STRUCTURE AND FUNCTION OF THE ERYTHROPOIETIN RECEPTOR

我们建议继续研究物理上的相互作用 促红细胞生成素受体(EPO-R)与Friend脾形成 病毒包膜蛋白，gp55(D‘Andrea，1989a；Li，1990)。这个 这两个多肽的相互作用导致了结构性 EPO-R的激活代表了逆转录病毒的一种新机制 白血病的发生。我们的工作与健康有关，因为其他逆转录病毒可能 通过类似的机制诱导恶性血液病。另外， 由EPO本身激活EPO-R已成为一种标准的治疗方法 各种疾病相关或治疗引起的贫血。在项目I期间 在建议的五年研究期内，我们会界定 EPO-R在信号转导和相互作用中起重要作用 Gp55。利用功能测定，野生型EPO-R的能力 为了使EPO依赖于IL-3依赖的细胞，我们将筛选 EPO-R的突变体，因为他们有能力赋予EPO依赖。使用 免疫共沉淀实验，我们将描绘所需的EPO-R结构域 用于与gp55结合。嵌合受体，包含 EPO-R和IL-2Rβ链或IL-3Rα的区域 链，将进一步定义这两个功能域。此外，使用 这些嵌合受体突变体我们将研究同源二聚化 EPO-R和参与这一过程的氨基酸残基。项目II 将侧重于gp55和EPO-R的功能相互作用。我们会 研究正常和异常的生物合成、碳水化合物的加工以及 配体诱导EPO-R的磷酸化及gp55的作用 有约束力的。EPO-R的其他亚基将通过交联法鉴定 和免疫共沉淀，以及各种克隆策略将被用于 分离编码这些其他亚单位的cDNA。

We propose to continue our studies of the physical interaction between the erythropoietin receptor (EPO-R) and the Friend Spleen Focus-Forming Virus envelope protein, gp55 (D'Andrea, 1989a; Li, 1990). The interaction of these two polypeptides results in the constitutive activation of the EPO-R and represents a novel mechanism of retroviral leukemogenesis. Our work is health related since other retroviruses may induce hematopoietic malignancies by analogous mechanisms. Also, the activation of the EPO-R by EPO itself has become a standard treatment for various disease related or treatment induced anemias. During Project I of the proposed 5 year study period we will delineate the domains of the EPO-R which are important in signal transduction and in interaction with the gp55. Using a functional assay, the ability of the wild-type EPO-R cDNA to confer EPO dependence on an IL-3 dependent cell, we will screen mutants of the EPO-R for their ability to confer EPO dependence. Using a coimmunoprecipitation assay, we will delineate the EPO-R domain required for binding to the gp55. Chimeric receptors, containing regions of the EPO-R and regions of either the IL-2R beta chain or the IL-3R alpha chain, will further define these two functional domains. Also, using these chimeric receptor mutants we will study the homodimerization of the EPO-R and the amino acid residues which mediate this process. Project II will focus on the functional interaction of gp55 and EPO-R. We will study the normal and abnormal biosynthesis, carbohydrate processing, and ligand-induced phosphorylation of the EPO-R and the effects of gp55 binding. Other subunits of the EPO-R will be identified by crosslinking and coimmunoprecipitation, and various cloning strategies will be used to isolate the cDNAs encoding these other subunits.