such as the beta subunit, and to determine the role of each receptor component in the IFNalpha signal transduction. In this application we propose to clone the alpha subunit of the Type I IFN-R. Different approaches for the cloning of the alpha subunit will be used: 1) purification of the alpha subunit using affinity chromatography on a wheat germ lectin column followed by affinity chromatography with specific anti-alpha subunit monoclonal antibody IFNaR3, microsequencing of the purified protein and screening of lambdagt11 and 3) eukaryotic expression libraries with the IFNaR3 monoclonal antibody. Once we clone the cDNA for the alpha subunit, we will express it in human and mouse cells alone and in association with the already cloned receptor subunit. This approach will allow us to determine the role of the alpha and cloned subunits in IFNalpha binding. Finally, we will characterize the structure of the gene encoding the alpha subunit at the genomic level. This part of the project will also conform the localization of the alpha subunit on human chromosome 21.