Blanchet Marie-Hélène, Le Good J Ann, Mesnard Daniel, Oorschot Viola, Baflast Stéphane, Minchiotti Gabriella, Klumperman Judith, Constam Daniel B
Ecole Polytechnique Fédérale de Lausanne (EPFL), Swiss Institute for Experimental Cancer Research (ISREC), Epalinges, Switzerland.
EMBO J. 2008 Oct 8;27(19):2580-91. doi: 10.1038/emboj.2008.174. Epub 2008 Sep 4.
The glycosylphosphatidylinositol (GPI)-anchored proteoglycan Cripto binds Nodal and its type I receptor Alk4 to activate Smad2,3 transcription factors, but a role during Nodal precursor processing has not been described. We show that Cripto also binds the proprotein convertases Furin and PACE4 and localizes Nodal processing at the cell surface. When coexpressed as in early embryonic cells, Cripto and uncleaved Nodal already associated during secretion, and a Cripto-interacting region in the Nodal propeptide potentiated the effect of proteolytic maturation on Nodal signalling. Disruption of the trans-Golgi network (TGN) by brefeldin A blocked secretion, but export of Cripto and Nodal to the cell surface was not inhibited, indicating that Nodal is exposed to extracellular convertases before entering the TGN/endosomal system. Density fractionation and antibody uptake experiments showed that Cripto guides the Nodal precursor in detergent-resistant membranes to endocytic microdomains marked by GFP-Flotillin. We conclude that Nodal processing and endocytosis are coupled in signal-receiving cells.
糖基磷脂酰肌醇（GPI）锚定蛋白聚糖Cripto与Nodal及其I型受体Alk4结合，以激活Smad2、3转录因子，但尚未描述其在Nodal前体加工过程中的作用。我们发现，Cripto还与前蛋白转化酶弗林蛋白酶和PACE4结合，并将Nodal加工定位在细胞表面。当如在早期胚胎细胞中那样共表达时，Cripto和未切割的Nodal在分泌过程中已经相互关联，并且Nodal前肽中的一个与Cripto相互作用的区域增强了蛋白水解成熟对Nodal信号传导的影响。布雷菲德菌素A破坏反式高尔基体网络（TGN）会阻断分泌，但Cripto和Nodal向细胞表面的输出未受抑制，这表明Nodal在进入TGN/内体系统之前就已暴露于细胞外转化酶。密度分级分离和抗体摄取实验表明，Cripto将Nodal前体引导至以绿色荧光蛋白标记的弗洛蒂林（GFP-Flotillin）为标记的耐去污剂膜中的内吞微区。我们得出结论，在信号接收细胞中，Nodal加工和内吞作用是偶联的。
