PRIMARY SIGNALING EFFECTORS IN THE EPO RECEPTOR SYSTEM

血细胞发育的主要调节是通过相互作用来实施的 可溶性或膜结合的造血素与谱系特异性细胞的关系 承诺的祖细胞表面受体。受体和细胞因子 克隆使激活的结构和功能分析成为可能 机制，以及最近发现的即刻胞浆 效应器。促红细胞生成素(EPO)受体系统是一个重要的 例子：促红细胞生成素促进红细胞祖细胞的增殖 红血球终末分化所必需的；有价值的 在治疗肾脏疾病、艾滋病方面。和骨髓移植。 拟议的研究旨在首先确定EPO受体的分子基础 与两个主要效应器联合并激活； 磷脂酰肌醇-3激酶(PL3-K)与Janus蛋白酪氨酸 激酶、JAK2。EPO受体/PL3-K相互作用的研究将从 介导p85/p13-K结合的受体基序的描绘 它们的定向突变及对P13-K可能作用的检测 促红细胞生成素诱导的有丝分裂中的激活。促红细胞生成素受体/JAK2的研究 相互作用将涉及识别EPO的保守特征 介导JAK2结合的受体必需胞质亚区，以及 激活。参与EPO受体结合的JAK2结构域也将 JAK2在P13-K激活中的潜在作用将是 通过显性否定形式的异位表达进行评估。直接 EPO受体的磷酸化和变构效应也 将被测试为PL3-K激活的机制。在相关实验中 旨在定义EPO作用的新的初级效应物--受体 介导诱导晚期红系基因激活的亚区将是 已定义。在这里，杂合受体由氨基末端结构域组成 G-CSF受体和EPO受体的远端胞质结构域将 在红系细胞中表达，并检测其介导性 诱导β-maj、E-γ和α-珠蛋白基因的转录。这 模型包括一个独特的研究EPO分子途径的系统。 激活了红细胞分化。拟议的研究应该推进 对班级调节激活关键机制的理解 L细胞因子受体，以及主要的下游效应因子。

Prime regulation of blood cell development is exerted through interaction of soluble or membrane-bound hematopoietins with lineage-specific cell surface receptors on committed progenitor cells. Receptor and cytokine cloning has allowed for structure-function analyses of activation mechanisms, and the recent identification of immediate cytosolic effectors. The erythropoietin (EPO) receptor system is an important example since EPO promotes the proliferation of red cell progenitors; is required for the terminal differentiation of erythrocytes; and is valuable in the treatment of renal disease, AIDS. and marrow transplantation. Proposed studies aim firstly to define molecular bases for EPO receptor association with, and activation of, two primary effectors; phosphatidylinositol-3 kinase (Pl3-K) and the Janus protein tyrosine kinase, JAK2. Studies of EPO receptor/Pl3-K interaction will proceed from the delineation of receptor motifs which mediate p85/P13-K binding, to their directed mutagenesis and assay of the putative role for P13-K activation in EPO-induced mitogenesis. Studies of EPO receptor/JAK2 interaction will involve identification of conserved features of the EPO receptor essential cytoplasmic subdomain which mediate JAK2 binding, and activation. Domains of JAK2 involved in EPO receptor association also will be delimited, and a potential role for JAK2 in P13-K activation will be assessed via ectopic expression of dominant negative forms. Direct phosphorylation and allosteric effects exerted by the EPO receptor also will be tested as mechanisms for Pl3-K activation. in related experiments aimed at defining novel primary effectors of EPO action, receptor subdomains which mediate induced late erythroid gene activation will be defined. Here, hybrid receptors comprised of amino-terminal domains of the G-CSF receptor and distal cytoplasmic domains of the EPO receptor will be expressed in erythroid J2E cells, and assayed for the ability to mediate induced transcription of beta-maj, E-gamma, and alpha-globin genes. This model comprises a unique system for studies of molecular pathways of EPO- activated red cell differentiation. Proposed studies should advance an understanding of mechanisms critical to the regulated activation of Class l cytokine receptors, and primary downstream effectors.