MOLECULAR BASIS OF HEMATOPOIETIC GROWTH FACTOR SIGNALING

造血是一个复杂的动态过程，在这个过程中，所有的血细胞 类型是从原始的祖细胞产生的。这个过程是 从多个层面进行监管。其中最早的一种机制 控制梯队是由一种名为白介素3的糖蛋白介导的 (IL-3)，刺激细胞的增殖和分化 原始祖先。由大爆炸引发的一连串细胞事件 IL-3与细胞表面的结合尚不完全清楚。虽然 一种潜在的细胞表面受体分子已经被生化 表征并分子克隆了编码该基因的cDNAs 提供高亲和力配体结合的受体 磷酸化和配体诱导的下调表明 生物上需要额外的多肽亚基才能形成 活性受体复合体。一种新型的抗髓系细胞的单抗， 命名为HIM-1，被发现抑制IL-3与各种 目标细胞，表明它在物理上链接到结构 它将配体结合在一起。证明这种抗体抑制了 IL-3对造血祖细胞及其结合的增殖作用 因子依赖的髓系细胞水平显著降低 随后的IL-3刺激进一步暗示了 His-1在IL-3与表面结合中识别的分子 髓系细胞。这项提案概述了对生物化学和 220kodalton糖蛋白作用的遗传学特征 (Gp220)在IL-3与细胞因子的特异性结合中被他识别 正常和白血病造血细胞的表面以及在 信号的转导以促进增殖。免疫沉淀 实验将使用保护内部的策略来进行 证明gp220直接与IL-3结合的分子联系 或者在物理上与结合IL-3的多肽有关。这个 Gp220参与具有生物活性的IL-3受体复合体 将由基因操纵其表达来决定。我们将测试 编码gp220的c DNA和低亲和力受体结合的能力 重建与IL-3高亲和力结合的结构和转导 它的生物活性。Gp220分子缺失的这种效应 高亲和力配体结合的表达及生物转导 活性，因为IL-3刺激诱导了酪氨酸特异性蛋白激酶 活性，该机制在配体诱导的降压中的参与。 将确定gp220的调制。总而言之，这些实验 应阐明控制病毒扩散的近期事件 正常的造血祖细胞并提供对分子的洞察 骨髓增生性疾病中异常增殖的基础 精神错乱。

Hematopoiesis is a complex, dynamic process in which blood cells of all types are produced from a primitive progenitor cell. This process is regulated at multiple levels. The mechanism for one of the earliest echelons of control is mediated by a glycoprotein called interleukin-3 (IL-3), which stimulates the proliferation and differentiation of these primitive progenitors. The cascade of cellular events elicited by the binding of IL-3 to the cell surface is incompletely understood. Although a potential cell surface receptor molecule has been biochemically characterized, and molecularly cloned, the inability of cDNA encoding the receptor to confer high affinity ligand binding, receptor phosphorylation, and ligand-induced down-modulation suggest that additional polypeptide subunits are required to form a biologically active receptor complex. A novel monoclonal antibody to myeloid cells, designated HIM-1, was found to inhibit the binding of IL-3 to various target cells, suggesting that it is physically linked to the structure that binds the ligand. The demonstration that this antibody inhibited the proliferative effect of IL-3 on hematopoietic progenitors and bound to factor-dependent myeloid cells in dramatically reduced levels following IL-3 stimulation further implicates the involvement of the molecule recognized by HIM-1 in the association of IL-3 with the surface of myeloid cells. This proposal outlines studies to biochemically and genetically characterize the role of the 220 kiloDalton glycoprotein (gp220) recognized by HIM-1 in the specific binding of IL-3 to the surface of normal and leukemic hematopoietic cells and in the transduction of the signal to proliferate. Immunoprecipitation experiments will be performed using strategies that preserve inter- molecular associations to demonstrate that gp220 binds directly to IL-3 or is physically associated with polypeptides that bind IL-3. The participation of gp220 in a biologically active IL-3 receptor complex will determined by genetically manipulating its expression. We will test the ability of cDNA encoding gp220 and the low affinity receptor to reconstitute a structure that binds IL-3 at high affinity and transduces its biological activity. This effect of molecular abrogation of gp220 expression on high affinity ligand binding and transduction of biologic activity, since IL-3 stimulation elicits tyrosine-specific protein kinase activity, the participation of this mechanism in ligand-induced down- modulation of gp220 will be determined. Together, these experiments should elucidate the proximal events that control the proliferation of normal hematopoietic progenitors and provide insight into the molecular basis for dysregulated proliferation as occurs in myeloproliferative disorders.