Erythropoietin receptor /Janus Kinase 2 complex

促红细胞生成素受体/Janus 激酶 2 复合物

• 批准号: 6463604
• 负责人: LILY JUNSHEN HUANG
• 金额: $ 10.34万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6463604
• 关键词: JAK kinase; SDS polyacrylamide gel electrophoresis; antibody; autoradiography; binding sites; biological signal transduction; chimeric proteins; conformation; cytokine receptors; enzyme activity; enzyme complex; erythropoietin; gene deletion mutation; growth factor receptors; immunoprecipitation; molecular site; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; receptor expression; recombinant proteins; transfection

DESCRIPTION (provided by applicant): The erythropoietin receptor (EpoR) is the primary regulator of mammalian erythropoiesis. The EpoR lacks intrinsic catalytic activity and relies on Janus Kinase 2 (JAK2) for signal transduction. Homo-dimerization of the EpoR in response to erythropoietin activates JAK2 kinase activity and in turn triggers downstream signal transduction. Aberrant activation of the EpoR can be oncogenic and lead to leukemia. Several lines of evidence show that JAK2 is an essential subunit of the EpoR and should be considered together with the EpoR as a functional entity. Unfortunately, little information is known about the structures of the cytoplasmic domain of any cytokine receptors, JAKs, or their complexes. This fellowship will probe the molecular structure of the EpoR/JAK2 complex. To accomplish this, biochemical, biophysical, and functional assays will be used. A novel functional assay will be used to study the functional interactions between the EpoR and JAK2. Deletion mutations on both the EpoR and on JAK2, and chimeric proteins between JAK1 and JAK2 will help map the minimal segments on the EpoR and on JAK2 for their interaction. Protein footprinting experiments will identify global interaction surfaces and potential conformational changes in the EpoR/JAK2 complex. Four specific residues in the cytoplasmic domain of the EpoR have been identified not for binding JAK2 but for its activation. These residues may interact with JAK2 specifically to switch on the kinase activity. These key residues will be mutated to cysteines on a cysteine deficient mutant EpoR and examined for protection against chemical modification upon binding JAK2 or domains of JAK2 using thio-specific reagents. Comparison between the accessibilities with or without JAK2 or domains of JAK2 will identify regions on JAK2 that interact with these specific amino acids. These results will help delineate the functional roles of specific key residues and also help construction of a three dimensional model for the EpoR/JAK2 complex. Understanding of the molecular structure of the EpoR/JAK2 complex will lead to a deeper and more comprehensive understanding of how EpoR-mediated signal transduction regulates the differentiation and proliferation of hematopoietic cells. This information will help to understand how an oncogenic EpoR induces leukemia in mice, and will shed considerable light on intracellular signal transduction pathways by other cytokines. Through this research proposal, the applicant will receive training in biophysical and structural biology and will gain the necessary skills and knowledge to become an independent investigator.

描述（由申请人提供）：促红细胞生成素受体（EpoR）是 哺乳动物红细胞生成的主要调节因子。 EpoR 缺乏内在的 催化活性并依赖 Janus 激酶 2 (JAK2) 提供信号 转导。 EpoR 响应促红细胞生成素的同二聚化 激活 JAK2 激酶活性，进而触发下游信号 转导。 EpoR 的异常激活可能致癌并导致 白血病。多项证据表明 JAK2 是 EpoR 应与 EpoR 一起视为功能 实体。不幸的是，人们对它的结构知之甚少。 任何细胞因子受体、JAK 或其复合物的胞质结构域。 该奖学金将探讨 EpoR/JAK2 复合物的分子结构。 为了实现这一目标，将进行生化、生物物理和功能测定 用过的。一种新颖的功能测定将用于研究功能 EpoR 和 JAK2 之间的相互作用。 EpoR 上的缺失突变 在 JAK2 上，JAK1 和 JAK2 之间的嵌合蛋白将有助于绘制 EpoR 和 JAK2 上相互作用的最小片段。蛋白质 足迹实验将识别全局交互表面和 EpoR/JAK2 复合物中潜在的构象变化。四具体 EpoR 胞质结构域中的残基已被鉴定为不用于 结合 JAK2 但激活其。这些残基可能与 JAK2 相互作用 专门用于开启激酶活性。这些关键残基将 在半胱氨酸缺陷突变体 EpoR 上突变为半胱氨酸并检查 结合 JAK2 或 JAK2 结构域时防止化学修饰 使用硫代特异性试剂。与 或 之间的可访问性比较 没有 JAK2 或 JAK2 域将识别 JAK2 上相互作用的区域 与这些特定的氨基酸。这些结果将有助于描绘 特定关键残基的功能作用，也有助于构建 EpoR/JAK2 复合体的三维模型。 了解 EpoR/JAK2 复合物的分子结构将导致 更深入、更全面地了解 EpoR 介导的信号如何 转导调节造血细胞的分化和增殖 细胞。这些信息将有助于了解致癌 EpoR 如何诱导 小鼠白血病，将为细胞内信号提供重要线索 其他细胞因子的转导途径。通过这项研究计划， 申请人将接受生物物理和结构生物学方面的培训，并将 获得成为独立调查员所需的技能和知识。