Erythropoietin receptor /Janus Kinase 2 complex

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，并依赖于Janus激酶2（JAK 2）的信号 转导促红细胞生成素诱导的EpoR同源二聚化 激活JAK 2激酶活性并反过来触发下游信号 转导EpoR的异常激活可能是致癌的，并导致 白血病一些证据表明JAK 2是一个重要的亚基， EpoR，应与EpoR一起视为功能性 实体不幸的是，我们对它们的结构知之甚少。 任何细胞因子受体、JAK或其复合物的胞质结构域。 该研究将探索EpoR/JAK 2复合物的分子结构。 为了实现这一点，将进行生物化学、生物物理和功能测定。 采用一种新的功能分析将被用来研究功能 EpoR和JAK 2之间的相互作用。EpoR基因上的缺失突变 JAK 1和JAK 2之间的嵌合蛋白将有助于绘制JAK 1和JAK 2之间的基因图谱。 EpoR和JAK 2上的最小片段用于它们的相互作用。蛋白 足迹实验将确定全球相互作用的表面， EpoR/JAK 2复合物的潜在构象变化。四个具体 EpoR的胞质结构域中的残基已经被鉴定为不适合于 结合JAK 2，但其激活。这些残基可能与JAK 2相互作用 特别是开启激酶活性。这些关键残留物将 在半胱氨酸缺陷突变体EpoR上突变为半胱氨酸，并检查 在结合JAK 2或JAK 2结构域时针对化学修饰的保护 使用硫特异性试剂。与带或不带附件的附件之间的比较 在没有JAK 2或JAK 2结构域的情况下，将鉴定JAK 2上与JAK 2相互作用的区域， 与这些特定的氨基酸结合这些结果将有助于描述 特定关键残基的功能作用，也有助于构建 EpoR/JAK 2复合物的三维模型。 理解EpoR/JAK 2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 转导调节造血干细胞的分化和增殖 细胞这些信息将有助于了解致癌EpoR如何诱导 小鼠白血病，并将揭示相当多的细胞内信号 其他细胞因子的转导途径。通过这项研究， 申请人将接受生物物理学和结构生物学的培训， 获得必要的技能和知识，成为一个独立的调查员。

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.