HUMAN IL-3 RECEPTOR

人类 IL-3 受体

• 批准号: 3198329
• 负责人: ROBERT A MUFSON
• 金额: $ 21.21万
• 依托单位: AMERICAN NATIONAL RED CROSS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1997-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3198329
• 关键词: antireceptor antibody; biological signal transduction; colony stimulating factor; complementary DNA; cytokine receptors; growth factor receptors; hematopoietic stem cells; human tissue; immunoprecipitation; interleukin 3; laboratory rabbit; lipolysis; molecular cloning; monoclonal antibody; monocyte; phosphorylation; polymerase chain reaction; protein kinase C; protein sequence; protein structure; radiotracer; receptor binding; receptor expression; synthetic peptide; transfection

Human interleukin-3 (IL-3) is a pluripotent colony stimulating factor (CSF) which has a crucial role in the development of early hematopoietic progenitor cells and a role in some forms of acute lymphocytic leukemia. In order to understand the mechanism of action of IL-3 in normal human hematopoiesis as well as in leukemogenesis it is important to obtain a full biochemical and molecular analysis of the IL-3 receptor and its associated transducing mechanism in normal hematopoietic cells. This application proposes to obtain such a characterization including the cloning of a cDNA(s) for this receptor in a normal human hematopoietic cell. Although the receptor appears in low abundance on human monocytes, the recent availability of expression cloning techniques has made it possible to clone such low abundance proteins. A cDNA library has been constructed from human monocytes in a high efficiency animal cell expression vector (pcDNA1) and pools of these clones will be transfected into COS-1 cells. The transfected cells will be screened for [125I] IL-3 binding. After identifying the cDNA clone coding for the IL-3 receptor the DNA sequence of the clone will be determined and compared to sequences of known genes to determine homologies. The affinity constant of the receptor transfected into COS cells will be compared to that of the natural receptor. The application also proposes to use polymerase chain reaction cDNA amplification as a supplement to expression cloning. If the expression cloning is successful this technique can also be used to identify unique unidentified but related family members to the IL-3 receptor in human monocytes. Amino acid sequence for the IL-3 receptor and the GM-CSF receptor beta subunit deduced from the cDNA sequence, will be used to synthesize peptides for production of monospecific polyclonal antibodies in rabbits. The ability of these antibodies to precipitate the natural and recombinant proteins will be assessed using [35S] methonine labelled cells. When we have an antibody that is successful in precipitating the [35S] methonine labelled IL-3 receptor we will also attempt to precipitate the receptor bound to IL-3. This experiment should reveal whether or not the binding of IL-3 to its receptor induces the association of any accessory proteins with the IL-3 receptor. Finally, based on preliminary data indicating a role for protein kinase C in IL-3 signal transduction, the application proposes to examine the relationship of protein kinase C to IL- 3 mediated signal transduction. Examination of protein phosphorylation and phospholipid hydrolysis in response to the stimulation of human monocytes by IL-3 will be conducted. Taken together these experiments should dramatically increase our understanding of the mechanism of action of human IL-3, and should suggest further experiments to identify molecular mechanisms fundamental to the regulation of normal human hematopoiesis and leukemogenesis.

人白细胞介素-3（IL-3）是一种多能集落刺激因子（CSF） 它在早期造血细胞的发育中起着至关重要的作用， 祖细胞和某些形式的急性淋巴细胞白血病的作用。 为了了解IL-3在正常人中的作用机制， 在造血以及白血病发生中，重要的是获得完整的 IL-3受体及其相关的生物化学和分子分析 正常造血细胞中的转导机制。 本申请 建议获得这样的特征，包括克隆一个 正常人造血细胞中该受体的cDNA。 虽然 这种受体在人单核细胞上的丰度很低， 表达克隆技术的可用性使得克隆 如此低丰度的蛋白质。 cDNA文库已经构建， 高效动物细胞表达载体（pcDNA 1）中的人单核细胞 并将这些克隆的库转染到COS-1细胞中。 的 筛选转染细胞的[125 I] IL-3结合。 后 鉴定编码IL-3受体的cDNA克隆的DNA序列 将确定克隆并与已知基因序列进行比较， 确定同源性。 转染受体的亲和常数 将与天然受体的情况进行比较。 的 本申请还提出使用聚合酶链反应cDNA 扩增作为表达克隆的补充。 如果表达式 克隆是成功的，这种技术也可以用来识别独特的 人IL-3受体未鉴定但相关的家族成员 单核细胞 IL-3受体和GM-CSF的氨基酸序列 从cDNA序列推导的受体β亚基，将用于 用于生产单特异性多克隆抗体合成肽 家兔 这些抗体沉淀天然和 将使用[35 S]甲硫氨酸标记的细胞评估重组蛋白。 当我们有一种抗体能够成功地沉淀[35 S] 我们还将尝试用甲醇标记的IL-3受体沉淀 受体结合IL-3。 这个实验应该能揭示 IL-3与其受体的结合诱导任何辅助的 蛋白质与IL-3受体。 根据初步数据， 表明蛋白激酶C在IL-3信号转导中的作用， 该申请提出检测蛋白激酶C与IL-10的关系。 3介导的信号转导。 蛋白质磷酸化和 磷脂水解对人单核细胞刺激的响应 IL-3将在 这些实验应该 大大增加了我们对人类行为机制的理解， IL-3，并应建议进一步的实验，以确定分子 调节正常人造血的基本机制， 白血病发生