Single chain TNF derivatives with individually mutated receptor binding sites reveal differential stoichiometry of ligand receptor complex formation for TNFR1 and TNFR2
Single chain TNF derivatives with individually mutated receptor binding sites reveal differential stoichiometry of ligand receptor complex formation for TNFR1 and TNFR2
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DOI：
10.1016/j.cellsig.2010.02.011
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发表时间：
2010-07-01
影响因子：
4.8
通讯作者：
Scheurich, Peter
中科院分区：
文献类型：
--
作者：
Boschert, Verena;Krippner-Heidenreich, Anja;Scheurich, Peter
肿瘤坏死因子配体家族的大多数成员形成非共价连接的同源三聚体，能够结合多达三个各自的膜受体分子。对于一些受体，已经确定了一个膜远端亲同性相互作用域，称为预配体结合组装域。因此，由典型的平衡结合研究确定的亲和值很可能受到亲和效应的影响。使用我们最近引入的共价稳定的TNF（单链TNF， scTNF），我们在这里研究了我们的表征良好的TNFR-Fas嵌合体系统中的受体-配体结合化学计量学。我们生产了具有功能缺失的单个受体结合位点的scTNF衍生物，导致TNF突变体只能结合一个或两个受体分子，而不是三个。冰与这些分子的平衡结合亲和研究表明，在单个受体结合位点被功能性删除后，没有明显的变化。相反，两个受体结合位点的功能缺失显示了TNFR2-Fas的亲和力和生物活性的强烈下降。相比之下，TNFR1-Fas配体结合和受体激活仅在所有三个受体结合位点的功能缺失后才受到影响。我们的数据证明了TNFR1- fas和TNFR2- fas之间配体/受体相互作用的关键差异，论证了对TNF结合和TNFR2的下游信号传导重要的亲和性效应，但对TNFR1的影响程度较小。这些结果得到了化学交联实验数据的支持，表明存在预形成的TNFR-Fas同型二聚体。(C) 2010爱思唯尔公司版权所有。
Most members of the tumor necrosis factor ligand family form noncovalently linked homotrimers, capable to bind up to three molecules of the respective membrane receptors. For several receptors a membrane distal homophilic interaction domain has been identified, called pre-ligand binding assembly domain. Accordingly, affinity values determined by typical equilibrium binding studies are likely to be influenced by avidity effects. Using our recently introduced covalently stabilized TNF (single chain TNF, scTNF), we have here investigated receptor-ligand binding stoichiometry in our well characterized system of TNFR-Fas chimeras. We produced scTNF derivatives with functionally deleted individual receptor binding sites, resulting in TNF mutants capable to only bind to one or two receptor molecules, rather than three. Equilibrium binding affinity studies on ice with these molecules revealed no significant changes after a single receptor binding site had been functionally deleted. In contrast, functional abrogation of two receptor binding sites showed a strong decrease in both, affinity and bioactivity on TNFR2-Fas. In contrast, TNFR1-Fas ligand binding and receptor activation was only affected after functional deletion of all three receptor binding sites. Our data demonstrate pivotal differences in ligand/receptor interactions between TNFR1-Fas and TNFR2-Fas, arguing for avidity effects important for TNF binding and downstream signaling of TNFR2, but to a lesser extent of TNFR1. These results are supported by data revealed from chemical crosslinking experiments suggesting the existence of preformed TNFR-Fas homodimers. (C) 2010 Elsevier Inc. All rights reserved.