HUMAN IL-3 RECEPTOR

人白细胞介素3(IL-3)是一种多能集落刺激因子 它在早期造血的发展中起着至关重要的作用 祖细胞和在某些形式的急性淋巴细胞性白血病中的作用。 为了解IL-3在正常人体内的作用机制 在造血和白血病发生过程中，获得一个完整的 IL-3受体及其相关分子的生化和分子分析 正常造血细胞的转导机制。此应用程序 建议获得这样的表征，包括克隆一个 该受体的c DNA(S)在正常的人造血细胞中。虽然 该受体在人类单核细胞上的丰度很低，最近 表达克隆技术的出现使克隆成为可能 如此低丰度的蛋白质。构建了一个cdna文库。 人单核细胞高效动物细胞表达载体(PcDNA1)的构建 这些克隆的池将被导入COS-1细胞。这个 将对转基因细胞进行[125I]IL-3结合筛查。之后 白介素3受体编码DNA序列的克隆鉴定 克隆将被确定，并与已知基因的序列进行比较 确定同源性。转染型受体的亲和常数 进入COS细胞将与天然受体进行比较。这个 应用程序还建议使用聚合酶链式反应c DNA 扩增作为表达克隆的补充。如果表达式 克隆是成功的这项技术也可以用来识别唯一的 人类IL-3受体的未知但相关的家族成员 单核细胞。IL-3受体和GM-CSF的氨基酸序列 从cdna序列推导出的受体β亚基将用于 合成多肽用于生产单特异性多克隆抗体 兔子。这些抗体能够沉淀天然的和 重组蛋白将使用[35S]蛋氨酸标记的细胞进行评估。 当我们有一种抗体成功地沉淀了[35S] 蛋氨酸标记的IL-3受体，我们还将尝试沉淀 受体与IL-3结合。这项实验应该会揭示出 IL-3与其受体的结合可诱导任何辅助成分的结合 与IL-3受体有关的蛋白质。最后，根据初步数据 表明蛋白激酶C在IL-3信号转导中的作用 应用建议研究蛋白激酶C与白介素2的关系 3介导的信号转导。蛋白质磷酸化和蛋白质磷酸化的检测 磷脂对人单核细胞刺激的反应 将通过IL-3进行。总而言之，这些实验应该 极大地提高了我们对人类活动机制的理解 IL-3，并应建议进行进一步的实验以识别分子 调节正常人类造血的基本机制和 白血病的发生。

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.