PRIMARY SIGNALING EFFECTORS IN THE EPO RECEPTOR SYSTEM

EPO 受体系统中的主要信号传导效应器

• 批准号: 2444015
• 负责人: DON Michael WOJCHOWSKI
• 金额: $ 14.94万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444015
• 关键词: allosteric site; biological signal transduction; cell cycle; chemical association; colony stimulating factor; enzyme activity; erythroid stem cell; erythropoiesis; erythropoietin; gene expression; gene induction /repression; genetic regulation; genetic transcription; globin; growth factor receptors; phosphatidylinositols; phosphorylation; protein structure function; protein tyrosine kinase; receptor binding; receptor expression; site directed mutagenesis; tissue /cell culture

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.

血细胞发育的主要调节是通过相互作用来实现的 可溶性或膜结合造血素与谱系特异性细胞 定向祖细胞表面受体。受体和细胞因子 克隆允许对激活进行结构-功能分析 机制，以及最近发现的直接细胞质 效应器促红细胞生成素（EPO）受体系统是一个重要的 例如，由于EPO促进红细胞祖细胞的增殖， 红细胞的终末分化所需;并且是有价值的 肾脏疾病和艾滋病的治疗和骨髓移植 建议的研究旨在首先确定EPO受体的分子基础 与两个主要效应物的关联和激活; 磷脂酰肌醇-3激酶（Pl 3-K）和Janus蛋白酪氨酸 激酶，JAK 2。EPO受体/P13-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， 它们的定向诱变和P13-K的推定作用的测定 在EPO诱导的有丝分裂中的激活。EPO受体/JAK 2的研究 相互作用将涉及鉴定EPO的保守特征 介导JAK 2结合的受体必需胞质亚结构域，和 activation.参与EPO受体结合的JAK 2结构域也将 JAK 2在P13-K激活中的潜在作用将是 通过显性阴性形式的异位表达进行评估。直接 由EPO受体发挥的磷酸化和变构作用也 将作为P13-K激活的机制进行测试。在相关实验中， 目的在于定义EPO作用的新的主要效应物，受体， 介导诱导的晚期红细胞基因活化的亚结构域将被 定义了在此，由多核苷酸的氨基末端结构域组成的杂合受体被称为杂合受体。 G-CSF受体和EPO受体的远端胞质结构域将被阻断。 在红系J2 E细胞中表达，并测定其介导 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系统，用于研究EPO的分子途径， 激活红细胞分化。拟议的研究应推动 理解对类的调节激活至关重要的机制 l细胞因子受体和初级下游效应物。