Signal Transduction Mechanisms of Erythropoietin

描述（由申请人提供）：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）在红细胞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TRPC 2调节钙内流，TRPC 2是参与非兴奋细胞中持续钙内流的TRP通道家族的成员。本更新的主要目标是了解Epo通过TRPC 2调节钙内流的机制以及TRPC 2在红系细胞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）通过其调节TRPC 2通道活化的机制。将在原代成红细胞中确定四种信号转导物在TRPC 2的Epo依赖性调节中的作用：衔接蛋白Nck 1/2和Grb 7、磷脂酶C/1，4，5-三磷酸肌醇、二酰基甘油和G蛋白亚基Gi（2.膜定位对TRPC的功能至关重要。我们将研究TRPC 2亚细胞定位，共定位与Epo-R和特定的换能器，使用共聚焦显微镜和免疫沉淀。我们还将确定TRPC 2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：鉴定参与钙内流的Epo调节的TRPC 2结构域。我们已经开发了一个模型系统来研究TRPC的调节，使用CHO细胞共转染EPO-R的GFP表达载体和TRPC连接到BFP。用Fura Red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TRPC 2结构域在钙内流中的作用：钙孔，C-末端钙调素和IP 3R结合位点，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TRPC 2 N-末端剪接变体的功能差异。具体目的3：确定TRPC 2在红系细胞增殖/分化中的功能。TRPC 2基因敲除小鼠将用于检测TRPC 2在原代红系细胞的活力、增殖、凋亡诱导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.