MOLECULAR BASIS OF HEMATOPOIETIC GROWTH FACTOR SIGNALING

造血是一个复杂的，动态的过程，其中所有的血细胞 类型是从原始祖细胞产生的。 这个过程是 在多个层面上进行监管。 最早的一种 一系列的控制由一种叫做白细胞介素-3的糖蛋白介导 （IL-3），其刺激这些细胞的增殖和分化。 原始祖先 细胞的级联反应是由 IL-3与细胞表面的结合还不完全清楚。 虽然 一种潜在的细胞表面受体分子已经被生化地 特征在于，并分子克隆，不能cDNA编码的 赋予高亲和力配体结合的受体，受体 磷酸化和配体诱导的下调表明， 需要另外的多肽亚基来形成生物学上 活性受体复合物 一种新的抗骨髓细胞的单克隆抗体， 被命名为HIM-1，被发现抑制IL-3与各种 靶细胞，这表明它与结构有物理联系， 与配体结合 证明这种抗体抑制了 IL-3对造血祖细胞增殖及结合功能影响 因子依赖性髓样细胞的水平显著降低 IL-3刺激后进一步暗示了 在IL-3与表面结合中被HIM-1识别的分子 骨髓细胞。 该提案概述了生物化学和 从遗传学上描述了220千道尔顿糖蛋白的作用 在IL-3特异性结合HIM-1所识别的gp 220蛋白时， 正常和白血病造血细胞的表面和 信号的传导以增殖。 免疫沉淀 实验将使用保持间- 证明gp 220与IL-3直接结合的分子关联 或者与结合IL-3的多肽物理结合。 的 gp 220参与生物活性IL-3受体复合物 意志是由基因操纵其表达决定的。 我们将测试 编码gp 220和低亲和力受体的cDNA对人的免疫应答的能力， 重组以高亲和力结合IL-3的结构并转导 其生物活性。 这种gp 220的分子消除的效果 高亲和力配体结合和生物转导的表达 活性，因为IL-3刺激激发酪氨酸特异性蛋白激酶 活动，这一机制的参与配体诱导的下降- 将确定GP 220的调制。 总之，这些实验 应该阐明控制增殖的近端事件， 正常的造血祖细胞，并提供深入了解的分子 骨髓增生性疾病中发生的增殖失调的基础 紊乱

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.