Schroers Andreas, Hecht Oliver, Kallen Karl-Josef, Pachta Michael, Rose-John Stefan, Grötzinger Joachim
Evotec Technologies GmbH, D-40225 Düsseldorf, Germany.
Protein Sci. 2005 Mar;14(3):783-90. doi: 10.1110/ps.041117105.
Cytokines of the interleukin-6 (IL-6)-type family all bind to the glycoprotein gp130 on the cell surface and require interaction with two gp130 or one gp130 and another related signal transducing receptor subunit. In addition, some cytokines of this family, such as IL-6, interleukin-11, ciliary neurotrophic factor, neuropoietin, cardiotrophin-1, and cardiotrophin-1-like-cytokine, interact with specific ligand binding receptor proteins. High- and low-affinity binding sites have been determined for these cytokines. So far, however, the stoichiometry of the signaling receptor complexes has remained unclear, because the formation of the cytokine/cytokine-receptor complexes has been analyzed with soluble receptor components in solution, which do not necessarily reflect the situation on the cellular membrane. Consequently, the binding affinities measured in solution have been orders of magnitude below the values obtained with whole cells. We have expressed two gp130 extracellular domains in the context of a Fc-fusion protein, which fixes the receptors within one dimension and thereby restricts the flexibility of the proteins in a fashion similar to that within the plasma membrane. We measured binding of IL-6 and interleukin-b receptor (IL-6R) by means of fluorescence-correlation spectroscopy. For the first time we have succeeded in recapitulating in a cell-free condition the binding affinities and dynamics of IL-6 and IL-6R to the gp130 receptor proteins, which have been determined on whole cells. Our results demonstrate that a dimer of gp130 first binds one IL-6/IL-6R complex and only at higher ligand concentrations does it bind a second IL-6/IL-6R complex. This view contrasts with the current perception of IL-6 receptor activation and reveals an alternative receptor activation mechanism.
白细胞介素-6(IL-6)家族的细胞因子都与细胞表面的糖蛋白gp130结合,并且需要与两个gp130相互作用,或者与一个gp130和另一个相关的信号转导受体亚基相互作用。此外,该家族的一些细胞因子,如IL-6、白细胞介素-11、睫状神经营养因子、神经营养素、心肌营养素-1和心肌营养素-1样细胞因子,与特定的配体结合受体蛋白相互作用。已经确定了这些细胞因子的高亲和力和低亲和力结合位点。然而,到目前为止,信号受体复合物的化学计量关系仍不清楚,因为细胞因子/细胞因子受体复合物的形成是用溶液中的可溶性受体成分进行分析的,这不一定反映细胞膜上的情况。因此,在溶液中测得的结合亲和力比用完整细胞获得的值低几个数量级。我们在Fc融合蛋白的背景下表达了两个gp130细胞外结构域,它在一个维度上固定受体,从而以类似于质膜内的方式限制蛋白质的灵活性。我们通过荧光相关光谱法测量了IL-6和白细胞介素-6受体(IL-6R)的结合。我们首次成功地在无细胞条件下重现了在完整细胞上测定的IL-6和IL-6R与gp130受体蛋白的结合亲和力和动力学。我们的结果表明,gp130二聚体首先结合一个IL-6/IL-6R复合物,只有在更高的配体浓度下才会结合第二个IL-6/IL-6R复合物。这一观点与目前对IL-6受体激活的认识形成对比,并揭示了一种替代的受体激活机制。