Kilander Michaela B C, Dahlström Jenny, Schulte Gunnar
Karolinska Institutet, Dept. of Physiology & Pharmacology, Sec. of Receptor Biology & Signaling, Nanna Svartz väg 2, S-17177 Stockholm, Sweden.
Karolinska Institutet, Dept. of Physiology & Pharmacology, Sec. of Receptor Biology & Signaling, Nanna Svartz väg 2, S-17177 Stockholm, Sweden; Institute of Experimental Biology, Masaryk University, 61137 Brno, Czech Republic.
Cell Signal. 2014 Sep;26(9):1943-9. doi: 10.1016/j.cellsig.2014.05.012. Epub 2014 May 27.
The WNT receptors of the Frizzled family comprise ten mammalian isoforms, bind WNT proteins and mediate downstream signaling to regulate stem cell fate, neuronal differentiation, cell survival and more. WNT-induced signaling pathways are either β-catenin-dependent or -independent, thereby dividing the 19 mammalian WNT proteins into two groups. So far hardly any quantitative, pharmacological information is available about WNT-FZD interaction profiles, affinities or mechanisms of signaling specification through distinct WNT/FZD pairings. This lack of knowledge originates from difficulties with WNT purification and a lack of suitable assays, such as ligand binding assays and FZD activity readouts. In order to minimize this gap, we employ fluorescence recovery after photobleaching (FRAP) to investigate WNT effects on the lateral mobility of FZD6-GFP in living cells. Pharmacological uncoupling of heterotrimeric G proteins by pertussis toxin and N-ethylmaleimide argues that changes in FZD6 mobility are related to putative precoupling of heterotrimeric Gi/o proteins to FZD6. We show that recombinant WNT-1, -2, 3A, -4, -5A, -7A, -9B and -10B affect FZD6 surface mobility and thus act on this receptor. WNT-5B and WNT-11, on the other hand, have no effect on FZD6 mobility and we conclude that they do not act through FZD6. We introduce here a novel way to assess WNT-FZD interaction by live cell imaging allowing further mapping of WNT-FZD interactions and challenging previous experimental limitations. Increased understanding of WNT-FZD selectivity provides important insight into the biological function of this crucial signaling system with importance in developmental biology, stem cell regulation oncogenesis, and human disease.
卷曲蛋白（Frizzled）家族的WNT受体包含十种哺乳动物亚型，可结合WNT蛋白并介导下游信号传导，以调节干细胞命运、神经元分化、细胞存活等。WNT诱导的信号通路分为β-连环蛋白依赖性或非依赖性，从而将19种哺乳动物WNT蛋白分为两组。到目前为止，几乎没有关于WNT-FZD相互作用谱、亲和力或通过不同WNT/FZD配对进行信号特异性传导机制的定量药理学信息。这种知识的匮乏源于WNT纯化的困难以及缺乏合适的检测方法，如配体结合检测和FZD活性读数。为了尽量缩小这一差距，我们采用光漂白后荧光恢复（FRAP）技术来研究WNT对活细胞中FZD6-GFP侧向迁移率的影响。百日咳毒素和N-乙基马来酰亚胺对异源三聚体G蛋白的药理学解偶联表明，FZD6迁移率的变化与异源三聚体Gi/o蛋白与FZD6的假定预偶联有关。我们表明，重组WNT-1、-2、-3A、-4、-5A、-7A、-9B和-10B会影响FZD6的表面迁移率，因此作用于该受体。另一方面，WNT-5B和WNT-11对FZD6迁移率没有影响，我们得出结论，它们不通过FZD6起作用。我们在此介绍一种通过活细胞成像评估WNT-FZD相互作用的新方法，这使得对WNT-FZD相互作用的进一步定位成为可能，并挑战了以往的实验局限性。对WNT-FZD选择性的深入了解为这个在发育生物学、干细胞调节、肿瘤发生和人类疾病中具有重要意义的关键信号系统的生物学功能提供了重要见解。
