STRUCTURE AND FUNCTION OF THE ERYTHROPOIETIN RECEPTOR

我们建议继续研究 促红细胞生成素受体（EPO-R）与Friend脾灶形成 病毒包膜蛋白，gp 55（D 'Andrea，1989 a; Li，1990）。 的 这两种多肽的相互作用导致组成性的 EPO-R的激活，代表了逆转录病毒的一种新机制。 白血病发生 我们的工作与健康有关，因为其他逆转录病毒可能 通过类似机制诱导造血系统恶性肿瘤。 还 通过EPO本身激活EPO-R已经成为一种标准治疗方法， 各种疾病相关或治疗诱导的贫血。 在项目I期间 在拟议的5年研究期内，我们将划定 EPO-R在信号转导和与EPO-R相互作用中起重要作用， GP55。 使用功能测定，野生型EPO-R的能力， cDNA赋予EPO对IL-3依赖性细胞的依赖性，我们将筛选 EPO-R突变体赋予EPO依赖性的能力。 使用 免疫共沉淀试验，我们将描绘EPO-R结构域所需 用于结合到GP 55。 嵌合受体，含有 EPO-R和IL-2 R β链或IL-3R α的区域 链，将进一步定义这两个功能域。 此外，使用 我们将研究这些嵌合受体突变体的同源二聚化， EPO-R和介导这一过程的氨基酸残基。 项目二 将集中在gp 55和EPO-R的功能相互作用。 我们将 研究正常和异常的生物合成，碳水化合物加工， 配体诱导的EPO-R磷酸化和gp 55的作用 约束力 EPO-R的其他亚基将通过交联来鉴定。 和免疫共沉淀，以及各种克隆策略将用于 分离编码这些亚基的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.