SIGNAL TRANSDUCTION MECHANISMS OF ERYTHROPOIETIN

这里提出的研究的长期目标是确定信号 生长因子通过其刺激 造血细胞增殖和分化。 这种知识是 对于理解造血调节障碍至关重要 包括再障和白血病。 这个项目的主要目标是 是了解促红细胞生成素（Epo） 在红细胞分化过程中调节离子通道。 该系统 一个模型来描述紧接着的信号事件， 促红细胞生成素与正常细胞上受体的相互作用。 的 钙通道的调节机制将在 正常人BFU-E衍生成红细胞的单细胞水平 不同分化阶段的质膜膜片钳 方法学、显微注射和定量荧光显微术 数字视频成像。 以下具体目标将是 地址： 具体目标1：Epo的电生理学表征- 可调节的钙通道 我们之前已经证明，Epo诱导 第10天成红细胞中胞质[Cai]增加，这是由于 Ca++内流。 钙电流将在第10天分离，BFU-E衍生 成红细胞与制霉菌素穿孔膜贴片，和 这些通道的电压依赖性和Epo对膜的影响 超极化测定。 无细胞，由内而外的补丁，细胞- 贴附贴片，并将采用制霉菌素穿孔囊泡 进一步表征单个Ca++通道并测量Ca++通道 密度的 具体目标2：确定信号传导机制， 促红细胞生成素调节钙通道。 A. 为了确定GTP- 结合蛋白调节钙通道GTP γ S、GDP β S或 将显微注射抗Gia α 1、2、3、Go α或p21 ras的抗体 进入第10天的细胞，以影响Epo刺激的[Cai]上升。 将适当的α亚单位或p21 ras显微注射至 重组促红细胞生成素反应 B。探讨EPO调节的 蛋白质磷酸化，成红细胞将用特异性 丝氨酸/苏氨酸或酪氨酸激酶或磷酸酶的抑制剂，和 [Cai]测定了 C. 为了确定磷酸肌醇水解 涉及，将显微注射IP 3和IP 4并测量[Cai]。 具体目标3：探索信号机制的差异 促红细胞生成素在红细胞成熟的不同阶段。 从一开始 7 BFU-E衍生的细胞不响应Epo而增加[Cai]， 将在第7天和第10天用免疫印迹法测量Gia α 1、2、3和Goalpha 细胞 第7天细胞上的Ca++通道密度将用 细胞附着或制霉菌素穿孔囊泡构型。 差异 在通道/受体偶联将探索与显微注射 激活的α亚单位或p21 ras。

The long term goal of studies proposed here is to determine the signal transduction mechanisms through which growth factors stimulate hematopoietic proliferation and differentiation. This knowledge is essential to understanding disorders of hematopoietic regulation including aplastic anemia and leukemia. The major goal of this project is to understand the mechanisms through which erythropoietin (Epo) regulates ion channels during erythroid differentiation. This system is a model to delineate the immediate signalling events which follow interaction of erythropoietin with its receptor on normal cells. The mechanisms of regulation of the calcium channel will be examined at the single cell level on normal human BFU-E derived erythroblasts at different stages of differentiation with plasma membrane patch-clamp methodology, microinjection, and quantitative fluorescence microscopy coupled digital video imaging. The following specific aims will be addressed: Specific Aim 1: Electrophysiologic characterization of the Epo- regulatable calcium channel. We have previously shown that Epo induces an increase in cytosolic [Cai] in day 10 erythroblasts which results from Ca++ influx. Calcium current will be isolated on day 10 BFU-E derived erythroblasts with the nystatin perforated membrane patch, and the voltage dependence of these channels and influence of Epo on membrane hyperpolarization determined. Cell-free, inside-out patches, cell- attached patches, and the nystatin perforated vesicle will be employed to further characterize single Ca++ channels and measure Ca++ channel density. Specific Aim 2: Determination of the signalling mechanisms through which erythropoietin regulates calcium channels. A. To determine whether GTP- binding proteins modulate the calcium channel GTPgammaS, GDPbetaS or antibodies to Gialpha1,2,3, Goalpha, or p21 ras will be microinjected into day 10 cells to influence the Epo-stimulated [Cai] rise. Appropriate alpha subunits or p21 ras will be microinjected to reconstitute the Epo response. B. To explore the role of EPo-modulated protein phosphorylation, erythroblasts will be treated with specific inhibitors for serine/threonine or tyrosine kinases or phosphatases and [Cai] measured. C. To determine whether inositol phosphate hydrolysis is involved, IP3 and IP4 will be microinjected and [Cai] measured. Specific Aim 3: Exploration of differences in the signalling mechanism of erythropoietin at different stages of erythroid maturation. Since day 7 BFU-E derived cells do not respond to Epo with an increase in [Cai], Gialpha1,2,3 ad Goalpha will be measured with immunoblot on day 7 and 10 cells. Ca++ channel density on day 7 cells will be measured with the cell-attached or nystatin perforated vesicle configuration. Differences in channel/receptor coupling will be explored with microinjection of activated alpha subunits or p21 ras.