ERYTHROPOIETIN--STRUCTURE FUNCTION RELATIONSHIPS

造血调控的重大悬而未决的问题之一 是可溶性生长因子与特定基因相互作用的机制 造血细胞上的受体。这项提案的重点是建立 促红细胞生成素(EPO)的结构特征 它的生物合成过程和功能。定点突变，两者都 删除和氨基酸替换，将被引入EPO 基因，以测试特定的功能特性。方法将是 是为在Cos7中高水平生产正常和突变的EPO而开发的， CHO和S.rugiperda细胞以及在E.Coli和无细胞条件下 翻译系统。重组EPO产品将被提纯到 同质性和生物化学特征。以下化验结果将 旨在评估正常的短期和长期影响 和突变产物在EPO反应细胞系HCD57中的平衡 结合、内化、钙内流、细胞内pH、[~3H]-胸腺嘧啶核苷 结合，和血红素合成。对于后3个测量，HCD57 细胞将与从猪脾中分离的红系细胞进行比较。 用苯肼处理的小鼠。我们将准备盒式磁带突变体 与从初级和次级预测的表面表位相对应 结构。这些突变体的特性将提供关于 促红细胞生成素与其受体结合的结构域以及 用于内部化。将选择感兴趣的特定表位 准备更多的突变体来更好地定义这些 具有重要功能的网站。还将使用定点突变体 研究POST-2的生物合成和功能作用 翻译修饰，包括N-末端加工，切割 C-末端精氨酸，以及N-和O-连接的糖基化。这 积累的信息将用于开发基于计算机的 三维结构的预测。同时，我们将尝试 制备正常EPO和所选突变体的晶体，如果成功， 它们将通过X射线衍射法进行分析。从这些研究中，我们希望 获取有关结构与功能关系的全面信息 EPO将有助于在分子水平上理解 受体结合、内化和信号转导。

One of the major unanswered questions in the regulation of hematopoiesis is the mechanism by which soluble growth factors interact with specific receptors on hematopoietic cells. This proposal focuses on establishing the structural features of erythropoietin (Epo) that are responsible for its biosynthetic processing and function. Site-directed mutations, both deletions and amino acid replacements, will be introduced into the Epo gene in order to test specific functional properties. Methods will be developed for high level production of normal and mutant Epo's in Cos7, CHO and S. frugiperda cells as well as in E. Coli and in a cell-free translation system. The recombinant Epo products will be purified to homogeneity and characterized biochemically. the following assays will be developed to assess both short-term and long-term effects of normal and mutant products in the Epo-responsive cell line HCD57: equilibrium binding, internalization, Ca2+ influx, intracellular pH, [3H]-thymidine incorporation, and heme synthesis. For the latter 3 measurements, HCD57 cells will be compared to erythroid cells isolated from the spleens of mice treated with phenylhydrazine. We will prepare cassette mutants that correspond to surface epitopes predicted from primary and secondary structure. The properties of these mutants will provide information on the domains of Epo responsible for binding to its receptor as well as for internalization. Particular epitopes of interest will be selected for preparation of additional mutants that will better define these functionally important sites. Site-directed mutants will also be used to investigate the biosynthetic and functional roles of post- translational modifications including N-terminal processing, cleavage of C-terminal Arg, as well as N- and O- linked glycosylation. This cumulative information will be used in developing a computer based prediction of 3-dimensional structure. Concurrently we will attempt to prepare crystals of normal Epo and selected mutants, and if successful, they will be analyzed by x-ray diffraction. From these studies we hope to gain comprehensive information on structure-function relationships of Epo that will contribute to an understanding, at the molecular level, of receptor binding, internalization and signal transduction.