Signal Transduction Mechanisms of Erythropoietin

描述(申请人提供)：我们的长期目标是了解生长因子调节造血细胞增殖和分化的信号转导机制。促红细胞生成素(EPO)在红系分化的特定阶段刺激细胞内钙的增加，我们现在已经证明EPO通过TRPC2调节钙内流，TRPC2是Trp通道家族的成员，参与不可兴奋细胞的持续钙内流。这次更新的主要目的是了解EPO通过TRPC2调节钙内流的机制，以及TRPC2在红系增殖和分化中的功能重要性。具体目的1：鉴定促红细胞生成素受体(EPO-R)调节TRPC2通道激活的机制。四种信号转导分子在EPO依赖的调节TRPC2中的作用将在原代红细胞中确定：适配蛋白Nck 1/2和Grb7，磷脂酶C/肌醇1，4，5三磷酸，二酰甘油和G蛋白亚基Gi(2.膜定位对TRPC的功能至关重要。我们将使用共聚焦显微镜和免疫沉淀来研究TRPC2的亚细胞定位，以及与EPO-R和特定转导子的共定位。我们还将确定TRPC2是否与红系细胞上表达的其他Trp通道形成功能性多聚体。具体目标2：鉴定参与EPO调节钙内流的TRPC2结构域。我们已经建立了一个模型系统来研究TRPC的调控，使用的是将EPO-R与GFP表达载体中的EPO-R共转染的CHO细胞和与BFP相连的TRPC。用富拉红和数字视频成像在相同的单个细胞中测量细胞内游离钙。我们将使用这个系统来确定四个特定的TRPC2结构域在钙内流中的作用：钙孔、C端钙调蛋白和IP3R结合部位、亮氨酸拉链基序和核定位信号。我们还将确定在红系细胞中表达的TRPC2 N末端剪接变体的功能差异。具体目标3：确定TRPC2在红系增殖/分化中的作用。TRPC2基因敲除小鼠将被用来检测TRPC2在原代红系细胞存活、增殖、诱导凋亡和细胞周期进展中的作用。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the signal transduction mechanisms through which growth factors regulate hematopoietic proliferation and differentiation. Erythropoietin (Epo) stimulates an increase in intracellular calcium at specific stages of erythroid differentiation, and we have now demonstrated that Epo modulates calcium influx through TRPC2, a member of the family of TRP channels involved in sustained calcium entry in non-excitable cells. The major goal of this renewal is to understand the mechanisms through which Epo regulates calcium influx through TRPC2 and the functional importance of TRPC2 in erythroid proliferation and differentiation. Specific Aim 1: Identification of the Mechanisms Throuqh Which the Erythropoietin Receptor (Epo-R) Modulates TRPC2 Channel Activation. The role of four signal transducers in Epo-dependent regulation of TRPC2 will be determined in primary erythroblasts: the adaptor proteins Nck1/2 and Grb7, phospholipase C/inositol 1,4,5 trisphosphate, diacylglycerol, and the G-protein subunit Gi(2. Membrane localization is critical to the function of TRPC. We will examine TRPC2 subcellular localization, and colocalization with Epo-R and specific transducers, using confocal microscopy and immunoprecipitation. We will also determine if TRPC2 forms functional multimers with other TRP channels expressed on erythroid cells. Specific Aim 2: Identification of the TRPC2 Domains Which are Involved in Epo Modulation of Calcium Influx. We have developed a model system to study regulation of TRPC using CHO cells cotransfected with EPO-R in a GFP-expressing vector and TRPC linked to BFP. Intracellular free calcium is measured in the same single cells with Fura Red and digital video imaging. We will use this system to identify the role of four specific TRPC2 domains in calcium influx: the calcium pore, the C-terminal calmodulin and IP3R binding sites, the leucine zipper motif and the nuclear localization signal. We will also identify functional differences in TRPC2 N-terminal splice variants expressed in erythroid cells. Specific Aim 3: Determination of the Function of TRPC2 in Erythroid Proliferation/Differentiation. TRPC2 knockout mice will be used to examine the rote of TRPC2 in viability, proliferation, apoptosis induction and cell cycle progression of primary erythroid cells.