In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130
In vitro reconstitution of recognition and activation complexes between interleukin-6 and gp130
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DOI：
10.1021/bi010192q
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发表时间：
2001-06-26
期刊：
影响因子：
2.9
通讯作者：
Garcia, KC
中科院分区：
文献类型：
--
作者：
Chow, DC;Ho, J;Garcia, KC
GP 130是一个四螺旋细胞因子家族的共有信号转导受体，白细胞介素-6是其中的原型成员。IL-6型细胞因子激活gp 130，通过形成异源寡聚受体复合物引发下游细胞内JAK/STAT信号级联。白细胞介素-6必须首先与其特异性α受体（R α）复合以结合并激活gp 130。我们已经解剖了人gp 130的细胞外激活途径，通过重建的可溶性复合物的IL-6/IL-6 R α/gp 130信号传导复合物的分级组装的中间和最终状态。为了分离这些异源复合物，我们应用了一种蛋白质工程策略，将IL-6与其R α共价连接，这导致了我们在大肠杆菌和昆虫细胞中表达的“超活性”单链复合物（超IL-6）。我们已经确定，n-6 m-R α和gp 130（D2 D3）的精氨酸结合同源区（D2 D）形成稳定的三分子“识别”复合物（三聚体），其由1 IL-6，1 IL-6 R α，将gp 130的N-末端（D1）Ig样结构域（IGD）添加到该cDNA导致转变为含有2个IL-130的六聚体“活化”复合物。6、2个IL-6 R α和2个gp 130。这些结果清楚地表明，识别和激活复合物是不同的异源寡聚分子种类，通过所有gp 130类细胞因子上存在的独特位点III表位招募gp 130 IGD而连接。这些研究的结果与gp 130-细胞因子的IL-6家族的其他成员相关，并解决了一个长期存在的问题，即gp 130 β和IGD在活性信号寡聚体组装中的各自作用。
Gp130 is a shared signal-transducing receptor for a family of four-helix cytokines, of which interleukin-6 is a prototypic member. IL-6-type cytokines activate gp130 to elicit downstream intracellular JAK/STAT signaling cascades through formation of hetero-oligomeric receptor complexes. Interleukin-6 must first complex with its specific alpha -receptor (R alpha) in order to bind and activate gp130. We have dissected the extracellular activation pathway of human gp130 by human IL-6 through reconstitution of soluble complexes representing intermediate and final states in the hierarchical assembly of the IL-6/IL-6R alpha/ gp130 signaling complex. To isolate these hetero-complexes, we have applied a protein engineering strategy of covalently linking IL-6 to its R alpha, which results in a "hyperactive" single-chain complex (hyper-IL-6) which we express in both Escherichia coli and insect cells. We have determined that n-6m-R alpha and the cytokine-binding homology region (CHR) of gp130 (D2D3) form a stable trimolecular "recognition" complex (trimer) consisting of 1IL-6,1 IL-6R alpha, and 1 gp130-CHR. Addition of the N-terrninal (D1) Ig-like domain (IGD) of gp130 to the CHR results in a transition to a hexameric "activation" complex containing 2 IL-6, 2IL-6R alpha, and 2 gp130. These results clearly demonstrate that the recognition and activation complexes are disparate hetero-oligomeric molecular species linked by the recruitment of the gp130 IGD by the unique site III epitope present on all gp130-class cytokines. The results of these studies are relevant to other members of the IL-6 family of gp130-cytokines and address a longstanding question concerning the respective roles of the gp130 CHR and IGD in assembly of the active signaling oligomer.