Duronio V, Clark-Lewis I, Federsppiel B, Wieler J S, Schrader J W
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1992 Oct 25;267(30):21856-63.
The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) consist of two polypeptides each belonging to a new class of molecules referred to as the hemopoietin receptor family. When expressed alone, receptor polypeptides of this family often bind their respective factors with lower affinity than the receptors identified in whole cells. Despite the lack of structural evidence for any enzymatic activity of the receptor polypeptides, both IL-3 and GM-CSF stimulate tyrosine phosphorylation of multiple intracellular substrates. We investigated IL-3 and GM-CSF receptor structure and signaling in a myeloid cell line, FDC-P1, which is dependent on either IL-3 or GM-CSF for growth. Antiphosphotyrosine antibodies were used to immunoprecipitate tyrosine-phosphorylated proteins from 32P-labeled cells or to probe immunoblots. Both IL-3 and GM-CSF stimulated the phosphorylation of a similar pattern of polypeptides on tyrosine. One tyrosine phosphorylated polypeptide migrated with M(r) = 135,000 and increased to 150,000 over a period of 10 min following stimulation of cells with IL-3 or GM-CSF. The M(r) = 135,000-150,000 polypeptide phosphorylated in response to IL-3 was shown to be primarily the Aic-2A polypeptide, the low affinity IL-3 receptor. Phosphatase treatment showed that the dramatic IL-3-induced shift in apparent molecular weight from M(r) = 125,000 in unstimulated cells was entirely due to phosphorylation. The closely related receptor, Aic-2B, was also tyrosine phosphorylated in response to IL-3, although to a lesser extent than Aic-2A. Treatment with GM-CSF resulted in tyrosine phosphorylation of the Aic-2B polypeptide exclusively. It was intriguing that GM-CSF treatment did affect the mobility of the Aic-2A polypeptide on polyacrylamide gels. Together, these results suggest that the Aic-2A polypeptide is part of the IL-3 receptor complex, but not the GM-CSF receptor. In contrast, the Aic-2B polypeptide is a component of the GM-CSF receptor, but it can also be utilized in an IL-3 receptor.
白细胞介素-3（IL-3）和粒细胞-巨噬细胞集落刺激因子（GM-CSF）的受体均由两条多肽组成，每条多肽都属于一类新的分子，即造血因子受体家族。当单独表达时，该家族的受体多肽与其各自因子的结合亲和力通常低于在完整细胞中鉴定出的受体。尽管缺乏受体多肽具有任何酶活性的结构证据，但IL-3和GM-CSF均能刺激多种细胞内底物的酪氨酸磷酸化。我们在一种髓样细胞系FDC-P1中研究了IL-3和GM-CSF受体的结构及信号传导，该细胞系的生长依赖于IL-3或GM-CSF。抗磷酸酪氨酸抗体用于从经32P标记的细胞中免疫沉淀酪氨酸磷酸化蛋白，或用于探测免疫印迹。IL-3和GM-CSF均刺激了相似模式的多肽酪氨酸磷酸化。一种酪氨酸磷酸化的多肽迁移率为M(r)=135,000，在用IL-3或GM-CSF刺激细胞后的10分钟内增加到150,000。响应IL-3而磷酸化的M(r)=135,000 - 150,000多肽被证明主要是Aic-2A多肽，即低亲和力IL-3受体。磷酸酶处理表明，IL-3诱导的未刺激细胞中表观分子量从M(r)=125,000的显著变化完全是由于磷酸化。密切相关的受体Aic-2B也会响应IL-3发生酪氨酸磷酸化，尽管程度低于Aic-2A。用GM-CSF处理仅导致Aic-2B多肽的酪氨酸磷酸化。有趣的是，GM-CSF处理确实影响了Aic-2A多肽在聚丙烯酰胺凝胶上的迁移率。总之，这些结果表明Aic-2A多肽是IL-3受体复合物的一部分，但不是GM-CSF受体的一部分。相反，Aic-2B多肽是GM-CSF受体的一个组分，但它也可用于IL-3受体。
